CN112773821A - Preparation method and application of gallnut polyphenol - Google Patents
Preparation method and application of gallnut polyphenol Download PDFInfo
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- CN112773821A CN112773821A CN202110042183.3A CN202110042183A CN112773821A CN 112773821 A CN112773821 A CN 112773821A CN 202110042183 A CN202110042183 A CN 202110042183A CN 112773821 A CN112773821 A CN 112773821A
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Abstract
The invention relates to a preparation method of Chinese gall polyphenol and application thereof, wherein the method takes water as a solvent and obtains a Chinese gall crude extract by an extraction mode of heating reflux; performing reverse column chromatography with polyamide resin, and performing gradient elution with 10%, 40% and 70% ethanol-water eluents respectively; collecting the eluent eluted by 40% ethanol, drying to obtain gallnut polyphenol, and verifying the anti-migration, proliferation and cell cycle arrest effects of the gallnut polyphenol obtained by the method on cervical cancer Siha cells in subsequent cell functional experiments; the up-regulation of oncoprotein E6 mRNA, E6 protein and concomitant tumor suppressor protein p53 is shown in the research on the mechanism of anticancer activity. And has antiviral and antitumor application values, and lays a foundation for the research of developing cervical cancer resistant medicaments with economy, low toxicity and low side effect.
Description
Technical Field
The invention relates to the field of medicine, in particular to a preparation method and application of gallnut polyphenol.
Background
Cervical cancer is the most common gynecological malignancy of female genital tract, HR-HPV infection is the most main cause of cervical cancer, and HPV16 and 18 types are closely related to cervical cancer occurrence. Despite the availability of approved HPV vaccines and drugs, there are still some difficulties with the spread of vaccines due to regional and economic differences. Tumor recurrence, metastasis and adverse drug reactions remain major problems. Some of the problems associated with chemotherapy, including acquired resistance or high rates of side effects on normal cells, remain significant. Thus, the availability of large quantities and low cost of natural product therapy is an alternative to the use of prophylactic vaccines in view of the ease with which natural product therapy is provided, and would greatly reduce the incidence and progression of cervical cancer in china and even worldwide.
Gallnuts are a very specific natural product characterized by the mutual parasitism of plants and insects. They are galls formed on the young shoots of plants due to the settlement of insect inhabitation and egg laying. The species are widely distributed in temperate, subtropical and tropical regions, including china, japan, malaysia and india. The gallnuts are usually 4-5X 1.5 cm in diameter, reddish brown in appearance, angular, and covered with soft fluff. Gallnut is usually harvested between september and october. The gallnut used in the traditional Chinese medicine is the dried and clean gallnut after larva is removed. The main components of Galla chinensis are phenolic compounds, gallic acid and gallic acid methyl ester, wherein gallic acid accounts for about 20%. It can up-regulate the pro-apoptotic protein Bax in cancer cells and induce caspase cascade activity [1 ]. Gallic acid, on the other hand, down-regulates anti-apoptotic proteins such as Bcl-2 and Xiap [2 ]; and has cell cycle arresting effect. It is also an important mechanism for effective anticancer of the polyphenol of gallnut. It has been reported that (DOI: 10.3969/j. issn.1672-7347.2012.09.002) gallic acid has preventive and therapeutic effects on various diseases (such as diarrhea, dysentery, rectal cancer and intestinal cancer, diabetes, sepsis, oral diseases and transfusion) [3,4], but the effects on HPV infection-related diseases and cervical cancer have not been reported. The invention carries out experimental verification on the anti-HPV virus efficacy of the gallnut polyphenol and lays a foundation for further overcoming various diseases caused by HPV infection.
Galla chinensis has been reported in literature (https:// doi.org/10.1002/ptr.3215) to have anticancer activity and antiviral (HIV, Anti-HCV and SARS-CoV) [4], and no study on the inhibitory effect of Galla chinensis on HPV has been reported. There are also patents (publication No.: CN101933916A) that show that gallic acid has anti-HPV efficacy: crushing dry fruits of brucea javanica, concentrating the water extract, extracting with ethyl acetate, and concentrating the extract to obtain an extract; separating with silica gel column chromatography, packing with wet method, and selecting components with single component and high content. Subsequently, in fluorescent quantitative PCR techniques, it was demonstrated that the copy number of HPV-DNA could be reduced [5 ].
Reference to the literature
1.Sariozlu,N.Y.and M.Kivanc,Chapter 60-Gallnuts(Quercus infectoria Oliv.and Rhus chinensis Mill.)and Their Usage in Health,in Nuts and Seeds in Health and Disease Prevention,V.R.Preedy,R.R.Watson,and V.B.Patel, Editors.2011,Academic Press:San Diego.p.505-511.
2.Kim,N.S.,et al.,Gallic acid inhibits cell viability and induces apoptosis in human monocytic cell line U937. J Med Food,2011.14(3):p.240-6.
3.Xiang,Q.,et al.,[Effect of gallnut extract on nasopharyngeal carcinoma 5-8F cells and its mechanism]. Zhong Nan Da Xue Xue Bao Yi Xue Ban,2012.37(9):p.871-5.
4.O,D.and Y.W,Rhus chinensis and Galla Chinensis--folklore to modern evidence:review.Phytotherapy research:PTR,2010.24(12):p.1739-47.
5. Application of gallic acid in preparation of anti-HPV drugs [ P ], poplar and willow, Chinese patent: CN101933916A,2011-01-05.
Based on the above, the preparation method and the application of the gallnut polyphenol provided by the invention have the following advantages:
firstly, the extraction process of the gallnut polyphenol is simple, the extraction process can be realized by utilizing the traditional extraction mode and separation technology, the operation is simple and convenient, and the obtained gallnut polyphenol has high purity and large content; secondly, the gallnut polyphenol has better effects on resisting migration and proliferation of cervical cancer cells, down-regulating cervical cancer E6 oncoprotein and up-regulating oncoprotein p53 than the positive drug pterostilbene.
Disclosure of Invention
The invention aims to provide a preparation method and application of Chinese gall polyphenol, wherein water is used as a solvent, and the Chinese gall polyphenol is extracted by a heating reflux extraction mode; performing reverse column chromatography by using polyamide resin, performing gradient elution by using 10% of ethanol to water, 40% of eluent and 70% of eluent respectively, collecting 40% of eluent, and drying to obtain the gallnut polyphenol; the up-regulation of oncoprotein E6 mRNA, E6 protein and concomitant tumor suppressor protein p53 is shown in the research on the mechanism of anticancer activity. Compared with the positive drug pterostilbene, the pterostilbene has better effect and antiviral and antitumor application values, and lays a foundation for the research of developing cervical cancer resistant drugs with economy, low toxicity and low side effects and the like.
The preparation method of the gallnut polyphenol provided by the invention comprises the following steps:
a. selecting gall cotton aphid, a type of gall which is parasitized on compound leaves of a plurality of trees of rhus of Anacardiaceae by aphid of Aphididae of fimbriatae as a gallnut medicinal material;
b. b, crushing the Chinese gall selected in the step a, sieving the crushed Chinese gall with a 60-mesh sieve, weighing 80g of medicinal material powder, performing reflux extraction for 3 times by using 240mL of distilled water as a solvent, extracting for 2 hours each time, and combining extracting solutions; concentrating the combined extract under reduced pressure to obtain 63.60g of nutgall crude extract;
c. and c, subjecting the nutgall crude extract obtained in the step b to polyamide resin reverse phase column chromatography, performing gradient elution by using eluents with the volume ratios of ethanol to water of 10%, 40% and 70%, performing liquid chromatography-mass spectrometry, collecting the eluent eluted by using 40% ethanol, and drying to obtain 35.2g of nutgall polyphenol containing di-O-galloyl glucose, digallic acid, tri-O-galloyl glucose, ethyl gallate, tetra-O-galloyl glucose, digallic acid ethyl ester and penta-O-galloyl glucose.
The application of the gallnut polyphenol obtained by the method in preparing a medicine for preventing cervical cancer.
The invention relates to a preparation method and application of Chinese gall polyphenol, wherein the analysis information of each component of the Chinese gall polyphenol is shown in table 1;
TABLE 1 analysis information of each component of Galla chinensis polyphenol
The invention relates to a preparation method and application of Chinese gall polyphenol, which is used for evaluating the effect of the Chinese gall polyphenol on HPV16(+) Siha cervical carcinoma cells:
siha cervical cancer cells were purchased from (American Type Culture Collection, Manassas, Va., USA); resuscitating the cells in 10% fetal calf serum, 0.1% penicillin streptomycin solution and DMEM (DMEM) culture medium, and incubating the cells in a thermostat at 37 ℃ and under the condition of 5% carbon dioxide; and carrying out passage;
screening of drug concentration and action time:
a blank control group, a gallnut polyphenol group and a positive control group (pterostilbene group) are set, cells are cultured in a 96-well plate, and gallnut polyphenol and pterostilbene (3 mu g/ml, 6 mu g/ml, 12 mu g/ml, 25 mu g/ml, 50 mu g/ml and 100 mu g/ml) are added with CCK-8 reagent for incubation to screen out the action time and half lethal concentration of the gallnut polyphenol and the pterostilbene on Siha cells, and the result indicates that the gallnut polyphenol has time-dependent concentration on the Siha cervical cancer cells and the cell survival rate is more than 90% when the gallnut polyphenol and the pterostilbene act for 24 hours at 6 mu g/ml;
the influence of the gallnut polyphenol on the cervical cancer cells is evaluated by a cell function biological experiment:
the influence of 603 on the migration, cloning, and cell cycle arrest of HPV16(+) cervical cancer Siha cells was evaluated by scratch test, plate cloning, and flow cytometry, and the results thereof showed: the Siha cervical carcinoma cells treated by the gallnut polyphenol have a relatively obvious anti-migration effect for 24 hours. We normalized the amount of untreated cell scratch (wound) to 100%. In contrast to this control, the gallnut polyphenol-treated cells migrated only 11.88% (3 μ g/ml), 9.56% (6 μ g/ml) and 8.23% (12 μ g/ml), while the cells treated with the positive pair (control pterostilbene group) at the same concentration migrated 12.17%, 9.03% and 8.80% (fig. 2, fig. 3). In plate cloning experiments, the gallnut polyphenol can effectively inhibit Siha cell aggregation (figure 4 and figure 5); the flow cytometry indicates that the gallnut polyphenol treated Siha cervical cancer cells have the cell cycle arrest effect and are mainly arrested in the S phase (figure 6, figure 7 and figure 8);
qRT-PCR detection of the regulation level of gallnut polyphenol on oncoprotein E6 mRNA:
inoculating SiHa cells in a 6-well plate and culturing overnight, treating the cells with gallnut polyphenol or pterostilbene For 24 hours, and designing a primer sequence, wherein the primer sequence is HPV 16E 6-For: 5'-CTGCGACGTGAGGTATATGAC-3' and Rev: 5'-TGATGATCTGCAACAAGACATAC-3'; GADPH-For: 5'-GAGTCAACGGATTTGGTCGT-3', Rev: 5'-TTGATTTTGGAGGGATCTCG-3', using equation 2-ΔΔCt calculates the relative mRNA expression level after GAPDH standardization according to the expression change, and the result indicates that the HPVE6 mRNA expression is in negative correlation with the drug concentration; e6 mR after 24h of action when the drug concentration is 12 mug/mlThe expression amount of NA is minimum;
verifying the influence level of the Chinese gall polyphenol on the oncoprotein E6 and the oncostatin p 53:
western Blot analysis: culturing Siha cells in a 6-pore plate, treating with gallnut polyphenol or pterostilbene, extracting total protein with protein lysate and phosphatase inhibitor, performing 10% SDS-PAGE gel electrophoresis, transferring to membrane, and exposing with an automatic exposure machine; image J analysis of grey values, mapping, WB analysis showed a significant increase in p53 protein levels in Siha cells treated with gallnut polyphenols 6 μ g/ml;
immunofluorescence analysis: siha cells are cultured in a 6-well plate, treated by gallnut polyphenol or pterostilbene for 24 hours, incubated with antibodies, stained, placed under a fluorescence inverted microscope to take fluorescence pictures of each group, Image J analyzes the fluorescence intensity of the pictures, and quantitative information is plotted, and the result shows that gallnut polyphenol can inhibit oncoprotein E6 and then up-regulate oncoprotein p53 (shown in figures 9-15).
Drawings
FIG. 1 is a liquid chromatogram of the water extract of Galla chinensis, wherein 1 is di-O-galloyl glucose, 2 is digallic acid, 3 is tri-O-galloyl glucose, 4 is ethyl gallate, 5 is tetra-O-galloyl glucose, 6 is penta-O-galloyl glucose and 7 is ethyl digallic acid;
FIG. 2 is a graph showing the anti-migration results of gallnut polyphenols on cervical cancer cells, the Siha cells are scratched, the cells treated with the sublethal concentrations (3. mu.g/mL, 6. mu.g/mL, 12. mu.g/mL) of the gallnut polyphenols and pterostilbene are monitored, the area of the cells migrating to the scratches is calculated after 48 hours, and the results show that the gallnut polyphenols have stronger anti-migration capability compared with the pterostilbene, wherein A is a control group, B is 3. mu.g/mL of gallnut polyphenols, C is 6. mu.g/mL of gallnut polyphenols, D is 12. mu.g/mL of gallnut polyphenols, E is 3. mu.g/mL of pterostilbene, F is 6. mu.g/mL of pterostilbene, and G is 12. mu.g/mL of pterostilbene;
FIG. 3 is a data diagram of three repeated sample experiments (mean% migrated cells + -SEM;. p <0.0001) with normalized cervical cancer cell migration resistance results to control of Galla chinensis polyphenol of the present invention, wherein A is the control, B is Galla chinensis polyphenol 3. mu.g/mL, C is Galla chinensis polyphenol 6. mu.g/mL, D is Galla chinensis polyphenol 12. mu.g/mL, E is pterostilbene 3. mu.g/mL, F is pterostilbene 6. mu.g/mL, and G is pterostilbene 12. mu.g/mL;
FIG. 4 is a graph comparing the effect of Galla chinensis polyphenols and pterostilbene on the ability of Siha cells to form clones in the cloning experiments of the present invention at different concentrations. Culturing cells treated by nutgall polyphenol and pterostilbene (3 mug/mL, 6 mug/mL and 12 mug/mL) and untreated cells for 15 days to form cell colonies, wherein A is a control group, B is 3 mug/mL of nutgall polyphenol, C is 6 mug/mL of nutgall polyphenol, D is 12 mug/mL of nutgall polyphenol, E is 3 mug/mL of pterostilbene, F is 6 mug/mL of pterostilbene, and G is 12 mug/mL of pterostilbene;
FIG. 5 is a graph showing that the gallnut polyphenols of the present invention inhibit cell clonogenic activity, and are more potent than pterostilbene. Data from three replicate sample experiments normalized to control (mean% survival ± SEM;. p <0.0001), where a is control, B is gallnut polyphenol 3 μ G/mL, C is gallnut polyphenol 6 μ G/mL, D is gallnut polyphenol 12 μ G/mL, E is pterostilbene 3 μ G/mL, F is pterostilbene 6 μ G/mL, G is pterostilbene 12 μ G/mL;
FIG. 6 is a graph showing the S-phase arrest and the decrease in G2/M cell number of Siha cells after treatment of cells with varying concentrations of Galla chinensis polyphenol according to the present invention; after detecting untreated cells and cells of a gallnut polyphenol (3 mug/mL, 6 mug/mL and 12 mug/mL) treatment group for 18 hours by using a flow cytometry technology, the cells show S-phase block and then the cell number of G2/M is reduced, wherein A is a control group, B is gallnut polyphenol 3 mug/mL, C is gallnut polyphenol 6 mug/mL, and D is gallnut polyphenol 12 mug/mL;
FIG. 7 is a graph showing the S-phase arrest and the decrease in G2/M cell number of Siha cells after treatment of cells with pterostilbene of various concentrations in accordance with the present invention. After detecting untreated cells and pterostilbene (3 mu G/mL, 6 mu G/mL and 12 mu G/mL) treatment group cells by using a flow cytometry technology for 18 hours, the cells show S-phase block and then the cell number of G2/M is reduced, wherein A is a control group, E is pterostilbene 3 mu G/mL, F is pterostilbene 6 mu G/mL, and G is pterostilbene 12 mu G/mL.
FIG. 8 is a diagram of the cell cycle effect induced by nutgall polyphenol and pterostilbene under different concentration conditions. Data (mean ± s.e.m.) of three groups of repeated sample experiments normalized to a control, wherein a is the control group, B is gallnut polyphenol 3 μ G/mL, C is gallnut polyphenol 6 μ G/mL, D is gallnut polyphenol 12 μ G/mL, E is pterostilbene 3 μ G/mL, F is pterostilbene 6 μ G/mL, and G is pterostilbene 12 μ G/mL;
FIG. 9 is a fluorescent image of E6 of cells treated with different concentrations of nutgall polyphenols and pterostilbene of the present invention. After treatment of cells with nutgall polyphenols and pterostilbene (3. mu.g/mL, 6. mu.g/mL, 12. mu.g/mL), Siha cell E6 protein (green) was immunofluorescent stained and counterstained with the nuclear dye 4', 6-diamino-2-phenylindole (DAPI) (blue). Adding 12 μ G/mL of nutgall polyphenol and pterostilbene leads to the loss of more E6 protein, wherein A is a control group, B is 3 μ G/mL of nutgall polyphenol, C is 6 μ G/mL of nutgall polyphenol, D is 12 μ G/mL of nutgall polyphenol, E is 3 μ G/mL of pterostilbene, F is 6 μ G/mL of pterostilbene, and G is 12 μ G/mL of pterostilbene;
FIG. 10 is a graph of E6 fluorescence data from the cell image analysis of the present invention, showing that 12 μ g/mL of Galla chinensis polyphenol treated cells resulted in approximately 70% reduction in E6 protein levels, 65% in the pterostilbene group. The five-fold subgroup down-regulation trend is more obvious than that of the pterostilbene group. Data from three replicate sample experiments normalized to control (mean ± s.e.m.;. p <0.0001), where a is control, B is gallnut polyphenol 3 μ G/mL, C is gallnut polyphenol 6 μ G/mL, D is gallnut polyphenol 12 μ G/mL, E is pterostilbene 3 μ G/mL, F is pterostilbene 6 μ G/mL, G is pterostilbene 12 μ G/mL;
FIG. 11 is a graph showing the change of E6 mRNA expression level detected by qRT-PCR after treatment of different concentrations of nutgall polyphenol and pterostilbene of the present invention. The nutgall polyphenol group and the pterostilbene group both show a down-regulation trend. Data from three replicate sample experiments normalized to control (mean ± s.e.m.;. p <0.0001), where a is control, B is gallnut polyphenol 3 μ G/mL, C is gallnut polyphenol 6 μ G/mL, D is gallnut polyphenol 12 μ G/mL, E is pterostilbene 3 μ G/mL, F is pterostilbene 6 μ G/mL, G is pterostilbene 12 μ G/mL;
FIG. 12 is a fluorescent graph of p53 of cells treated with different concentrations of polyphenols and pterostilbene of Galla chinensis according to the present invention. After treatment of cells with nutgall polyphenol and pterostilbene (3. mu.g/mL, 6. mu.g/mL, 12. mu.g/mL), Siha cell p53 protein (green) was immunofluorescent stained and counterstained with the nuclear dye 4', 6-diamidino-2-phenylindole (DAPI) (blue). Adding gallnut polyphenol of 12 mu G/mL and pterostilbene to activate the expression of more p53 protein, wherein A is a control group, B is gallnut polyphenol of 3 mu G/mL, C is gallnut polyphenol of 6 mu G/mL, D is gallnut polyphenol of 12 mu G/mL, E is pterostilbene of 3 mu G/mL, F is pterostilbene of 6 mu G/mL, and G is pterostilbene of 12 mu G/mL;
FIG. 13 is a graph of p53 fluorescence data from image analysis of cells of the present invention, showing that the expression of p53 was increased by nearly 5-fold in cells treated with 12. mu.g/mL of Galla chinensis polyphenol as compared to the control group; the pterostilbene group increased 4.5 times. Data from three replicate sample experiments normalized to control (mean ± s.e.m.;. p <0.05), where a is control, B is gallnut polyphenol 3 μ G/mL, C is gallnut polyphenol 6 μ G/mL, D is gallnut polyphenol 12 μ G/mL, E is pterostilbene 3 μ G/mL, F is pterostilbene 6 μ G/mL, G is pterostilbene 12 μ G/mL;
FIG. 14 is a Western blot (Western blot) of cells treated with nutgall polyphenol and pterostilbene of the present invention at different concentrations, showing that the expression of p53 in the treated cells at different concentrations is up-regulated, wherein A is a control group, B is nutgall polyphenol 3. mu.g/mL, C is nutgall polyphenol 6. mu.g/mL, D is nutgall polyphenol 12. mu.g/mL, and E is pterostilbene 3. mu.g/mL;
FIG. 15 is a data graph of three repeated sample experiments normalized to a control by Western blot experiment (mean + -S.E.M.;. p <0.05), wherein A is the control, B is gallnut polyphenol 3. mu.g/mL, C is gallnut polyphenol 6. mu.g/mL, D is gallnut polyphenol 12. mu.g/mL, and E is pterostilbene 3. mu.g/mL.
Detailed Description
The technical solution of the present invention will be described in further detail below.
Example 1
a. Selecting a kind of gall which is formed by parasitizing aphids of Aphis gossypii Miq and Aphis quinquefolius of Aphidae on compound leaves of several trees of Rhus of Anacardiaceae as a gallnut medicinal material;
b. b, crushing the Chinese gall selected in the step a, sieving the crushed Chinese gall with a 60-mesh sieve, weighing 80g of medicinal material powder, performing reflux extraction for 3 times by using 240mL of distilled water as a solvent, extracting for 2 hours each time, and combining extracting solutions; concentrating the combined extract under reduced pressure to obtain 63.60g of nutgall crude extract;
c. and (c) separating components in the gallnut from the crude extract of the gallnut obtained in the step (b) by polyamide resin reverse column chromatography, wherein polyamide molecules contain rich amide groups and can be combined with phenolic hydroxyl groups in the polyphenol compounds to form hydrogen bonds to be adsorbed, so that certain substances in a certain polarity range are separated, and the specific operation steps are as follows: first is the activation of the polyamide: soaking polyamide in 90-95 vol% ethanol overnight, stirring, removing bubbles, and loading into column; eluting with 3 times volume of 90-95% ethanol until the eluate is transparent and has no residue (or little residue) after evaporation; then sequentially eluting with 2.5 times of 5% NaOH aqueous solution, 1 time of distilled water and 2 times of 10% acetic acid aqueous solution, and finally eluting with distilled water until the pH is neutral for later use; secondly, filling activated polyamide into a column by a wet method, wherein each 100ml of polyamide powder can be loaded with 2.0g, and the total loading is 60.0 g; then gradient elution is carried out by eluent with the volume ratio of ethanol to water being respectively 10 percent, 40 percent and 70 percent; and (3) measuring by liquid chromatography-mass spectrometry, collecting the eluent eluted by 40% ethanol, and drying to obtain 35.2g of nutgall polyphenol containing di-O-galloyl glucose, digallic acid, tri-O-galloyl glucose, ethyl gallate, tetra-O-galloyl glucose, digallic acid ethyl ester and penta-O-galloyl glucose.
Example 2
The application of the gallnut polyphenol obtained by the method in preparing the cervical cancer prevention medicine is as follows:
in the experiment, a blank control group, a gallnut polyphenol group and a positive control group (pterostilbene group) are established;
1. the material and the method are as follows:
1.1 cell culture:
human cervical cancer SiHa cell lines were obtained from the Korean Cell Line Bank (KCLB), and cells were cultured in Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 0.1% penicillin streptomycin solution (Biosharp), and modified by Dulbecco corporation; incubating the cells in an incubator at 37 ℃ and with 5% carbon dioxide;
1.2CCK-8 (cell proliferation-toxicity assay):
digesting cells in logarithmic phase with trypsin to prepare cell suspension, adding 100 mu l of the cell suspension into each hole to enable the cell density of each hole to be 4000, and filling the marginal holes with sterile PBS; 5% CO2Culturing at 37 ℃ until cells are attached to the wall and spread to the bottom of a full hole, diluting the medicine gallnut polyphenol and pterostilbene in a DMEM complete culture medium according to 0-100 mu g, setting 4 multiple holes for each concentration treatment group, respectively adding 10 mu l of CCK-8 solution into each hole after treatment is finished, then incubating a 96-hole plate in an incubator for 1 hour, monitoring absorbance at 450nm by using a microplate reader, and analyzing the obtained result by using GraphPad Prism 8 software to determine the half lethal concentration of the gallnut polyphenol and the pterostilbene;
1.3 scratch test:
culturing 25 ten thousand cells on a 6-well plate until the cells are completely attached to the wall; scratching each well in the center of the medium with a sterile p200 probe, washing the cell debris with 1 × PBS and marking the bottom of the plate, taking images at the same location, then treating the cells with different concentrations (3 μ g/ml, 6 μ g/ml and 12 μ g/ml) of nutgall polyphenol or pterostilbene, and taking images after 24 and 48 hours, measuring the migration residual gap between SiHa cells at three random intervals under each experimental condition, comparing the percentage of the original scratch width as comparative data;
1.4 plate cloning experiments:
25 ten thousand cells were cultured on a 6-well plate, treated with nutgall polyphenol or pterostilbene (3 μ g/ml, 6 μ g/ml, 12 μ g/ml) for 24 hours, after 24 hours, the cells were trypsinized into single cell suspensions, after cell counting, 200 live cells of each treatment group were plated from the 6-well plate into one well and grown in complete DMEM medium for 15 days, after the above time period, the cells were washed three times with 1 × PBS, fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 30 minutes, the cells were briefly rinsed with single distilled water and allowed to air dry, images of each well were taken using a digital camera and the number of colonies (cell number-clone number) × 100% were calculated;
1.5 real-time quantitative reverse transcription PCR (qRT-PCR) Gene expression analysis:
inoculating SiHa cells in 6-well plate and culturing overnight, treating cells with Galla chinensis polyphenol or pterostilbene for 24 hr, and separating total RNA from used primary cells
Total RNA Miniprep kit (New England Biolabs, MA, USA) and by quantification using a NanoDrop spectrophotometer (Thermo Scientific), all samples showed a ratio of optical densities A260/A280 and A260/A230 of about 2.0; reverse transcription of RNA into cDNA using iScriptTM, cDNA Synthesis kit (Bio-Rad, Hercules, Calif., USA); using SsoAdvancedTMUniversal SYBR Green Supermix (Bio-Rad) in Bio-Rad CFX96 Touch for qRT-PCR; the PCR cycles were as follows: the temperature is 95 ℃ for 15min, the temperature is 95 ℃ for 15s and the temperature is 60 ℃ for 1min, and 40 cycles are carried out; then, carrying out melting curve analysis, and increasing each period from the temperature of 65-95 ℃ to 0.5 ℃; the primers, nucleotide sequence listing, equation 2-ΔΔCt calculates relative mRNA expression level after GAPDH normalization from the change of expression; primer sequences for qRT-PCR analysis;
TABLE 2 qRT-PCR primer related sequences
1.6 flow cytometry:
culturing 25 ten thousand cells on a 6-well culture plate, treating the cells for 24 hours by using nutgall polyphenol or pterostilbene (3 mu g/ml, 6 mu g/ml and 12 mu g/ml), treating the cells by trypsin, centrifuging, washing the cells in 1 XPBS solution, resuspending the cells in 70% ethanol at the temperature of-20 ℃, dripping ethanol while shaking, fixing the samples at the temperature of 4 ℃ overnight, washing the samples twice in 1 XPBS, preparing RNase A and PI working solution into staining working solution according to the volume of 1:9 before use, staining the working solution for 30 minutes before flow cytometry, incubating 500 mu l of the working solution per sample, measuring PI fluorescence by using an Accuri-C6 flow cytometer (BD), and analyzing the DNA content of the cells;
1.7 Western Blot analysis:
protein lysate was extracted from Siha cells using RIPA lysis buffer (H8080, Solarbio biotechnology, china), phosphatase inhibitor (P1261-100T, Solarbio, china); the concentration of protein lysates was quantified by BCA analysis (#23227, Thermo, USA), then the samples were incubated in boiling water for 10 min, then the samples were separated by 10% SDS-PAGE, then transferred to PVDF middle membranes; after blocking with 5% Bovine Serum Albumin (BSA) in TBS + Tween Buffer (TBST) for 2 hours, membranes were incubated with HR primary antibody (1:1000 dilution, abcam, China); p53(1:1000 dilution, TA336582, China fir Jinqiao, China); at 4 ℃ overnight, at the end of primary antibody incubation, horseradish peroxidase-conjugated secondary antibody was diluted 1:10000, incubated for 2 hours, and specific bands were visualized by ECL (Advansta, usa); bands were detected using the ChemiDoc XRS + system (Bio-Rad, Hercules, Calif., USA) and the ImageJ program was used to quantify the proteins of interest;
1.8 immunofluorescence:
8000 SiHa cells were cultured in 6-well plates, allowed to grow for 24 hours, then treated with varying concentrations of nutgall polyphenols (3. mu.g/ml, 6. mu.g/ml, 12. mu.g/ml) or pterostilbene (3. mu.g/ml, 6. mu.g/ml, 12. mu.g/ml) for 22 hours (for studies E6 and p53), after which the cells were fixed in 4% paraformaldehyde at room temperature, washed with 1 XPBS, then osmotically blocked with 0.5% polyethylene glycol octylphenyl ether (Triton X-100) in Phosphate Buffered Saline (PBS) for 1h, then incubated overnight with primary antibody in blocking buffer, after which the cells were washed, then incubated with the corresponding Fluorescein Isothiocyanate (FITC) -conjugated secondary antibody for 3h, then with 4', 6-diamino-2-phenylindole (DAPI) (100. mu.L/well), and washed 3 times with 1 × PBS; the slides were then mounted with coverslips, images of the cells were taken with a fluorescence microscope, images of different randomly selected fields were collected from each well, quantified with the same exposure time, fluorescence intensity was measured with ImageJ, fluorescence intensity of E6 and P53 antibodies was normalized to DAPI intensity (blue), and antibodies used: e6 antibody (abcam), p53 antibody (TA336582, sequoia china, sequoia chinensis), secondary antibody (ZF-0321, sequoia chinensis, china);
and (4) conclusion: the influence of 603 on the migration, cloning, and cell cycle arrest of HPV16(+) cervical cancer Siha cells was evaluated by scratch test, plate cloning, and flow cytometry, and the results thereof showed: siha cervical carcinoma cells treated by gallnut polyphenol have a relatively obvious anti-migration effect for 24 hours; the amount of untreated cell scratch (wound) was normalized to 100%, relative to this control, gallnut polyphenol treated cells migrated only 11.88% (3 μ g/ml), 9.56% (6 μ g/ml) and 8.23% (12 μ g/ml), while cells treated with positive pairs (control pterostilbene group) at the same concentration 12.17%, 9.03% and 8.80% (fig. 2, fig. 3); in plate cloning experiments, the gallnut polyphenol can effectively inhibit Siha cell aggregation (figure 4 and figure 5); the flow cytometry indicates that the gallnut polyphenol treated Siha cervical cancer cells have the cell cycle arrest effect and are mainly arrested in the S phase (figure 6, figure 7 and figure 8); therefore, the gallnut polyphenol has better effects on resisting migration and proliferation of cervical cancer cells, down-regulating cervical cancer E6 oncoprotein and up-regulating oncoprotein p53 than the positive drug pterostilbene.
Claims (2)
1. The preparation method of the gallnut polyphenol is characterized by comprising the following steps:
a. selecting a kind of gall which is formed by parasitizing aphids of Aphis gossypii Miq and Aphis quinquefolius of Aphidae on compound leaves of several trees of Rhus of Anacardiaceae as a gallnut medicinal material;
b. b, crushing the Chinese gall selected in the step a, sieving the crushed Chinese gall with a 60-mesh sieve, weighing 80g of medicinal material powder, performing reflux extraction for 3 times by using 240mL of distilled water as a solvent, extracting for 2 hours each time, and combining extracting solutions; concentrating the combined extract under reduced pressure to obtain 63.60g of nutgall crude extract;
c. subjecting the crude extract of Galla chinensis obtained in step b to polyamide resin reverse phase column chromatography, performing gradient elution with 10%, 40% and 70% ethanol-water volume ratio, respectively, determining by liquid chromatography-mass spectrometry, collecting 40% ethanol-eluted eluate, and drying to obtain crude extract containing di-substituted organic acidsOGalloylglucose, digallic acid, tris-O-galloylglucose, ethyl gallate, tetrakis-OGalloylglucose, ethyl digallate and penta-O35.2g of nutgall polyphenol of galloylglucose.
2. Use of the polyphenols of Galla rhois obtained by the method of claim 1 in the preparation of a medicament for preventing cervical cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109125309A (en) * | 2018-07-12 | 2019-01-04 | 石河子大学 | With the active component and preparation method thereof of anti-inflammatory effect in galla turcica |
| CN115105513A (en) * | 2022-08-16 | 2022-09-27 | 新乡医学院 | Pharmaceutical composition for adjuvant therapy of cervical cancer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1559392A (en) * | 2004-03-10 | 2005-01-05 | 牛凤兰 | Applicaton of gallic acid in prepn. of medicine for antitumor |
| CN103690567A (en) * | 2012-12-28 | 2014-04-02 | 中国医学科学院药用植物研究所 | Gallnut ethyl acetate extract as well as preparation and detection methods and application thereof |
| CN108864236A (en) * | 2018-08-09 | 2018-11-23 | 中国林业科学研究院林产化学工业研究所 | A kind of 1,2,3,4,6-O- Penta-O-galloyl-D-glucopyranose germanium complex and its preparation method and application |
| CN111574571A (en) * | 2020-06-11 | 2020-08-25 | 石河子大学 | Separation method and application of effective part of gallnut |
-
2021
- 2021-01-13 CN CN202110042183.3A patent/CN112773821A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1559392A (en) * | 2004-03-10 | 2005-01-05 | 牛凤兰 | Applicaton of gallic acid in prepn. of medicine for antitumor |
| CN103690567A (en) * | 2012-12-28 | 2014-04-02 | 中国医学科学院药用植物研究所 | Gallnut ethyl acetate extract as well as preparation and detection methods and application thereof |
| CN108864236A (en) * | 2018-08-09 | 2018-11-23 | 中国林业科学研究院林产化学工业研究所 | A kind of 1,2,3,4,6-O- Penta-O-galloyl-D-glucopyranose germanium complex and its preparation method and application |
| CN111574571A (en) * | 2020-06-11 | 2020-08-25 | 石河子大学 | Separation method and application of effective part of gallnut |
Non-Patent Citations (4)
| Title |
|---|
| SUN-YOUNG YOON, ET AL: "Identification of Vaccinia-H1 Related Phosphatase as an Anticancer Target for 1, 2, 3, 4, 6-O-Pentagalloylglucose", CHEM BIODIVERS., vol. 17, no. 2, pages 1900414 * |
| 杨杰: "现代临床专科护理新进展", 河南大学出版社, pages: 142 * |
| 陈静岚, 刘妹, 张国平: "五倍子注射液治疗早期宫颈癌24例", 黑龙江中医药, no. 01 * |
| 高洁莹等: "盐肤木属植物研究进展", 中国实验方剂学杂志, vol. 21, no. 8, pages 216 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109125309A (en) * | 2018-07-12 | 2019-01-04 | 石河子大学 | With the active component and preparation method thereof of anti-inflammatory effect in galla turcica |
| CN115105513A (en) * | 2022-08-16 | 2022-09-27 | 新乡医学院 | Pharmaceutical composition for adjuvant therapy of cervical cancer |
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