CN116531423A - Application of pharbitis seed and extract protein thereof in preparation of antitumor drugs - Google Patents
Application of pharbitis seed and extract protein thereof in preparation of antitumor drugs Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/39—Convolvulaceae (Morning-glory family), e.g. bindweed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
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- Water Supply & Treatment (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses application of pharbitis seed and extract protein thereof in preparing antitumor drugs, and in particular relates to a method for extracting protein from pharbitis seed, so that the pharbitis seed and extract protein thereof are researched in application aspect to obtain new progress.
Description
Technical Field
The invention belongs to the application field of pharbitis seed and an extract thereof, and particularly relates to application of pharbitis seed and an extract protein thereof in preparation of antitumor drugs.
Background
Semen Pharbitidis is dry mature seed of Pharbitidis nilotica (L.) Choisy or Pharbitidis pulcure (L.) Voigt of Convolvulaceae, and is classified into Pharbitidis and Pharbitidis. The traditional Chinese herbs of the past generation are bitter in taste, cold in nature and toxic, and have the effects of purging water, relaxing bowels, resolving phlegm, removing fluid retention, killing parasites and eliminating accumulation. The researches show that the pharbitis seed mainly contains components such as pharbitis glycoside, phenolic acid compounds, alkaloids, saccharides, proteins, sterol compounds, pigments, fatty oil and the like. Modern pharmacological researches have shown that pharbitis seed has the effects of relieving constipation, promoting urination, resisting inflammation, enhancing immunity, exciting uterus and resisting tumor. The antitumor active ingredients reported by the current pharbitis seed are mainly concentrated in an alcohol extract, and the alcohol extract can inhibit the growth of tumor cells such as colorectal cancer, colon cancer, liver cancer, breast cancer, lung cancer and the like. Proteins are a type of chemical components which are commonly existing in medicinal materials of subclasses and have high content, and play an important role in the processes of plant growth, development and metabolism, signal transduction and the like. The research shows that the protein content in the pharbitis seed is higher, and how to extract the protein and the application of the extract thereof are needed to be researched.
Disclosure of Invention
The invention aims to provide application of pharbitis seed and extract protein thereof in preparing antitumor drugs, so that the pharbitis seed is extracted.
In order to achieve the technical effects, the invention is realized by the following technical scheme.
Application of semen Pharbitidis and its extract protein in preparing antitumor drugs is provided.
Preferably, the pharbitis seed and the extracted protein is a polypeptide component after proteolysis.
In the technical scheme, the selected Pharbitis seed is specifically seed of Pharbitidis nilotica (L.) Choisy of Convolvulaceae, and the extracted protein is protein polypeptide component.
Preferably, the extraction method of the extract is further included, and specifically includes the following steps:
pulverizing semen Pharbitidis;
degreasing in a water bath;
ultrasonic extracting the defatted semen Pharbitidis powder, and centrifuging to collect supernatant to obtain semen Pharbitidis leaching solution;
adding the pharbitis seed extract into a salt solution, precipitating and centrifuging, dialyzing the precipitate, and freeze-drying to obtain pharbitis seed protein;
adding corresponding amount of alkaline protease and pepsin into semen Pharbitidis protein, performing enzymolysis and centrifugation to obtain supernatant, filtering and separating with sephadex G-15, collecting protein polypeptide, freeze drying, and preserving at-20deg.C to obtain extract protein polypeptide.
In the technical scheme, in the extraction of the pharbitis seed protein, the protein polypeptide is well extracted by selecting enzymolysis, so that the research and the application are further carried out.
Preferably, in the step of water bath degreasing, petroleum ether is added into crushed pharbitis seed medicinal materials and extracted by a Soxhlet extractor, and the extract is degreased by water bath at 50-60 ℃.
In the technical scheme, petroleum ether and a Soxhlet extractor are used for extraction, the boiling point range of the petroleum ether is 30-60 ℃, the solubility of the extract is increased and is lost when the boiling point range is higher than 60 ℃, and the degreasing time is increased when the temperature is lower than 50 ℃.
Preferably, before the ultrasonic extraction, the method further comprises dissolving salt of defatted pharbitis seed, specifically: taking defatted semen Pharbitidis, and dissolving salt with 0.1-0.2 mol/L salt solution at a mass ratio of 1:20-1:80, wherein the pH value of the salt solution is 5-9.
In the technical scheme, the salt dissolution method is adopted to help maintain the stability of the active protein, and as the protein is extracted from the pharbitis seed, according to the research on the solubility of DNA-nucleoprotein and RNA-nucleoprotein, the solubility of the DNA-nucleoprotein is only 1% in water when the NaCl concentration is 0.14mol/L, and the solubility of the DNA-nucleoprotein is 2 times greater than that in water when the concentration is increased to 1 mol/L. Thus, 0.1mol/LNaCl-0.2mol/LNaCl, preferably 0.14mol/L, is selected.
Preferably, in the ultrasonic extraction in the step, the ultrasonic extraction temperature is 20-60 ℃, and the ultrasonic extraction time is 20-40 min.
In the technical scheme, the ultrasonic temperature is 20-60 ℃ and the ultrasonic time is 20-40 min for condition optimization, and multiple experiments prove that the ultrasonic temperature is 50 ℃ and the ultrasonic time is 30min for maximum protein extraction.
Preferably, the steps are to add the pharbitis seed extract into a salt solution for precipitation and centrifugation, specifically: the salt solution is added into the pharbitis seed leaching solution at a speed of 0.5g/min in a volume of 1ml, and the pH value of precipitation centrifugation is 5-9.
In the technical scheme, the method is characterized in that the adding speed is that 1ml of solution is added into the pharbitis seed leaching solution at the speed of 0.5g/min, the pH value is in the range of 5-9, and the protein extraction amount is maximum when the pH value is 8.
In the technical scheme, the salinity effect of the ammonium sulfate solution is utilized to regulate the protein hydration layer, so that the hydrophobic part of the protein is exposed, and the solubility of the protein is reduced to precipitate. The fractional precipitation of the pharbitis seed protein can remove the impurity protein, and can maintain the biological activity of the protein, thus obtaining purer protein. Other substances such as magnesium sulfate, sodium chloride and the like can be used for replacing the salt, but the most common salt is ammonium sulfate, and the main advantages are small temperature coefficient and high solubility, and the ammonium sulfate has better fractional salting-out effect than other salts and is not easy to cause protein denaturation. The ammonium sulfate is added with stirring, and the adding speed is too high, so that protein coprecipitation occurs. The stirring is slow and the protein solution after foaming causes denaturation of the protein due to the surface tension effect.
Preferably, the centrifugation of adding the pharbitis seed extract to the salt solution to precipitate the core is specifically: centrifuging at 7000-9000 r/min for 8-12min at 3-5deg.C.
In the technical scheme, the stability of the protein is facilitated at low temperature, and then the protein precipitate is separated out through centrifugation.
Further, in order to achieve better stabilization of the protein, stirring is further included before centrifugation, the stirring time is 3-4h, and the stirring speed is 500-800rpm.
Preferably, the tumor is a tongue squamous cell carcinoma.
Preferably, the tongue squamous cell carcinoma is in particular SCC-9 human tongue squamous cell carcinoma and/or SCC25 human tongue squamous cell carcinoma.
Drawings
FIG. 1 is a flow chart of the extraction of the extract provided by the invention;
FIG. 2 is a graph showing the comparison of pH factors in extraction of pharbitis seed provided by the present invention;
FIG. 3 is a graph showing comparison of feed-liquid ratio factors in extraction of semen Pharbitidis provided by the invention;
FIG. 4 is a graph showing the comparison of the extraction temperature factors in the extraction of pharbitis seed provided by the present invention;
FIG. 5 is a diagram showing the change of polypeptide components in pharbitis seed extraction;
FIG. 6 is a graph showing the inhibition of MCF7 cell activity by GA of different concentrations of the protein polypeptide component of the pharbitis seed extract provided by the present invention;
FIG. 7 is a graph showing the change in inhibition of MCF7 cell activity by different concentrations of the pharbitis seed extract protein polypeptide component GB;
FIG. 8 is a graph showing the inhibition of the activity of A549 cells by GA in different concentrations of the protein polypeptide component of the pharbitis seed extract;
FIG. 9 is a graph showing the inhibition of different concentrations of the pharbitis seed extract protein polypeptide component GB on the activity of A549 cells;
FIG. 10 is a graph showing the change in inhibition of SCC-9 human tongue squamous cell activity by different concentrations of the pharbitis seed extract protein polypeptide component GA provided by the present invention;
FIG. 11 is a graph showing the change in inhibition of SCC-9 human tongue squamous cell activity by different concentrations of pharbitis seed extract protein polypeptide component GB provided by the present invention;
FIG. 12 is a graph showing the change in inhibition of SCC-25 human tongue squamous cell activity by different concentrations of the pharbitis seed extract protein polypeptide component GA provided by the present invention;
FIG. 13 is a graph showing the change in inhibition of SCC-25 human tongue squamous cell activity by different concentrations of pharbitis seed extract protein polypeptide component GB provided by the present invention;
fig. 14 is a diagram showing tumor changes of mice in a blank group in a pharbitis seed extract protein pharmacological experiment provided by the invention.
Fig. 15 is a diagram showing tumor changes of a group of mice with GA group in pharbitis seed extract protein polypeptide composition in a pharbitis seed extract protein pharmacological experiment provided by the invention.
Fig. 16 is a diagram showing tumor changes of mice in group GB of pharbitis seed extract protein polypeptide components in a pharbitis seed extract protein pharmacological experiment provided by the invention.
Fig. 17 is a graph showing tumor changes of each group of mice in the pharbitis seed extract protein pharmacological experiment provided by the invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
First, the present invention is carried out by obtaining various raw materials:
the raw materials in this example were obtained as follows:
seed of Pharbitis nil (l.) Choisy, a seed of Pharbitis seed of the family Convolvulaceae, which is purchased from Hebei Chu wind decoction piece company. Petroleum ether, sodium chloride, ammonium sulfate, sodium dodecyl sulfate, ammonium persulfate, sodium hydroxide, hydrochloric acid, and absolute ethanol (national pharmaceutical systems chemical agents limited); pepsin, alkaline protease (BIOSHARP), sephadex G-25 (Solarbio), dimethyl sulfoxide (DMSO), 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) (Bomei Biotechnology Co., ltd.).
Cell lines in the feedstock
Human squamous tongue carcinoma cells SCC9, SCC25 were purchased from ATCC, USA and cultured in DMEM/F12 medium containing 10% FBS, 100U/mL penicillin and 100g/mL streptomycin at 5% CO 2 Culturing in a constant temperature incubator at 37 ℃. Human breast cancer cells MCF7 and lung cancer human alveolar basal epithelial cells A549 were both purchased from ATCC, america, and cultured in DMEM medium containing 10% FBS, 100U/mL penicillin and 100g/mL streptomycin at 5% CO 2 Constant temperature of 37 DEG CCulturing in an incubator.
Next, the instrument used in this embodiment will be described
High-speed universal pulverizer (Test instruments Co., ltd.), ultrasonic cleaner instrument (Yingyingyishihua instruments Co., ltd.), enspire multifunctional enzyme-labeled instrument (Perkinelmer Co., ltd.), PH meter (Shanghai electric science instruments Co., ltd.), HF151UV carbon dioxide incubator (Shanghai Likang biological Co., ltd.)
Next, the experimental method in this embodiment will be mainly described
Preparation of pharbitis seed protein
Pulverizing appropriate amount of semen Pharbitidis to 200 mesh, adding petroleum ether, passing through Soxhlet extractor, and heating in water bath at 60deg.C for degreasing. Adding proper amount of 0.14mol/LNaCl solution into 1g of defatted semen Pharbitidis powder, ultrasonically extracting for 30min, centrifuging at 4deg.C for 10min at 8000r/min, collecting supernatant, repeating the operation twice, and mixing the supernatants to obtain semen Pharbitidis leaching solution. And (3) weighing ammonium sulfate with the corresponding mass with the volume of 35% of the leaching liquid, slowly adding the ammonium sulfate into the leaching liquid, stirring for 4 hours, centrifuging at 8000r/min at 4 ℃ for 10 minutes, discarding the supernatant, dissolving the precipitate in normal saline, dialyzing for 48 hours, changing clear water every 4 hours, freeze-drying to obtain the pharbitis seed protein, and preserving at-20 ℃ for later use. And measuring the protein concentration by ultraviolet spectrophotometry.
Single factor test design
And selecting factors which can influence the extraction effect of the pharbitis seed protein, wherein the extraction temperature (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃), the pH (5.0, 6.0, 7.0, 8.0 and 9.0) and the feed-liquid ratio (1:10, 1:20, 1:30, 1:40 and 1:50) are likely to influence the protein content of different varieties of the pharbitis seed, and carrying out a single factor experiment to determine the influence of related factors on the protein content of different varieties of the pharbitis seed, thereby providing the value range of each factor for the response surface experiment.
Response surface method optimized extraction process of total protein of pharbitis seed
On the basis of a single-factor test result, three factors of solution pH (A), feed-liquid ratio (B) and extraction temperature (C) are used as independent variables of an optimization object, total protein content Y is used as a response value, design expert software is used for designing, three-factor three-level response surface analysis experiments are carried out, and the extraction process of total protein of the pharbitis seed is optimized.
Enzymolysis of pharbitis seed protein
Dissolving the completely dialyzed pharbitis seed protein in normal saline, regulating the pH value of a protein solution to 8.5, adding a corresponding amount of alkaline protease (the ratio of enzyme to protein is 3:1), and placing on a shaking table for shaking and enzymolysis for 4 hours at 55 ℃ for inactivation; adjusting pH to 2.0-3.0, adding pepsin with corresponding amount, placing on a shaking table, shaking for enzymolysis at 37deg.C for 2 hr, inactivating enzyme for 10min after enzymolysis, centrifuging at 8000r/min for 10min, collecting supernatant, lyophilizing, and preserving at-20deg.C.
Sephadex G-15 filtration separation
And loading the column after the dextran gel G-15 is treated, introducing ultrafiltration water, and loading the column after the chromatographic column volume is stable. The liquid level is kept at a height of 0.5-1 cm, the sample loading amount is about 1mL each time, the flow rate of the mobile phase is regulated to be 2.2mL/min, and the polypeptide sample is collected according to the absorption peak. The freeze-dried concentrated polypeptide sample is put into a refrigerator at the temperature of minus 80 ℃ for preservation.
MTT assay
Compounding cells in logarithmic growth phase to 3 x 10 4 Cell suspension per ml. 100 μl each was incubated in 96-well plates at 37deg.C for 24 hours. The pharbitis seed polypeptide with the concentration of 0, 0.05, 0.1, 0.5 and 1.0 mug/mug is added into each hole respectively, 20 mug of MTT solution with the concentration of 5mg/ml is added after 24 hours and 48 hours of treatment, the culture medium is discarded after 4 hours of incubation at 37 ℃, 100 mug of DMSO is added into each hole for incubation for 30 minutes in a dark place, and an enzyme-labeled instrument is arranged for measuring the optical density value (OD) of each hole at 490 nm. Cell viability = (experimental OD value-blank OD value)/(negative control OD value-blank OD value) ×100%.
Experimental results
Single factor experimental results
Referring to fig. 2, it can be seen that as the pH of the solution increases, the extraction amount of pharbitis protein tends to increase and then decrease, and at pH 8, the extraction amount of protein is maximum, mainly because the protein affects its dotted property with the increase of pH, changes the ionization of functional groups of protein, affects the interaction between proteins, and thus affects the solubility of protein, and further, in the extraction, pH 8 is preferred. Referring to fig. 3, it can be seen that the protein extraction amount of the feed liquid ratio is in a trend of increasing and then steadily decreasing, when the feed liquid ratio is 1: the maximum protein extraction amount is 40, and when the solution volume is smaller, the extraction of the medicinal materials is incomplete, and the protein extraction amount is low, so that the ratio of the simple aunt to the solution is 1:40. referring to FIG. 4, it can be seen that the extraction amount of pharbitis seed protein increases with increasing extraction temperature, and the protein extraction amount peaks when the temperature is 50 ℃, because the increased temperature causes the cell wall to be destroyed, accelerating the release of protein, but the temperature excessively high causes the deposited protein to be denatured, resulting in the decrease of the extraction amount, so that the extraction temperature is preferably 50 ℃.
In the whole extraction step, the salt-dissolving method is adopted to help maintain the stability of the active protein, and since the protein is extracted from the pharbitis seed, according to the research on the solubility of DNA-nucleoprotein and RNA-nucleoprotein, the solubility of the DNA-nucleoprotein is only 1% in water when the NaCl concentration is 0.14mol/L, and when the concentration is increased to 1mol/L, the solubility is 2 times higher than that in water. Thus, 0.1mol/LNaCl-0.2mol/LNaCl, preferably 0.14mol/L, is selected. In ultrasonic extraction, multiple experiments prove that the protein extraction amount is maximum at the ultrasonic temperature of 50 ℃ for 30 min.
In this example, the protein hydration layer was adjusted by utilizing the salinity effect of the ammonium sulfate solution to expose the hydrophobic portion of the protein, and the solubility of the protein was reduced to precipitate. The fractional precipitation of the pharbitis seed protein can remove the impurity protein, and can maintain the biological activity of the protein, thus obtaining purer protein. Other substances such as magnesium sulfate, sodium chloride and the like can be used for replacing the salt, but the most common salt is ammonium sulfate, and the main advantages are small temperature coefficient and high solubility, and the ammonium sulfate has better fractional salting-out effect than other salts and is not easy to cause protein denaturation. The ammonium sulfate is added with stirring, and the adding speed is too high, so that protein coprecipitation occurs. The stirring is slow and the protein solution after foaming causes denaturation of the protein due to the surface tension effect.
Response surface method optimization test results
Response surface experimental results
Three factors of solution pH (A), feed-liquid ratio (B) and extraction temperature (C) are used as independent variables of an optimization object, total protein content Y is used as a response value, a Box-Behnken center combination is used for designing a three-level response surface analysis experiment (table 1), the extraction process of total protein of pharbitis seed is optimized, and statistical and data analysis is carried out on experimental data by using Design-expert8.0.6 software, wherein the results are shown in table 2.
TABLE 1 response surface test design factors and level tables
| A solution pH | B ratio of feed to liquid (g/ml) | Extraction temperature (. Degree. C.) | |
| －1 | 7 | 1:30 | 40 |
| 0 | 8 | 1:40 | 50 |
| 1 | 9 | 1:50 | 60 |
TABLE 2Box-Behnken experiment design and results
Performing multiple regression fitting on the results of table 2 by using design experet8.0.6 data statistical software and taking the total protein extraction as a response value to obtain a quadratic multiple regression equation of the total protein extraction of the pharbitis seed: 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.
TABLE 3 response surface quadratic regression equation model analysis of variance results
Note that: * P <0.01 is very significant; * P <0.05 is significant.
Table 1 is a three-factor three-level design table of extraction conditions of pharbitis seed, and it is not concluded that table 2 is the extraction amount of the pharbitis seed protein obtained according to the extraction conditions designed in table 1; table 3 shows the results of variance analysis of the response surface quadratic regression equation model, which can be used for analyzing and predicting the total protein extraction of the pharbitis seed, and the primary and secondary orders of the influence of each factor on the total protein extraction of the pharbitis seed are feed-liquid ratio > temperature > pH.
As can be seen from Table 3, model P<0.001, which shows that the model has obvious difference and simulates the correlation coefficient R 2 0.9916, correction coefficient R 2 adj=0.9807, which shows that the extraction amount of the pharbitis seed protein has better fitting degree with the model, and can well reflect the relation between each factor and response value. Loose item P<0.05, the test has errors, but the factors are combined,the model can be used for analyzing and predicting the total protein extraction amount of the pharbitis seed. The factor contribution rate is checked by F to be: b (B)>C>A, the interaction of AC and BC has obvious influence on the extraction of total protein of pharbitis seed. In conclusion, the major and minor orders of the influence of each factor on the extraction amount of the pharbitis seed protein are the ratio of feed to liquid>Temperature (temperature)>pH。
Response surface factor interactions and validation tests
The extraction process of the pharbitis seed protein is optimized by adopting a response surface analysis method, and fig. 2 shows the influence of the interaction of solution pH (A), feed-liquid ratio (B) and extraction temperature (C) on the protein extraction amount. The 3D curved surface graph can intuitively see the influence of each factor on the extraction amount of the pharbitis seed protein. The steeper the slope of the response surface, the greater the effect this factor has on the result. Contour plots visually see the interaction between two independent variables. The closer the contour shape is to an ellipse, the greater the correlation, and the closer to a normal line circle, the smaller the correlation. From the response surface graph, it can be seen that the order of factors affecting the protein extraction amount is: feed to liquid ratio > extraction temperature > pH.
Under the optimized condition of pH 8.13, feed-liquid ratio of 1:50 and temperature of 57.3 ℃, the theoretical extraction amount of the pharbitis seed protein is 549.175mg. In order to further confirm the reliability of the response surface result and fully consider the actual operability, the optimal extraction process parameters are modified as follows: the pH value is 8, the feed-liquid ratio is 1:50, the temperature is 57 ℃, the actual extraction amount is 544.608 +/-1.13 mg (n=3) according to the condition, the deviation from the theoretical predicted value is not large, and the model established by the test can well predict the change relation between each factor and the response surface.
Isolation of pharbitis seed polypeptides
And (3) performing double enzymolysis on the extracted pharbitis protein by pepsin and alkaline protease, and performing G-15 gel filtration on the obtained pharbitis protein polypeptide mixture.
It can be seen in fig. 5 that the polypeptide is separated into two components and that the separation of the two components is relatively complete and the separation effect is relatively ideal. The isolated first component of the pharbitis polypeptide was designated GA and the second component was designated GB. And freeze-drying and storing the collected GA and GB.
MTT detection of the Effect of pharbitis seed Polypeptides on tumor Activity
Human breast cancer cells MCF7, human squamous tongue carcinoma cells SCC9 and SCC25 and lung cancer human alveolar basal epithelial cells A549 are selected, and the influence of different components of pharbitis protein on the activities of three tumor cells is observed.
FIGS. 6 and 7 show that the administration of the pharbitis seed components GA and GB at concentrations of 0.05, 0.1, 0.5, 1.0 μg/μL had no significant inhibitory effect on MCF7 cell activity, and that the cellular activity was significantly promoted at concentrations above 0.1 μg/μL for 48 hours;
FIGS. 8 and 9 show that administration of the pharbitis seed components GA and GB at concentrations of 0.05, 0.1, 0.5 μg/μL had no significant inhibitory effect on A549 cell activity, and that the cellular activity was significantly promoted at concentrations above 0.1 μg/μL for 48 hours;
as can be seen from fig. 10 to 13, GA treatment for 24 and 48 hours was found to have an inhibitory effect on the growth and proliferation of SCC9 and SCC25 cells in human squamous tongue carcinoma cells SCC9, SCC25, with a significant difference at a concentration of 0.1 μg/μl, and a low cell survival rate at a concentration of 1 μg/μl; the GB component has obvious inhibition on the activity of human squamous tongue carcinoma cells when the concentration reaches 0.5 mug/mu L, and the result shows that the GA has better effect on inhibiting the activity of human squamous tongue carcinoma than GB and has low use concentration; as can be seen from fig. 4, the effect of different fractions of pharbitis seed on tumor activity.
There are studies reporting that pharbitis seed is predicted to have anti-tumor effects based on network pharmacology and molecular docking techniques. The invention optimizes the extraction process of total protein of pharbitis seed by using a response surface method, and obtains two polypeptide components GA and GB after enzymolysis and G-15 gel chromatography separation, has different inhibition effects on different tumor cell activities, can inhibit the growth of human squamous tongue cancer cells, but has no obvious inhibition effect on human breast cancer cells and lung cancer human alveolar basal epithelial cells, thus indicating that the pharbitis seed polypeptide has a certain specific inhibition effect on tumors and has the potential of preparing anti-tumor drugs for treating human squamous tongue cancer. The tongue squamous cell carcinoma is one of malignant tumors with higher incidence rate of oral and maxillofacial tumors, and has the advantages of quick growth, high malignant degree, strong wettability, survival rate of less than 15% in 5 years and unsatisfactory treatment effect of the traditional cancers. Further separating and purifying the GA and the GB components, and identifying the amino acid sequence, thereby providing a basis for clinical application of pharbitis seed in treating tumor and research and development of anti-tumor drugs.
The pharmacological experiments of the mice of the invention are studied as follows:
24 BALB/c nude mice with the weight of 18-22g are purchased and are fed to the affiliated experimental animal center of the university of science and technology, university of China, the free diet is carried out, the temperature is controlled to be room temperature, and the humidity is controlled to be 45-55%. SCC25 tongue cancer cells were counted after being resuspended in PBS and then cell concentration was adjusted to 5 x 10 7 0.15mL of the mice were subcutaneously injected under the left forelimb axilla, and after 72 hours, the mice were randomly divided into a control group of DMSO, a morning glory seed administration group (GA 125mg/Kg.day,250 mg/Kg.day) and a group of (GB 125mg/Kg.day,250 mg/Kg.day), 6 animals each were directly administered by solid tumor injection, 1 time per day, 10 days after continuous administration, and all mice were sacrificed on the 11 th dislocation. Tumors were dissected and completely dissected, photographed, recorded, and tumor volumes and weights were measured (see fig. 14-16 in particular). As can be seen from various comparisons of FIGS. 14-17, the results show that tumor volumes were reduced by 45.6.+ -. 8.9% and 51.+ -. 4.3% after administration of 125mg/kg GA and GB; after 250mg/kg GA and GB administration, the tumor volume was significantly reduced by 72.3.+ -. 3.6% and 81.+ -. 2.3%. Furthermore, the pharbitis seed extract protein polypeptide component GA and polypeptide component GB can be seen to have obvious inhibition effect on SCC25 tongue cancer cells (namely human tongue squamous cells).
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. Application of semen Pharbitidis and its extract protein in preparing antitumor drugs is provided.
2. The use according to claim 1, wherein the pharbitis seed and the extract protein is a proteolytically processed polypeptide component.
3. The use according to claim 1, further comprising a method of extraction of said extract, comprising in particular the steps of:
pulverizing semen Pharbitidis;
degreasing in a water bath;
ultrasonic extracting the defatted semen Pharbitidis powder, and centrifuging to collect supernatant to obtain semen Pharbitidis leaching solution;
adding the pharbitis seed extract into a salt solution, precipitating and centrifuging, dialyzing the precipitate, and freeze-drying to obtain pharbitis seed protein;
adding corresponding amount of alkaline protease and pepsin into semen Pharbitidis protein, performing enzymolysis and centrifugation to obtain supernatant, filtering and separating with sephadex G-15, collecting protein polypeptide, freeze drying, and preserving at-20deg.C to obtain extract protein polypeptide.
4. The use according to claim 3, wherein in the step of degreasing in a water bath, petroleum ether is added to the crushed pharbitis seed medicinal material and extracted by a soxhlet extractor, and the extract is degreased by a water bath at 50-60 ℃.
5. The use according to claim 3, characterized in that it further comprises the dissolution of the salt of the defatted pharbitis seed, before said ultrasonic extraction, in particular: taking defatted semen Pharbitidis, and dissolving salt with 0.1-0.2 mol/L salt solution at a mass ratio of 1:20-1:80, wherein the pH value of the salt solution is 5-9.
6. The use according to claim 3, wherein in the step of ultrasonic extraction, the ultrasonic extraction is carried out at a temperature of 20-60 ℃ for a time of 20-40 min.
7. Use according to claim 3, characterized in that said step consists in adding the pharbitis seed extract to a salt solution for precipitation centrifugation, in particular: the salt solution is added into the pharbitis seed leaching solution at a speed of 0.5g/min in a volume of 1ml, and the pH value of precipitation centrifugation is 5-9.
8. The use according to claim 3, characterized in that the centrifugation of the pharbitis seed extract in the precipitation core added to the salt solution is in particular: centrifuging at 7000-9000 r/min for 8-12min at 3-5deg.C.
9. The use according to claim 1, wherein the tumour is a tongue squamous cell carcinoma.
10. The use according to claim 9, characterized in that the tongue squamous cell carcinoma is in particular SCC-9 human tongue squamous cell carcinoma and/or SCC25 human tongue squamous cell carcinoma.
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