CN110950973B - Strychnos nux-vomica polysaccharide and application thereof in preparing radiotherapy sensitization medicine for bladder cancer - Google Patents
Strychnos nux-vomica polysaccharide and application thereof in preparing radiotherapy sensitization medicine for bladder cancer Download PDFInfo
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Abstract
The invention relates to strychnos nux-vomica polysaccharide and application thereof in preparing a radiotherapy sensitization medicine for bladder cancer. The activity test research result shows that the strychnos nux-vomica polysaccharide provided by the invention has no proliferation inhibition effect on the bladder cancer cells, but can increase the radiosensitivity of the bladder cancer cells, and the mechanism of the radiosensitization effect of the strychnos nux-vomica polysaccharide on the bladder cancer cells is probably related to the inhibition of the expression of Survivin protein under the condition of radiation stimulation. Therefore, the strychnos nux-vomica polysaccharide provided by the invention can be used for preparing a radiotherapy sensitizing medicament for bladder cancer.
Description
Technical Field
The invention relates to the field of medicines, in particular to strychnos polysaccharide and application thereof in preparing a radiotherapy sensitizing medicine for bladder cancer.
Background
Tumors are diseases with extremely high mortality rate worldwide, malignant tumors become one of the major diseases seriously threatening the physical and mental health of people of all countries worldwide, and the incidence of the diseases is increased year by year. According to GLOBOCAN 2018, about 956 ten thousand cases of death are shown, and both morbidity and mortality are ranked in the front of various diseases.
Natural drugs are one of the important routes for the source of antitumor drugs, and thus natural products are valuable resources for identifying and developing new cancer treatment regimens. The natural product resources in China are rich, wherein the natural product resources with anti-tumor activity are more various, and a large number of scientific researchers in China are continuously exploring the development of natural anti-tumor drugs.
Semen Strychni is dry mature seed of semen Strychni of Loganiaceae, has long medicinal history, bitter taste, warm nature, and strong toxicity, and has pharmacological effects of exciting central nerve, relieving pain and inflammation, resisting thrombi, tonifying heart, and improving blood microcirculation. Semen Strychni mainly contains alkaloids, glycosides, acids, alcohols, etc., wherein the alkaloids mainly comprise brucine and strychnine, which are effective components and toxic components.
At present, the nux vomica polysaccharide is rarely researched, and the application of the nux vomica polysaccharide in the aspect of tumor treatment is not seen.
Disclosure of Invention
The invention aims to provide strychnos polysaccharide and application thereof in preparing a radiotherapy sensitization medicine for bladder cancer.
In order to achieve the purpose, the invention provides the following technical scheme:
a strychnos polysaccharide is prepared by the following method: taking a proper amount of nux vomica powder, adding deionized water, soaking at normal temperature for 6-10 hours, carrying out thermal reflux extraction for 2-4 times, carrying out 1-3 hours each time, filtering, combining filtrates, concentrating under reduced pressure to 1/10 of the original volume, adding 4 times of volume of anhydrous ethanol, carrying out alcohol precipitation for 11-13 hours, centrifuging, collecting precipitate, washing the precipitate with anhydrous ethanol, diethyl ether and acetone in sequence, volatilizing the solvent, re-dissolving with deionized water, deproteinizing by a Sevag method, and carrying out freeze drying to obtain the nux vomica powder.
Preferably, soaking for 8h at normal temperature.
Preferably, the extraction is carried out 3 times with hot reflux for 2h each time.
Preferably, absolute ethanol is added for alcoholysis for 12 hours.
Preferably, the centrifugation conditions are 4500r/min for 15 min.
The application of the nux vomica polysaccharide in preparing the radiotherapy sensitization medicine of bladder cancer.
The invention provides strychnos nux-vomica polysaccharide, and researches show that although the strychnos nux-vomica polysaccharide has no proliferation inhibition effect on bladder cancer cells, the strychnos nux-vomica polysaccharide can increase the radiosensitivity of the bladder cancer cells, and the mechanism of the radiosensitization effect of the strychnos nux-vomica polysaccharide on the bladder cancer cells is probably related to the inhibition of the expression of Survivin protein under the condition of radiation stimulation.
Drawings
FIG. 1 shows the results of the Westernblot test.
Detailed Description
Example 1: polysaccharide preparation
The semen Strychni is purchased from the traditional Chinese medicine market of Bozhou, Anhui, and is dried mature seeds of Strychnos nux-vomica L. of Strychnos of Loganiaceae, and is sieved with a 40-mesh sieve for later use after being crushed.
Taking a proper amount of nux vomica powder, adding deionized water according to a material-liquid ratio of 1:25(kg: L), soaking at normal temperature for 8h, extracting for 3 times by heating and refluxing, each time for 2h, filtering, combining filtrates, concentrating under reduced pressure to an original volume of 1/10, adding 4 times of volume of anhydrous ethanol, performing alcohol precipitation for 12h, centrifuging at 4500r/min for 15min, collecting precipitate, sequentially washing the precipitate with anhydrous ethanol, diethyl ether and acetone according to a material-liquid ratio of 1:50(g: mL), volatilizing a solvent, re-dissolving with deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the nux vomica polysaccharide.
Example 2: activity assay
First, experimental material
Human bladder cancer cells T24, 5637 and J82 were purchased from ATCC, cryopreserved with liquid nitrogen, and used for testing after recovery according to a conventional method.
DMEM medium, FBS and streptomycin were purchased from Gibco.
The nux vomica polysaccharide is prepared into mother liquor by deionized water, and is stored at 4 ℃ for later use.
Second, Experimental methods
1. Cell culture
Human bladder cancer cells T24, 5637 and J82 were incubated in DMEM medium (complete medium) containing 10% FBS and 1% double antibody at 37 ℃ and 5% CO, respectively2The incubator of (2) and taking the cells in logarithmic growth phase for subsequent experiments.
2. MTT method for determining proliferation inhibition effect of nux vomica polysaccharide on tumor cells
Human bladder cancer cells in logarithmic growth phase T24, 5637 or J82 were inoculated into a 96-well culture plate at a density of 5000 cells/well, and after 24 hours of culture, the cells were replaced with complete media containing curdlan at different concentrations (1, 2, 5mg/mL), and a control group containing no curdlan was set, and 3 duplicate wells were set for each group. After further culturing for 48 hours, 20. mu.L of 5mg/mL MTT solution was added to each well and treated for 4 hours, and the culture was terminated. Centrifugation was performed, the culture supernatant was carefully aspirated from the wells, and finally 150. mu.L of DMSO was added to each well, followed by shaking for 10 minutes to dissolve the crystals sufficiently. Measuring the absorbance OD value of each hole by using an enzyme-linked immunosorbent instrument at the wavelength of 490nm, calculating the result and calculating the cell growth inhibition rate according to the formula: the growth inhibition ratio (%) (control OD value-administration OD value)/control OD value × 100%. All experiments were repeated 3 times and averaged.
3. Method for determining radiosensitization of nux vomica polysaccharide on tumor cells by colony formation method
Inoculating human bladder cancer cells T24, 5637 or J82 in logarithmic growth phase into a 12-hole culture plate at a density of 200 cells/hole, culturing for 24h, replacing a drug group with a complete culture medium containing 2 and 5mg/mL of curdlan, simultaneously setting a control group containing no curdlan, and setting 3 multiple holes in each group. After 24h, the 12-well plate was placed under a 6MV linear accelerator at a dose rate of 400cGy/min and gamma-irradiated at a dose of 4Gy for 1 min. Then, 12-well culture plates were placed in an incubator for normal culture for 12d, the culture solution was carefully aspirated and fixed with methanol for 15min, 1mL of GIMSA staining solution was added to each well, allowed to stand for 10min, the solution was aspirated and washed with PBS for 3 times, the plates were observed under a light microscope, and the number of colonies with >50 cell numbers was counted.
4. Western blot method for determining influence of combined action of nux vomica polysaccharide and radiation on protein Survivin expression
Taking human bladder cancer cells in logarithmic growth phase T24, 5637 or J82 at the ratio of 1.5X 104Inoculating the mixture into 6-well culture plate, culturing for 24 hr, replacing the drug group with complete culture medium containing 2, 5mg/mL strychnos polysaccharide, and setting control group containing no strychnos polysaccharide, wherein each group has 2 multiple wells. After 48h, the 6-well plate was placed under a 6MV linear accelerator at a dose rate of 400cGy/min and gamma-irradiated at a dose of 4Gy for 1 min. Cells were collected, total protein extracted, and total protein concentration determined by BCA method. Taking a proper amount of protein from each group, carrying out SDS-PAGE gel electrophoresis, transferring the protein to a mold, sealing with 5% skimmed milk powder for 1h, carrying out primary antibody to Survivin and GAPDH overnight at 4 ℃, washing the membrane for 3 times and 10min each time by TBST, incubating the secondary antibody for 1h at room temperature, washing the membrane for 3 times and 10min each time by TBST, and carrying out development and photographing by a gel imaging system.
5. Statistical method
Data were processed using SPSS 19.0, mean. + -. standard deviation, and pairwise comparisons between groups using t-test, P < 0.05 representing differences of statistical significance.
Third, experimental results
1. Effect of Strychnos nux-vomica polysaccharide on tumor cell proliferation
The inhibition rate of the proliferation of bladder cancer cells by different concentrations of strychnos polysaccharide is shown in table 1. The results show that the nux vomica polysaccharide has no obvious proliferation inhibition effect on the bladder cancer cells.
TABLE 1 inhibition of proliferation of bladder cancer cells (%). by Strychnos nux-vomica polysaccharides at various concentrations
| Concentration of | Bladder cancer T24 | Bladder cancer 5637 | Bladder cancer J82 |
| 1mg/mL | 1.9±0.7 | 3.3±1.2 | 2.6±1.0 |
| 2mg/mL | 2.4±1.1 | 3.5±1.3 | 2.5±1.2 |
| 5mg/mL | 2.1±0.8 | 2.7±0.9 | 3.1±0.9 |
2. Radiosensitization of nux vomica polysaccharide on tumor cells
The colony numbers of the groups are shown in table 2, compared with the control group, the colony numbers of the drug groups are obviously reduced, and the obvious dose dependence is shown, and the colony forming capacity of the human bladder cancer cells T24, 5637 or J82 is obviously reduced, which shows that the strychnine polysaccharide can obviously increase the radiation sensitivity of the bladder cancer cells.
TABLE 2 cell colony counts for each group
| Bladder cancer T24 | Bladder cancer 5637 | Bladder cancer J82 | |
| Control group | 46±2 | 52±3 | 42±2 |
| 2mg/mL polysaccharide | 36±2 | 35±2 | 21±2 |
| 5mg/mL polysaccharide | 20±3 | 21±3 | 13±2 |
3. Influence of the combined action of strychnos nux-vomica polysaccharide and radiation on protein Survivin expression
The Survivin is a member of an apoptosis-inhibiting protein family, has tumor specificity, is only expressed in tumor and embryonic tissues and is closely related to differentiation, proliferation and infiltration and metastasis of tumor cells, and the tumor cells are not easy to die when the Survivin expression is increased. As shown in the result of the Western blot test in figure 1, compared with the control group, the expression level of the protein Survivin in the bladder cancer cells of the drug group is obviously reduced, and the obvious dose dependence is presented.
The activity test research result shows that the strychnos nux-vomica polysaccharide provided by the invention has no proliferation inhibition effect on the bladder cancer cells, but can increase the radiosensitivity of the bladder cancer cells, and the mechanism of the radiosensitization effect of the strychnos nux-vomica polysaccharide on the bladder cancer cells is probably related to the inhibition of the expression of Survivin protein under the condition of radiation stimulation. Therefore, the strychnos nux-vomica polysaccharide provided by the invention can be used for preparing a radiotherapy sensitizing medicament for bladder cancer.
Claims (1)
1. The application of strychnos nux-vomica polysaccharide in preparing a bladder cancer radiotherapy sensitization medicine is characterized in that the strychnos nux-vomica polysaccharide is prepared by the following steps: taking a proper amount of nux vomica powder, adding deionized water according to the material-liquid ratio of 1kg:25L, soaking at normal temperature for 8h, extracting for 3 times by heating and refluxing, each time for 2h, filtering, combining filtrates, concentrating under reduced pressure to the original volume of 1/10, adding 4 times of volume of anhydrous ethanol for alcohol precipitation for 12h, centrifuging at 4500r/min for 15min, collecting precipitate, sequentially washing the precipitate with anhydrous ethanol, diethyl ether and acetone according to the material-liquid ratio of 1g:50mL, volatilizing the solvent, re-dissolving with deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the nux vomica powder.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105233281A (en) * | 2015-10-28 | 2016-01-13 | 陈填烽 | Application of nano-selenium as iodine-125 particle radiotherapy sensitizing agent |
| CN109306022A (en) * | 2017-07-26 | 2019-02-05 | 晋燕君 | A kind of extracting method of Rui drupe reality polysaccharide |
| CN110194808A (en) * | 2019-06-12 | 2019-09-03 | 陈敏 | A kind of plant essence polysaccharide and preparing the application in skin-lightening cosmetic |
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- 2020-01-02 CN CN202010001503.6A patent/CN110950973B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105233281A (en) * | 2015-10-28 | 2016-01-13 | 陈填烽 | Application of nano-selenium as iodine-125 particle radiotherapy sensitizing agent |
| CN109306022A (en) * | 2017-07-26 | 2019-02-05 | 晋燕君 | A kind of extracting method of Rui drupe reality polysaccharide |
| CN110194808A (en) * | 2019-06-12 | 2019-09-03 | 陈敏 | A kind of plant essence polysaccharide and preparing the application in skin-lightening cosmetic |
Non-Patent Citations (1)
| Title |
|---|
| "POLYSACCHARIDES FROM SEEDS OF STRYCHNOS SPECIES";M. M. CORSARO等;《PHYTOCHEMISTRY》;19950831;第39卷(第6期);第1377-1380页 * |
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