Application of Pharbitis nil and its extract protein in the preparation of anti-tumor drugs

Abstract

The present invention discloses the application of Pharbitis nil and its extract protein in the preparation of anti-tumor drugs, specifically relating to the method for extracting protein from Pharbitis nil, so as to make new progress in the research on the application of Pharbitis nil and its extract protein.

Description

Technical Field

[0001] The present invention belongs to the application field of Pharbitidis Semen and its extracts, and particularly relates to the application of Pharbitidis Semen and its extract protein in the preparation of anti-tumor drugs. Background Art

[0002] Pharbitidis Semen is the dry and mature seed of Pharbitis nil (L.) Choisy or Pharbitis purpurea (L.) Voigt of the Convolvulaceae family, which can be divided into black Pharbitidis Semen and white Pharbitidis Semen. In past dynasties, most herbal medicines considered it bitter in taste, cold in nature, and toxic, with the effects of promoting defecation by purging water, eliminating phlegm and removing fluid-retention, and killing parasites and attacking accumulation. Research has found that Pharbitidis Semen mainly contains components such as pharbitin, phenolic acid compounds, alkaloids, saccharides, proteins, sterol compounds, pigments, and fatty oils. Modern pharmacological research shows that Pharbitidis Semen has activities such as promoting defecation by purging, diuresis, anti-inflammation, enhancing immunity, exciting the uterus, and anti-tumor. Currently, the reported anti-tumor active components of Pharbitidis Semen mainly focus on the alcohol extract, and its alcohol extract can inhibit the growth of tumor cells such as colorectal cancer, colon cancer, liver cancer, breast cancer, and lung cancer. Protein is a type of chemical component that is commonly present and has a relatively high content in sub-category medicinal materials, and it plays an important role in the processes of plant growth, development, metabolism, and signal transduction. Research has found that the protein content in Pharbitidis Semen is relatively high, and how to extract the protein and its application of the extract remain to be studied. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of Pharbitidis Semen and its extract protein in the preparation of anti-tumor drugs, so that the extract from Pharbitidis Semen...

[0004] In order to achieve the above technical effects, the present invention is realized through the following technical solutions.

[0005] The application of Pharbitidis Semen and its extract protein in the preparation of anti-tumor drugs.

[0006] Preferably, the Pharbitidis Semen is Pharbitidis Semen seeds, and the extract protein is the polypeptide component after protein enzymolysis.

[0007] In this technical solution, the selected Pharbitidis Semen is specifically the seed of Pharbitis nil (L.) Choisy of the Convolvulaceae family, and the extract protein is the protein polypeptide component.

[0008] Preferably, it further includes the extraction method of the extract, which specifically includes the following steps:

[0009] Crush the Pharbitidis Semen medicinal material;

[0010] Degrease by water bath;

[0011] The defatted Pharbitis nil powder is ultrasonically extracted and then centrifuged to collect the supernatant, obtaining the Pharbitis nil extract;

[0012] The Pharbitis nil extract is added to a salt solution, precipitated by centrifugation, and then the precipitate is dialyzed and freeze-dried to obtain Pharbitis nil protein;

[0013] The Pharbitis nil protein is added with corresponding amounts of alkaline protease and pepsin for enzymatic hydrolysis, centrifuged to take the supernatant, filtered and separated through Sephadex G-15, the protein polypeptide is collected, freeze-dried, and stored at -20 °C for standby, and the extract protein polypeptide can be obtained.

[0014] In this technical solution, during the extraction of Pharbitis nil protein, enzymatic hydrolysis is selected to well extract the protein polypeptide, and then research and application are carried out.

[0015] Preferably, in the step of water bath degreasing, specifically, petroleum ether is added to the pulverized Pharbitis nil medicinal material and extracted through a Soxhlet extractor, and the extract is degreased by a water bath at 50 °C - 60 °C.

[0016] In this technical solution, petroleum ether and a Soxhlet extractor are used for extraction. The boiling point range of petroleum ether is 30 - 60 °C. If the temperature is higher than 60 °C, the solubility of the extract will increase and there will be losses. If the temperature is lower than 50 °C, the degreasing time will increase.

[0017] Preferably, before the ultrasonic extraction, it also includes salting out the defatted Pharbitis nil. Specifically: Take the defatted Pharbitis nil, and use a salt solution with a concentration of 0.1 mol / L - 0.2 mol / L for salting out. The mass ratio of the salt solution to Pharbitis nil is 1:20 - 1:80, and the pH value during salting out is 5 - 9.

[0018] In this technical solution, the method of salting out helps to maintain the stability of active proteins. Since proteins are extracted from Pharbitis nil seeds, according to the research on the solubility of DNA-nucleoprotein and RNA-nucleoprotein, when the NaCl concentration is 0.14 mol / L, the solubility of deoxyribonucleoprotein in water is only 1%, and when its concentration increases to 1 mol / L, its solubility is 2 times greater than that in water. Therefore, 0.1 mol / L NaCl - 0.2 mol / L NaCl is selected, preferably 0.14 mol / L.

[0019] Preferably, in the step of ultrasonic extraction, the temperature of ultrasonic extraction is 20 - 60 °C, and the time of ultrasonic extraction is 20 min - 40 min.

[0020] In this technical solution, the ultrasonic temperature is optimized under the conditions of 20 - 60 °C and the ultrasonic time of 20 min - 40 min. Multiple experiments have proved that when the ultrasonic temperature is 50 °C and the ultrasonic time is 30 min, the protein extraction amount is the largest.

[0021] Preferably, in the step of adding the Pharbitis nil extract into a salt solution for precipitation and centrifugation, specifically: adding the salt solution into the Pharbitis nil extract at a rate of 0.5 g / min for a solution volume of 1 ml, and the pH value for precipitation and centrifugation is 5 - 9.

[0022] In this technical solution, when adding at a rate of 0.5 g / min for a solution volume of 1 ml into the Pharbitis nil extract within the pH range of 5 - 9, the maximum amount of protein is extracted at 8.

[0023] In this technical solution, by utilizing the salinity effect of the ammonium sulfate solution, the protein hydration layer is adjusted, the hydrophobic part of the protein is exposed, and the solubility of the protein decreases and precipitates. Fractional precipitation of Pharbitis nil protein can remove impurity proteins and maintain the biological activity of the protein, obtaining a relatively pure protein. Other substances can be used instead, such as magnesium sulfate, sodium sulfate, sodium chloride, etc., but the most common salt is ammonium sulfate. The main advantages are a small temperature coefficient and high solubility, and the fractional salting - out effect of ammonium sulfate is better than that of other salts and is not likely to cause protein denaturation. When adding ammonium sulfate, it should be added while stirring. If the adding speed is too fast, it will cause co - precipitation of proteins. The stirring should be slow. After the protein solution foams, protein denaturation will occur due to the surface tension effect.

[0024] Preferably, the centrifugation in the step of adding the Pharbitis nil extract into a salt solution for precipitation and centrifugation is specifically: centrifuging at 7000 r / min - 9000 r / min for 8 - 12 min at 3°C - 5°C.

[0025] In this technical solution, low temperature helps to stabilize the protein, and then the protein precipitate is separated by centrifugation.

[0026] Furthermore, in order to achieve better protein stability, stirring is included before centrifugation. The stirring time is 3 - 4 h and the stirring speed is 500 - 800 rpm.

[0027] Preferably, the tumor is tongue squamous cell carcinoma.

[0028] Preferably, the tongue squamous cell carcinoma is specifically SCC - 9 human tongue squamous cell carcinoma and / or SCC25 human tongue squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a flowchart of the extraction of the extract provided by the present invention;

[0030] Figure 2 is a comparison chart of pH factors in the Pharbitis nil extraction provided by the present invention;

[0031] Figure 3 is a comparison chart of solid - to - liquid ratio factors in the Pharbitis nil extraction provided by the present invention;

[0032] Figure 4 It is a comparison chart of the extraction temperature factor in the extraction of Pharbitis seeds provided by the present invention;

[0033] Figure 5 It is a change chart of the polypeptide components in the extraction of Pharbitis seeds provided by the present invention;

[0034] Figure 6 It is a change chart of the inhibition of the activity of MCF7 cells by the protein polypeptide component GA of the Pharbitis seed extract with different concentrations provided by the present invention;

[0035] Figure 7 It is a change chart of the inhibition of the activity of MCF7 cells by the protein polypeptide component GB of the Pharbitis seed extract with different concentrations provided by the present invention;

[0036] Figure 8 It is a change chart of the inhibition of the activity of A549 cells by the protein polypeptide component GA of the Pharbitis seed extract with different concentrations provided by the present invention;

[0037] Figure 9 It is a change chart of the inhibition of the activity of A549 cells by the protein polypeptide component GB of the Pharbitis seed extract with different concentrations provided by the present invention;

[0038] Figure 10 It is a change chart of the inhibition of the activity of SCC-9 human tongue squamous cells by the protein polypeptide component GA of the Pharbitis seed extract with different concentrations provided by the present invention;

[0039] Figure 11 It is a change chart of the inhibition of the activity of SCC-9 human tongue squamous cells by the protein polypeptide component GB of the Pharbitis seed extract with different concentrations provided by the present invention;

[0040] Figure 12 It is a change chart of the inhibition of the activity of SCC-25 human tongue squamous cells by the protein polypeptide component GA of the Pharbitis seed extract with different concentrations provided by the present invention;

[0041] Figure 13 It is a change chart of the inhibition of the activity of SCC-25 human tongue squamous cells by the protein polypeptide component GB of the Pharbitis seed extract with different concentrations provided by the present invention;

[0042] Figure 14 It is a change chart of the tumors of the blank group of mice in the pharmacological experiment of the Pharbitis seed extract protein provided by the present invention.

[0043] Figure 15 It is a change chart of the tumors of the mice in the group of the protein polypeptide component GA of the Pharbitis seed extract in the pharmacological experiment of the Pharbitis seed extract protein provided by the present invention.

[0044] Figure 16This is the tumor change diagram of the white mice in the GB group of the protein polypeptide component of the Pharbitis semen extract in the pharmacological experiment of the Pharbitis semen extract provided by the present invention.

[0045] Figure 17 This is the tumor change diagram of the white mice in each group in the pharmacological experiment of the Pharbitis semen extract provided by the present invention. Specific embodiments

[0046] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0047] First, obtain various raw materials in the present invention:

[0048] The raw materials in this embodiment are obtained as follows:

[0049] The seeds of Pharbitis nil (L.) Choisy of Convolvulaceae, namely black Pharbitis semen, are purchased from Hebei Chufeng Chinese Medicine Decoction Pieces Co., Ltd. Petroleum ether, sodium chloride, ammonium sulfate, sodium dodecyl sulfate, ammonium persulfate, sodium hydroxide, hydrochloric acid, absolute ethanol (Sinopharm Chemical Reagent Co., Ltd.); pepsin, alkaline protease (BIOSHARP), Sephadex G-25 (Solarbio), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Bomei Biotechnology Co., Ltd.).

[0050] Cell lines in the raw materials

[0051] Human squamous epithelial tongue cancer cells SCC9 and SCC25 are purchased from ATCC, USA, and cultured in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in a 5% CO2, 37 °C constant temperature incubator. Human breast cancer cells MCF7 and human lung alveolar basal epithelial cells A549 are both purchased from ATCC, USA, and cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in a 5% CO2, 37 °C constant temperature incubator.

[0052] Secondly, introduce the instruments used in this embodiment

[0053] High-speed universal grinder (Test Instrument Co., Ltd.), ultrasonic cleaner (Gongyi Yinglue Yuhua Instrument Factory), EnSpire multifunctional microplate reader (PerkinElmer), pH meter (Shanghai Yidian Scientific Instrument Co., Ltd.), HF151UV carbon dioxide incubator (Shanghai Likon Biotech Co., Ltd.)

[0054] Secondly, the experimental method in this embodiment is mainly introduced

[0055] Preparation of Pharbitis nil protein

[0056] Take an appropriate amount of Pharbitis nil medicinal materials and crush them to 200 meshes. Add petroleum ether and use a Soxhlet extractor to heat and degrease in a water bath at 60 °C. Take 1 g of the degreased Pharbitis nil powder, add an appropriate amount of 0.14 mol / L NaCl solution, perform ultrasonic extraction for 30 min, then centrifuge at 8000 r / min at 4 °C for 10 min to collect the supernatant. Repeat the operation twice and combine the supernatants to obtain the Pharbitis nil extract. Weigh ammonium sulfate corresponding to 35% of the volume of the extract and slowly add it to the extract. Stir for 4 h, then centrifuge at 8000 r / min at 4 °C for 10 min and discard the supernatant. Dissolve the precipitate in physiological saline and dialyze for 48 hours, changing the water every 4 hours. Freeze-dry to obtain Pharbitis nil protein and store it at -20 °C for later use. Determine the protein concentration by ultraviolet spectrophotometry.

[0057] Single-factor experimental design

[0058] Select factors that may affect the extraction effect of Pharbitis nil protein, such as extraction temperature (20 °C, 30 °C, 40 °C, 50 °C, 60 °C), pH (5.0, 6.0, 7.0, 8.0, 9.0), and solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50), to conduct single-factor experiments to determine the influence of relevant factors on the protein content in different varieties of Pharbitis nil and provide the value range of each factor for the response surface experiment.

[0059] Optimization of the extraction process of total Pharbitis nil protein by response surface methodology

[0060] On the basis of the single-factor experimental results, use the solution pH (A), solid-liquid ratio (B), and extraction temperature (C) as the independent variables for optimization, and the total protein content Y as the response value. Use DesignExpert software to design and conduct a response surface analysis experiment with three factors and three levels to optimize the extraction process of total Pharbitis nil protein.

[0061] Enzymatic hydrolysis of Pharbitis nil protein

[0062] Dissolve the fully dialyzed Pharbitis nil protein in physiological saline, adjust the pH of the protein solution to 8.5, add the corresponding amount of alkaline protease (the ratio of enzyme to protein is 3:1), place it on a shaker and incubate at 55 °C for 4 hours to inactivate the enzyme; adjust the pH to 2.0 - 3.0, add the corresponding amount of pepsin, place it on a shaker and incubate at 37 °C for 2 hours, inactivate the enzyme for 10 min after the enzymatic hydrolysis is completed, centrifuge at 8000 r / min for 10 min, take the supernatant and freeze-dry it, and store it at -20 °C for later use.

[0063] Separation by Sephadex G-15 filtration

[0064] After treatment, Sephadex G-15 is packed into a column, and ultrafiltration water is passed through. After the volume of the chromatography column is stable, the column packing is completed. Keep the liquid level at a height of 0.5 - 1 cm. The sample loading volume for each time is about 1 mL. Adjust the flow rate of the mobile phase to 2.2 mL / min, and collect the polypeptide samples according to the absorption peaks. Freeze-dry and concentrate the polypeptide samples and store them in a -80 °C refrigerator.

[0065] MTT assay

[0066] Prepare a cell suspension of 3×10 4 cells / ml with cells in the logarithmic growth phase. Take 100 μl each and culture them in a 96-well plate at 37 °C for 24 hours. Add Pharbitis nil polypeptides with concentrations of 0, 0.05, 0.1, 0.5, and 1.0 μg / μL to each well. After treatment for 24 and 48 h, add 20 μl of MTT solution with a concentration of 5 mg / ml, incubate at 37 °C for 4 h, then discard the culture medium. Add 100 μl of DMSO to each well and incubate in the dark for 30 min. Measure the optical density value (OD) of each well at 490 nm using an enzyme-linked immunosorbent assay reader. Cell survival rate = (OD value of the experimental group - OD value of the blank group) / (OD value of the negative control group - OD value of the blank group) × 100%.

[0067] Experimental results

[0068] Results of single-factor experiments

[0069] Refer to Appendix Figure 2 It can be seen that as the pH of the solution increases, the extraction amount of Pharbitis nil protein shows a trend of first increasing and then decreasing. The maximum protein extraction amount is obtained at pH 8. This is mainly because the charge properties of the protein are affected as the pH increases, which changes the ionization of the functional groups of the protein and affects the interaction between proteins, thus affecting the solubility of the protein. Therefore, in the extraction, a pH of 8 is preferably selected. Refer to Appendix Figure 3 As shown, it can be seen that the ratio of material to liquid has a trend of first increasing and then steadily decreasing on the protein extraction amount. When the ratio of material to liquid is 1:40, the maximum protein extraction amount is obtained. When the volume of the solution is small, the medicinal materials are not completely extracted and the protein extraction amount is low. Therefore, the ratio of material to liquid is preferably 1:40. Refer to Appendix Figure 4As shown, it can be seen that the extraction amount of Pharbitis nil protein increases with the increase of extraction temperature. When the temperature is 50°C, the protein extraction amount reaches the peak. The reason is that the increased temperature will cause the cell wall to be damaged, accelerating the release of protein. However, when the temperature is too high, the precipitated protein will denature, resulting in a decrease in the extraction amount. Therefore, the preferred extraction temperature is 50°C.

[0070] During the entire extraction process, the method of salting-in helps to maintain the stability of active proteins. Since proteins are extracted from Pharbitis nil seeds, according to the research on the solubility of DNA-nucleoprotein and RNA-nucleoprotein, when the NaCl concentration is 0.14 mol / L, the solubility of deoxyribonucleoprotein in water is only 1%, and when its concentration increases to 1 mol / L, its solubility is 2 times greater than that in water. Therefore, 0.1 mol / L NaCl - 0.2 mol / L NaCl is selected, preferably 0.14 mol / L. In ultrasonic extraction, multiple experiments have shown that when the ultrasonic temperature is 50°C and the ultrasonic time is 30 min, the protein extraction amount is the largest.

[0071] In this example, by utilizing the salinity effect of ammonium sulfate solution, the protein hydration layer is adjusted, the hydrophobic part of the protein is exposed, and the solubility of the protein decreases and precipitates. Fractional precipitation of Pharbitis nil protein can remove impurity proteins and maintain the biological activity of the protein, obtaining relatively pure protein. Other substances can be used instead, such as magnesium sulfate, sodium sulfate, sodium chloride, etc. However, the most common salt is ammonium sulfate, and its main advantages are small temperature coefficient and large solubility, and the effect of fractional salting-out with ammonium sulfate is better than that of other salts, and it is not easy to cause protein denaturation. When adding ammonium sulfate, it should be added while stirring. If the addition speed is too fast, it will cause co-precipitation of proteins. The stirring should be slow. After the protein solution foams, protein denaturation will occur due to the surface tension effect.

[0072] Results of optimization experiment by response surface method

[0073] Results of response surface experiment

[0074] Taking the three factors of solution pH (A), solid-liquid ratio (B), and extraction temperature (C) as independent variables for optimization, and the total protein content Y as the response value, a response surface analysis experiment with three factors and three levels using Box-Behnken central composite design (Table 1) was carried out to optimize the extraction process of Pharbitis nil total protein. The Design-Expert 8.0.6 software was used to statistically analyze and analyze the experimental data, and the results are shown in Table 2.

[0075] Table 1 Factor and level table of response surface test design

[0076] A: Solution pH B: Ratio of material liquid (g / ml) C: Extraction temperature (°C) －1 7 1:30 40 0 8 1:40 50 1 9 1:50 60

[0077] Table 2 Box-Behnken experimental design and results

[0078]

[0079]

[0080] Using the DesignExpert 8.0.6 data statistical software, with the total protein extraction amount as the response value, the results in Table 2 were subjected to multiple regression fitting to obtain the quadratic multiple regression equation for the total protein extraction amount of Pharbitis seeds: Y = 464.57 - 2.7A + 43.79B - 7.36C - 0.42AB + 12.86AC + 31.01BC - 53.47A 2 + 28.67B 2 - 8.74C 2 . Analysis of variance was performed on the experimental data model, and the results are shown in Table 3.

[0081] Table 3 Analysis of variance results of the response surface quadratic regression equation model

[0082]

[0083]

[0084] Note: **P < 0.01 indicates extremely significant difference; *P < 0.05 indicates significant difference.

[0085] Table 1 is the design table of three factors and three levels for the extraction conditions of Pharbitis seeds, without conclusions. Table 2 is the protein extraction amount of Pharbitis seeds obtained according to the extraction conditions designed in Table 1. Table 3 is the analysis of variance results of the response surface quadratic regression equation model. The model can be used to analyze and predict the total protein extraction amount of Pharbitis seeds. The primary and secondary order of the influence of each factor on the protein extraction amount of Pharbitis seeds is solid-liquid ratio > temperature > pH.

[0086] As can be seen from Table 3, the model P < 0.001, indicating that the model has a significant difference. The simulation correlation coefficient R 2 = 0.9916, and the corrected determination coefficient R 2 adj = 0.9807, indicating that the protein extraction amount of Pharbitis seeds has a good fitting degree with the model and can well reflect the relationship between each factor and the response value. The lack-of-fit term P < 0.05, indicating that there are experimental errors. However, considering all factors, the model can be used to analyze and predict the total protein extraction amount of Pharbitis seeds. Through the F-test, the factor contribution rates were found to be: B > C > A. At the same time, the AC and BC interaction effects have a significant impact on the total protein extraction of Pharbitis seeds. In summary, the primary and secondary order of the influence of each factor on the protein extraction amount of Pharbitis seeds is solid-liquid ratio > temperature > pH.

[0087] Interaction effects of each factor on the response surface and verification experiments

[0088] The response surface analysis method was used to optimize the protein extraction process of Pharbitis seeds.Figure 2 It is the influence of the pairwise interaction of solution pH (A), solid-liquid ratio (B), and extraction temperature (C) on the protein extraction amount. The 3D surface plot can visually show the influence of each factor on the protein extraction amount of Pharbitis nil. The steeper the slope of the response surface, the greater the influence of this factor on the result. The contour plot can directly show the interaction between two independent variables. The closer the contour shape is to an ellipse, the greater the correlation; the closer it is to a normal circle, the smaller the correlation. It can be seen from the response surface plot that the order of factors affecting the protein extraction amount is: solid-liquid ratio > extraction temperature > pH.

[0089] Under the optimized conditions of pH 8.13, a solid-liquid ratio of 1:50, and a temperature of 57.3 °C, the theoretical extraction amount of Pharbitis nil protein is 549.175 mg. To further confirm the reliability of the response surface results and fully consider the actual operability, the optimal extraction process parameters were corrected to: pH 8, a solid-liquid ratio of 1:50, and a temperature of 57 °C. According to these conditions, the actual extraction amount is 544.608 ± 1.13 mg (n = 3), with a small deviation from the theoretical predicted value, indicating that the model established in the experiment can well predict the variation relationship between each factor and the response surface.

[0090] Separation of Pharbitis nil polypeptides

[0091] The extracted Pharbitis nil protein was digested with pepsin and alkaline protease, and the obtained mixture of Pharbitis nil protein polypeptides was subjected to G-15 gel filtration.

[0092] Figure 5 It can be observed that the polypeptides were divided into two components and these two components were completely separated, and the separation effect was ideal. The first component of the separated Pharbitis nil polypeptides was named GA, and the second component was named GB. The collected GA and GB were freeze-dried and stored.

[0093] MTT assay for the effect of Pharbitis nil polypeptides on tumor activity

[0094] Human breast cancer cells MCF7, human squamous epithelial tongue cancer cells SCC9, SCC25, and human lung alveolar basal epithelial cells A549 were selected to observe the effects of different components of Pharbitis nil protein on the activities of three types of tumor cells.

[0095] Figure 6 and 7 It shows that the concentrations of 0.05, 0.1, 0.5, and 1.0 μg / μL of Pharbitis nil components GA and GB have no obvious inhibitory effect on the activity of MCF7 cells, and have a significant promoting effect on cell activity at concentrations above 0.1 μg / μL for 48 hours;

[0096] Figure 8 and 9It is shown that the components GA and GB of Pharbitidis Semen at the concentrations of 0.05, 0.1, and 0.5 μg / μL have no obvious inhibitory effect on the activity of A549 cells, and have a significant promoting effect on cell activity at a concentration above 0.1 μg / μL for 48 hours;

[0097] It can be seen from Figures 10 - 13 that GA treatment for 24 and 48 hours has an inhibitory effect on the growth and proliferation of SCC9 and SCC25 cells in human squamous epithelial tongue cancer cells SCC9 and SCC25. There is a significant difference when the concentration reaches 0.1 μg / μL, and the cell survival rate is low when the concentration reaches 1 μg / μL; the GB component has an obvious inhibitory effect on the activity of human squamous epithelial tongue cancer cells when its concentration reaches 0.5 μg / μL. The results show that GA has a better inhibitory effect on human squamous cell carcinoma activity than GB and has a lower use concentration; It can be seen from Figure 4 the effects of different components of Pharbitidis Semen on tumor activity.

[0098] There are research reports predicting that Pharbitidis Semen has anti-tumor effects based on network pharmacology and molecular docking technology. The present invention optimizes the extraction process of total protein from Pharbitidis Semen by the response surface method. After enzymatic hydrolysis and separation by G-15 gel chromatography, two polypeptide components GA and GB are obtained. They have different inhibitory effects on the activity of different tumor cells, can inhibit the growth of human squamous epithelial tongue cancer cells, but have no obvious inhibitory effect on human breast cancer cells and human alveolar basal epithelial cells of the lung. This shows that Pharbitidis Semen polypeptides have a certain specific inhibitory effect on tumors and have the potential to be anti-tumor drugs for treating human squamous epithelial tongue cancer. Squamous cell carcinoma of the tongue is one of the malignant tumors with a relatively high incidence in oral and maxillofacial tumors. It grows fast, has a high degree of malignancy, strong invasiveness, and the 5-year survival rate is less than 15%. The traditional cancer treatment effect is not ideal. Subsequently, the two components GA and GB will be further separated and purified, and the amino acid sequence will be identified to provide a basis for the clinical application of Pharbitidis Semen in treating tumors and the research and development of anti-tumor drugs.

[0099] The pharmacological experiment of the present invention on mice is as follows:

[0100] Twenty-four BALB / c nude mice weighing 18 - 22 g were purchased and raised in the Experimental Animal Center Affiliated to Tongji Medical College of Huazhong University of Science and Technology, with free diet, the temperature controlled at room temperature, and the humidity controlled at 45 - 55%. The SCC25 tongue cancer cells were resuspended with PBS and counted, and then the cell concentration was adjusted to 5×10 7, take 0.15 mL and inject it subcutaneously under the axilla of the left forelimb of the mice. After 72 h, the mice are randomly divided into a blank control group with DMSO, a Pharbitis nil Choisy administration group (GA 125 mg / Kg.day, 250 mg / Kg.day) and a (GB 125 mg / Kg.day, 250 mg / Kg.day) group, with 6 mice in each group. Directly perform injection administration for solid tumors, administer the drug once a day for 10 consecutive days, and dislocate and sacrifice all the mice on the 11th day. Dissect and completely peel off the tumors, take pictures for record, and measure the volume and weight of the tumors (see specifically Figures 14 - 16 ). It can be seen from Figures 14 - 17 various comparisons that the results show that after administering 125 mg / kg GA and GB, the tumor volume is reduced by 45.6 ± 8.9% and 51 ± 4.3%; after administering 250 mg / kg GA and GB, the tumor volume is significantly reduced by 72.3 ± 3.6% and 81 ± 2.3%. Furthermore, it can be seen that the protein polypeptide component GA and the polypeptide component GB of the Pharbitis nil Choisy extract have obvious inhibitory effects on SCC25 tongue cancer cells (i.e., human tongue squamous cells).

[0101] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of Pharbitis nil protein polypeptide in the preparation of anti-tumor drugs, specifically including the following steps: Take an appropriate amount of Pharbitis nil medicinal materials, crush them, add petroleum ether, and heat and degrease them in a Soxhlet extractor at a water bath temperature of 50 - 60 °C; take 1 g of the defatted Pharbitis nil powder, add an appropriate amount of 0.14 mol / L NaCl solution, perform ultrasonic extraction for 30 min, then centrifuge at 8000 r / min at 4 °C for 10 min to collect the supernatant. Repeat the operation twice and combine the supernatants to obtain the Pharbitis nil extract; the extraction process parameters are: pH = 8, solid-liquid ratio = 1:50, and temperature = 57 °C; Weigh ammonium sulfate corresponding to 35% of the volume of the Pharbitis nil extract, slowly add it to the extract, stir for 4 h, then centrifuge at 8000 r / min at 4 °C for 10 min, discard the supernatant, dissolve the precipitate in physiological saline, dialyze for 48 hours, change the water every 4 hours, and freeze-dry to obtain Pharbitis nil protein; Dissolve the Pharbitis nil protein in physiological saline, adjust the pH of the protein solution to 8.5, add the corresponding amount of alkaline protease, with the enzyme-to-protein ratio of 3:1, place it on a shaker and shake at 55 °C for enzymatic hydrolysis for 4 hours to inactivate; adjust the pH to 2.0 - 3.0, add the corresponding amount of pepsin, place it on a shaker and shake at 37 °C for enzymatic hydrolysis for 2 hours. After the enzymatic hydrolysis is completed, inactivate the enzyme for 10 min, centrifuge at 8000 r / min for 10 min, take the supernatant and freeze-dry. The obtained Pharbitis nil protein polypeptide mixture is separated by filtration through Sephadex G-15, collect the protein polypeptide, freeze-dry, and store it at -20 °C for standby to obtain the said protein polypeptide; The tumor is tongue squamous cell carcinoma.

2. The application according to claim 1, wherein The tongue squamous cell carcinoma is SCC9 human tongue squamous cell carcinoma or SCC25 human tongue squamous cell carcinoma.

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