CN114642714A - Anti-tumor traditional Chinese medicine composition extract, preparation method and application thereof in inhibiting breast cancer lung metastasis - Google Patents
Anti-tumor traditional Chinese medicine composition extract, preparation method and application thereof in inhibiting breast cancer lung metastasis Download PDFInfo
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Abstract
The invention relates to an anti-tumor traditional Chinese medicine composition extract, a preparation method and application thereof in inhibiting lung metastasis of breast cancer, wherein the traditional Chinese medicine composition extract can obviously inhibit lung metastasis of breast cancer tumor cells; in addition, the cell activity can be inhibited through synergistic induction of apoptosis, and the sensitivity of breast cancer cell radiotherapy can be enhanced.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an anti-tumor traditional Chinese medicine composition extract, a preparation method and application thereof in inhibiting breast cancer lung metastasis.
Background
The breast cancer is a phenomenon that mammary epithelial cells generate uncontrolled proliferation under the action of various carcinogenic factors. The early stage of the disease often shows symptoms of breast lumps, nipple discharge, axillary lymphadenectasis and the like, and the later stage of the disease can generate distant metastasis due to cancer cells to generate multi-organ lesion, thereby directly threatening the life of a patient. The lung is a common metastatic site of breast cancer, often manifesting as bilateral multiple nodules. The patient may have symptoms of cough, dyspnea, hemoptysis, chest pain, etc. The invention provides a traditional Chinese medicine composition extract capable of inhibiting breast cancer cell proliferation and breast cancer lung metastasis.
Disclosure of Invention
The invention provides a traditional Chinese medicine composition (for inhibiting breast cancer lung metastasis), which is characterized by comprising the following components in parts by weight: 1-5 parts of radix curcumae, 1-5 parts of hairyvein agrimony, 1-5 parts of trogopterus dung, 1-5 parts of alum, 1-5 parts of saltpeter, 0.5-3 parts of prepared dried lacquer, 3-7 parts of bran-fried fructus aurantii and 0.5-4 parts of nux vomica powder. Preferably, the following components are used: 2-4 parts of curcuma aromatica, 2-4 parts of agrimony, 2-4 parts of trogopterus dung, 2-4 parts of alum, 2-4 parts of saltpeter, 0.5-2 parts of prepared lacquer, 4-6 parts of bran-fried fructus aurantii and 1-3 parts of nux vomica powder. The best is as follows: 3 parts of curcuma aromatica, 3 parts of agrimony, 2.5 parts of trogopterus dung, 3 parts of alum, 3 parts of saltpeter, 1 part of prepared lacquer, 5 parts of bran-fried bitter orange and 2 parts of nux vomica powder.
The invention provides a traditional Chinese medicine composition extract (for inhibiting breast cancer lung metastasis), which is characterized in that a preparation method of the traditional Chinese medicine composition extract comprises the following steps:
(1) pulverizing the prepared dried lacquer, trogopterus dung, alum and saltpeter according to the formula amount, sieving by a 80-mesh sieve, adding water, ultrasonically extracting for 1-3 times, mixing the extracting solutions, and concentrating to obtain thick paste a; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract b;
(2) percolating radix Curcumae and fructus Aurantii parched with bran with 70% ethanol, collecting percolate and residue, and concentrating percolate to obtain extract c;
(3) weighing herba et Gemma Agrimoniae according to the formula, mixing with the residues collected in step (2), decocting with water for 1-3 times, mixing decoctions, and concentrating to obtain soft extract d;
(4) weighing semen Strychni Pulveratum according to formula, extracting with water under reflux for 1-3 times, mixing extractive solutions, and concentrating to obtain soft extract e; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract f;
(5) and (4) combining the thick paste a-f obtained in the steps (1) to (4), re-dissolving the thick paste with water, and volatilizing the mixture to obtain the traditional Chinese medicine composition extract.
The ultrasonic extraction in the steps (1) and (4) adopts conventional ultrasonic extraction equipment in the field, and the ultrasonic extraction time is 15-30min each time. The extraction times is 1-3 times, and the dosage of the extraction solvent is preferably 4-6 times (more preferably 5 times) of the medicinal material; the dosage of the extraction solvent (including percolation, decoction, reflux and ultrasound) in the steps (1) to (4) can be reasonably selected by a person skilled in the art according to the physicochemical properties of the medicinal materials, and is preferably 4-6 times (more preferably 5 times) of the mass of the medicinal materials; the extraction time and times can be selected reasonably by those skilled in the art according to physicochemical properties of medicinal materials, preferably 15-120min, and preferably 1-3 times.
Another embodiment of the present invention provides a method for preparing the above-mentioned extract of the Chinese medicinal composition, which is characterized by comprising the following steps:
(1) pulverizing the prepared dried lacquer, trogopterus dung, alum and saltpeter according to the formula amount, sieving by a 80-mesh sieve, adding water, ultrasonically extracting for 1-3 times, mixing the extracting solutions, and concentrating to obtain thick paste a; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract b;
(2) according to the formula, weighing radix curcumae and bran-fried fructus aurantii, percolating the radix curcumae and the bran-fried fructus aurantii with 70% ethanol, collecting percolate and dregs, and concentrating the percolate to obtain an extract c;
(3) weighing herba et Gemma Agrimoniae according to the formula, mixing with the residues collected in step (2), decocting with water for 1-3 times, mixing decoctions, and concentrating to obtain soft extract d;
(4) weighing semen Strychni Pulveratum according to formula, extracting with water under reflux for 1-3 times, mixing extractive solutions, and concentrating to obtain soft extract e; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract f;
(5) and (4) combining the thick paste a-f obtained in the steps (1) to (4), re-dissolving the thick paste with water, and volatilizing the mixture to obtain the traditional Chinese medicine composition extract.
Another embodiment of the invention provides application of the traditional Chinese medicine composition and/or the traditional Chinese medicine composition extract in preparing a breast cancer radiotherapy sensitizing drug.
The other embodiment of the invention provides application of the traditional Chinese medicine composition and/or the traditional Chinese medicine composition extract in preparing a medicine with sensitization effect on X-ray induced apoptosis. In particular to the application in preparing the medicine with the sensitization effect on the apoptosis of X-ray induced MDA-MB-231 and MDA-MB-468 cells.
Another embodiment of the present invention provides an application of the above traditional Chinese medicine composition and/or traditional Chinese medicine composition extract in preparing a medicine for inhibiting breast cancer lung metastasis.
Another embodiment of the present invention provides a medicament for inhibiting lung metastasis of breast cancer, wherein the medicament comprises the above-mentioned Chinese medicinal composition and/or Chinese medicinal composition extract as an active ingredient. Also comprises pharmaceutically acceptable auxiliary materials. The dosage form of the medicament is selected from solid preparation, liquid preparation or semisolid preparation.
Compared with the prior art, the invention has the advantages that: (1) the traditional Chinese medicine composition extract and the medicine-containing serum can obviously inhibit breast cancer cells, and the proliferation inhibition effect on the three-negative breast cancer cells is superior to that of other typed breast cancer cells. (2) The Chinese medicinal composition extract has certain inhibiting effect on MDA-MB-231 and MDA-MB-468 cell migration and invasion. (3) The research of a mouse tumor lung metastasis model proves that the traditional Chinese medicine composition extract can obviously inhibit the lung metastasis of tumor cells. (4) Through the investigation on the radiotherapy sensitization, the traditional Chinese medicine composition extract can inhibit the cell activity by synergistically inducing the apoptosis and enhance the sensitivity of the breast cancer cell radiotherapy.
Drawings
FIG. 1 is a graph of the protein expression of ER α and HER2 in individual breast cancer cells;
fig. 2 is a graph of the inhibitory effect of the extract of the traditional Chinese medicine composition on different types of breast cancer cells (x ± s, n ═ 5); a, the inhibition rate of the traditional Chinese medicine composition after 72 hours of action; b: the inhibition rate of the traditional Chinese medicine composition after 96 hours of action; the administration concentration is as follows: increasing from left to right, wherein the concentration is respectively 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 mu g/mL;
FIG. 3 is a graph showing the inhibitory effect of drug-containing serum of the herbal composition extract on different types of breast cancer cells (x + -s, n-5);
fig. 4 is a graph of the effect of herbal extracts on breast cancer cell clonality (x ± s, n ═ 3); a, MDA-MB-231 cells; b, MDA-MB-468 cells;
fig. 5 is a graph of the inhibitory effect of the extract of the Chinese medicinal composition on the migration of different breast cancer cells (x ± s, n ═ 3);
fig. 6 is a graph of the inhibition effect (x ± s, n ═ 3) of the extracts of the Chinese medicinal composition on the invasion of different breast cancer cells;
FIG. 7 is a graph of HE staining (100X) of lung metastases of 4T1 nude mice transplanted tumors; a is Blank; b, control;
FIG. 8 is a graph of HE staining (x. + -.s, n ═ 6) of lung transverse metastases from 4T1 nude mice transplantable tumors; a, HE staining pattern, b, lung cross section metastasis nodule quantification; p <0.05, p <0.01 compared to control group;
FIG. 9 is a graph showing the sensitizing effect (X + -s, n ═ 3) of extracts of Chinese medicinal compositions on the inhibition of cell proliferation by MDA-MB-231 and MDA-MB-468 after X-ray irradiation;
FIG. 10 is a graph showing the sensitizing effect (X + -s, n-3) of extracts of a Chinese medicinal composition on MDA-MB-468 apoptosis after X-ray irradiation;
FIG. 11 is a graph showing the effect of the herbal extracts on the enhancement of MDA-MB-231 apoptosis after X-ray irradiation (X + -s, n-3).
Detailed Description
Example 1 preparation of extract of the Chinese medicinal composition of the present invention
The formula is as follows: 3 parts of curcuma aromatica, 3 parts of hairyvein agrimony, 2.5 parts of trogopterus dung, 3 parts of alum, 3 parts of saltpeter, 1 part of prepared lacquer, 5 parts of bran-fried fructus aurantii and 2 parts of nux vomica powder (each part in the embodiment represents 20 g).
(1) Crushing the prepared dried lacquer, trogopterus dung, the alum and the saltpeter according to the formula amount, sieving by a 80-mesh sieve, adding 5 times of water by mass, carrying out ultrasonic extraction for 2 times, each time for 30min, combining the extracting solutions, and concentrating to obtain thick paste a; ultrasonically extracting the residue with 5 times of chloroform for 2 times (30 min each time), mixing extractive solutions, and concentrating to obtain soft extract b;
(2) according to the formula, weighing radix curcumae and bran-fried fructus aurantii, percolating and extracting the radix curcumae and the bran-fried fructus aurantii with 5 times of ethanol with the volume fraction of 70% by mass, collecting percolate and dregs, and concentrating the percolate to obtain extract c;
(3) weighing herba et Gemma Agrimoniae according to the formula, mixing with the residues collected in step (2), decocting with 5 times of water for 2 times, mixing the decoctions, and concentrating to obtain soft extract d;
(4) weighing nux vomica powder according to a formula, carrying out reflux extraction for 2 times by using water with the mass of 5 times, carrying out extraction for 2 hours each time, combining extracting solutions, and concentrating to obtain thick paste e; ultrasonically extracting the residue with 5 times of chloroform for 2 times (30 min each time), mixing extractive solutions, and concentrating to obtain soft extract f;
(5) and (4) combining the thick pastes a-f obtained in the steps (1) to (4), re-dissolving the thick pastes with water, and volatilizing the thick pastes to obtain the traditional Chinese medicine composition extract.
Example 2 study of the Targeted Effect of extracts of Chinese medicinal composition on different types of Breast cancer
3.1 study of Targeted Effect of different types of Breast cancer cells
3.1.1 measurement of expression levels of ER α and HER2
Culturing breast cancer cells MCF-7, T47D, SK-BR-3, MDA-MB-453, BT-474, MDA-MB-361, MDA-MB-231 and MDA-MB-468, and determining the cell typing by adopting western blotting to examine the expression conditions of ER alpha and HER2 in the cells.
3.1.2 preparation of herbal extract stock solution and medicated serum
(1) Preparation of stock solution: the extract of the Chinese medicinal composition prepared in example 1 was precisely weighed, dissolved in DMSO, and a 1g/ml stock solution was prepared for use.
(2) Preparation of serum containing medicine:
about 10 male 8-week-old rats (about 200 g) were collected, and 5 of them were kept as a blank serum group, and the other 5 were used as a drug-containing serum group. The fasting time before administration is about 12-16 h. Rats were given a gavage dose of 0.767g/d 1 time per day for 7 consecutive days. After the last intragastric administration for 1h, the animal is anesthetized by ether inhalation, the heart or abdominal aorta is subjected to blood sampling, and the animal is kept stand for 8h at 4 ℃ under the condition of ensuring the sterility and centrifuged for 10min at 5000r/min to prepare serum. Inactivating at 56 deg.C for 30min, filtering with 0.22 μm microporous membrane for sterilization, and storing at-80 deg.C; blank serogroup: an equal volume of saline was administered.
3.1.3 cell viability assay
Culturing breast cancer cells MCF-7 and T47D (ER alpha +/HER 2-); SK-BR-3, BT-474(ER α -/HER2 +); MDA-MB-453, MDA-MB-361(ER alpha +/HER2+) and MDA-MB-231(ER alpha-/HER 2-), MDA-MB-468(ER-/HER2-) and normal mammary epithelial cells MCF-10A, inoculating the cells into a 96-well plate, respectively adding traditional Chinese medicine composition extracts and drug-containing serum with different concentrations for drug intervention for 72h and 96h, detecting the cell viability by adopting a CCK-8 method, and comparing the difference of the traditional Chinese medicine composition extracts on the influence of the different types of breast cancer cell viability.
The administration concentration of the traditional Chinese medicine composition extract is as follows: the final concentrations were 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125. mu.g/mL.
Administration concentration of drug-containing serum: different volume fractions of drug-containing serum (final volume fraction of 5%, 10%, 15%) were compared to blank serum (final volume fraction of 5%, 10%, 15%).
3.1.4 plate cloning experiments
Culturing the breast cancer cells, adding the traditional Chinese medicine composition extracts with different concentrations respectively for intervention for 7-14d, and calculating the difference of the sizes and the number of clones after the traditional Chinese medicine composition extracts with different concentrations are treated. And (3) inspecting the influence of the traditional Chinese medicine composition extract on the formation of breast cancer cell colonies.
3.1.5Transwell Chamber experiments
Culturing the breast cancer cells, respectively adding the traditional Chinese medicine composition extracts with different concentrations for intervention, respectively acting for 96h, then digesting the cells, inoculating the cells into a transwell chamber without matrigel (migration investigation) or with matrigel (invasion investigation), and investigating the influence of the traditional Chinese medicine composition extracts on the migration and invasion of the breast cancer cells.
3.2 evaluation of antitumor Effect of extracts of Chinese medicinal composition in vivo
(1) Mouse in situ breast tumor model: selecting different types of human breast cancer cell strains SK-BR-3 and MDA-MB-231, and inspecting the inhibition effect of the traditional Chinese medicine composition extract on in vivo tumors by adopting a nude mouse transplantation tumor model.Culturing cells in cell factory, digesting and collecting cells when the cells are in logarithmic phase, suspending in physiological saline to obtain 2.0 × 108200. mu.L of each cell suspension was inoculated under the second pair of papillary fat pads of nude mice by subcutaneous injection when the tumor volume reached 100mm3In this case, nude mice were randomly divided into five groups of 6 mice each, namely, a control group, a PX-S group (375mg/kg), a PX-M group (750mg/kg), a PX-L group (1500mg/kg), and Paclitaxel (20mg/kg), according to tumor volumes. The specific animal group administration table is shown in table 1. During this period, tumor volume changes were monitored and mouse body weight was measured every other day to monitor mouse health. After the administration, the mice were sacrificed and the tumor inhibition rate was calculated. Inhibition of breast cancer growth in vivo was examined.
TABLE 1 animal grouping table for studying effect of Chinese medicinal composition extract on human breast cancer cell tumor-bearing nude mouse transplantation model
Note: n animals, i.g. gavage, i.p intraperitoneal injection, PX Chinese medicinal composition extract, Pac paclitaxel
After the administration, the mice were sacrificed by dislocation of cervical vertebrae, tumor tissues, liver, spleen and kidney were peeled off, and the tumor inhibition rate and organ coefficient were weighed and calculated. The stripped tumor tissues and organs are washed by normal saline to remove residual blood stains on the surfaces, and are respectively stored in 4% paraformaldehyde solution and a refrigerator at minus 80 ℃ for later use. The tumor inhibition rate, the organ index and the relative tumor volume are calculated according to the following formulas respectively.
Tumor inhibition rate (%) (tumor weight of control group-tumor weight of administration group)/tumor weight of control group × 100%
Organ coefficient is organ weight/body weight
Relative tumor volume ═ Vn/V0N: the number of days of administration; 0: day of administration
(2) Mouse tail vein injection tumor metastasis model: culturing 4T1 mouse breast cancer cell, digesting and collecting the cell when it is growing, suspending in physiological saline to prepareInto 2X 107Cell suspension per mL. 200 μ L of cell suspension was used to inoculate balb/c mice by tail vein injection. One week after inoculation, the mice were randomized into 5 groups of 6 mice, control, PX-S (375mg/kg), PX-M (750mg/kg), PX-L (1500mg/kg), and Paclitaxel (20 mg/kg). The other group of healthy mice, which were not inoculated with tumors and administered with no drug, was set as a blank control group. The specific animal group administration table is shown in table 2. During this period, the body weight of the mice was measured every other day to monitor the health of the mice. After the administration, the mice were sacrificed by dislocation of cervical vertebrae, lungs, livers, spleens, and kidneys were peeled off, weighed, photographed, the metastatic foci conditions of each part were observed, and the organ coefficients were calculated. The stripped organs are washed away by normal saline to remove residual bloodstains on the surfaces, and are respectively stored in 4% paraformaldehyde solution and a refrigerator at minus 80 ℃ for later use. The organ index is calculated according to the following formula:
organ coefficient is organ weight/body weight
TABLE 2 animal group table for studying effect of Chinese medicinal composition extract on lung transplantation model of breast cancer
Note: n animals, i.g. gavage, i.p intraperitoneal injection, PX Chinese medicinal composition extract, Pac paclitaxel
3.3 statistical treatment
Data were analyzed using SPSS18.0 statistical software. The results are expressed as mean. + -. standard deviation (x. + -.s). The comparison between groups was performed by two-by-two t test in anova, P <0.05 indicates significant difference, and P <0.01 indicates significant difference.
4. Results of the experiment
4.1 expression levels of ER α and HER2 in breast cancer cells
Breast cancer cells MCF-7, T47D, SK-BR-3, MDA-MB-453, BT-474, MDA-MB-361, MDA-MB-231 and MDA-MB-468 were cultured, and expression of ER alpha, HER2 in the cells was examined. Westernblotting results are shown in FIG. 1, ER α positive breast cancer cells are T47D, MCF-7, MDA-MB-361 and MDA-MB-453, HER2 high expression cells are SK-BR-3 and BT474, MDA-MB-453 and MDA-MB-361, and MDA-MB-231 and MDA-MB-468 are cells that are both ER α and HER2 negative. This result is consistent with literature reports. Based on this, the following experiment was performed with the above different types of cells.
4.2 Effect of extracts of Chinese medicinal composition on Breast cancer cell viability
The inhibition effect of the traditional Chinese medicine composition extract on the proliferation of the breast cancer cells is examined by adopting a cell viability experiment, as shown in figure 2, the traditional Chinese medicine composition extract has a certain inhibition effect on the proliferation of the breast cancer cells, wherein the traditional Chinese medicine composition extract has a remarkable inhibition effect on two types of triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells, and is superior to other types of breast cancer cells. In addition, the traditional Chinese medicine composition extract does not show a remarkable inhibition effect on normal breast cell MCF-10A.
4.3 Effect of drug-containing serum extracted from Chinese medicinal composition on Breast cancer cell viability
The inhibition effect of the drug-containing serum of the traditional Chinese medicine composition extract on the proliferation of breast cancer cells is examined by adopting a cell viability experiment, as shown in figure 3, the drug-containing serum of the traditional Chinese medicine composition has a remarkable inhibition effect on two types of triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells, and is superior to other types of breast cancer cells and normal breast cells. The results show that the traditional Chinese medicine composition extract can obviously inhibit the proliferation of triple negative breast cancer cells and has strong specificity.
From the research results, the traditional Chinese medicine composition extract and the drug-containing serum have obvious inhibition effect on the triple negative breast cancer cells. Therefore, in the following experiments, the triple negative breast cancer MDA-MB-231 and MDA-MB-468 cells were used as target cells for further research.
4.4 Effect of extracts of Chinese medicinal composition on clone formation of Breast cancer cells
The influence of the extract of the Chinese medicinal composition on the clone formation of MDA-MB-231 and MDA-MB-468 cells was examined by using a plate clone formation experiment, as shown in FIG. 4, the result obtained by 14d continuous culture shows that the number of clone formation of the administration group is remarkably reduced and the number of cells in a single clone is also remarkably reduced compared with that of the blank control group. The results show that the extract of the Chinese medicinal composition can effectively inhibit the clone formation of two cells.
4.5 Effect of herbal extracts on Breast cancer cell migration and invasion
The cell migration experiment is adopted to examine the influence of the Chinese medicinal composition extract on the cell migration ability of MDA-MB-231 and MDA-MB-468. As can be seen in FIG. 5, the number of cells migrating to the lower part of the Millicell cell gradually decreased with the increase of the administered concentration, indicating that the extract of the Chinese medicinal composition can inhibit the migration of breast cancer cells.
The cell invasion test is adopted to examine the influence of the Chinese medicinal composition extract on the invasion capacity of MDA-MB-231 and MDA-MB-468 cells. As can be seen from FIG. 6, the number of cells invading under the Millicell cell decreased gradually with the increase of the administered concentration, indicating that the extract of the Chinese medicinal composition can inhibit the invasion of breast cancer cells.
4.6 the inhibitory action of the extract of the Chinese medicinal composition on the pulmonary metastasis of the breast cancer of mice
The influence of the traditional Chinese medicine composition extract on breast cancer metastasis is examined by establishing a mouse breast cancer cell 4T1 tail vein injection tumor metastasis model. The experimental result shows that after 4T1 cells are inoculated to a mouse for 15 days, 6 control group mice all have lung metastasis, lung tissues are subjected to HE staining, tumor cell metastasis in the lung can be observed under a microscope, the metastasis cells are closely arranged, the nucleus is large and deep stained, the nuclear membrane is clear, the nucleolus is obvious, the division phase is multiple, and the pathological histology is diagnosed as an invasive cancer nest. No lung metastasis was observed in any of the 6 placebo mice, indicating successful molding (FIG. 7). Compared with the control group, after the extracts of the traditional Chinese medicine composition of 375mg/kg, 750mg/kg and 1500mg/kg are respectively treated for 14 days, the number of the pulmonary metastasis nodules of the mice is greatly reduced, and the mice are even better than the positive control group in the case of large dose and have statistical difference (figure 8).
The inhibition effect of the pingxiao capsules (commercially available products produced by the applicant company) on lung metastasis of breast cancer in mice was tested according to the method described in this example, and the results showed that the number of lung metastasis nodules in mice was reduced after the respectively-treated pingxiao capsules of 375mg/kg, 750mg/kg and 1500mg/kg for 14 days, but the degree of reduction was not as good as that of the extract of the Chinese medicinal composition, and was lower than that of the positive control group at a large dose, compared to the control group.
EXAMPLE 3 study of sensitization of extracts of Chinese medicinal composition
3.1 study of sensitization of Chinese medicinal composition extract on cell viability
And digesting the MDA-MB-231 cells and the MDA-MB-468 cells to prepare single cell suspensions, counting, inoculating the single cell suspensions into a 96-well plate, and culturing in a cell culture box overnight. Adding the traditional Chinese medicine composition extract after the cells adhere to the wall, wherein the final concentration is 1mg/mL and 0.5mg/mL respectively, incubating for 24h, then changing into a fresh complete culture medium, and performing ionizing radiation treatment on the cells, wherein the irradiation dose is 8 Gry. After irradiation, the cells were returned to the incubator and incubated for another 48 hours. After 48h, the CCK8 method detects the activity change of each group of cells.
3.2 study of sensitization of Chinese medicinal composition extract on apoptosis induced by X-ray
After being digested, MDA-MB-231 cells and MDA-MB-468 cells are prepared into single cell suspensions, and after counting, the single cell suspensions are respectively inoculated into a 6-well plate and cultured in a cell incubator overnight. Adding the traditional Chinese medicine composition extract after the cells adhere to the wall, wherein the final concentration is 1mg/mL and 0.5mg/mL respectively, incubating for 24h, then changing into a fresh complete culture medium, and performing ionizing radiation treatment on the cells, wherein the irradiation dose is 8 Gry. After irradiation, the cells were returned to the incubator and incubated for another 48 hours. After 48h, the apoptosis of each group of cells was detected by flow cytometry.
3.3 statistical treatment
Data were analyzed using SPSS18.0 statistical software. The results are expressed as mean. + -. standard deviation (x. + -.s). The comparison between groups was performed by two-by-two t test in anova, P <0.05 indicates significant difference, and P <0.01 indicates significant difference.
4. Results of the experiment
4.1 Effect of extracts of Chinese medicinal composition on survival of X-ray irradiated MDA-MB-231 cells and MDA-MB-468 cells
In order to examine the sensitization effect of the traditional Chinese medicine composition extract on X-ray irradiation treatment, the study detects the inhibition rate of the traditional Chinese medicine composition extract on cells pretreated for 24 hours and irradiated by 8Gry rays. The experimental results are shown in fig. 9, compared with the simple radiotherapy group, the survival rates of MDA-MB-468 cells after the cells pretreated by the extracts of the traditional Chinese medicine composition of 0.5mg/mL and 1.0mg/mL are irradiated by 8Gry rays are both obviously reduced; after the cells pretreated by the extract of the traditional Chinese medicine composition with the concentration of 1.0mg/mL are irradiated by 8Gry rays, the survival rate of MDA-MB-231 cells is obviously reduced. The Chinese medicinal composition extract can enhance the killing effect of X-ray irradiation.
4.2 Effect of extracts of Chinese medicinal composition on apoptosis of X-ray irradiated MDA-MB-231 and MDA-MB-468 cells
In order to examine the sensitization effect of the traditional Chinese medicine composition extract on X-ray irradiation treatment, the study detects the apoptosis rate of the traditional Chinese medicine composition extract after the cells are pretreated and the cells are irradiated by 8Gry rays. Treating MDA-MB-231 and MDA-MB-468 cells with 0.5mg/mL and 1.0mg/mL Chinese medicinal composition extract for 24h, changing culture solution for 48h, and detecting apoptosis with flow cytometer. For MDA-MB-231 cells, the apoptosis rate of the Chinese medicinal composition extract groups with the concentration of 0.5mg/mL and 1.0mg/mL is increased from 3.39% to 5.17% and 6.11% compared with the control group. Treating MDA-MB-468 cells with 0.5mg/mL and 1.0mg/mL Chinese medicinal composition extract for 24h, irradiating with 8Gy intensity, culturing in changed solution for 48h, and detecting apoptosis with flow cytometer. Compared with the group only irradiated by 8Gry, the apoptosis rate of the traditional Chinese medicine composition extract pretreatment group is increased from 3.97% to 11.33% and 18.04%. Compared with the Control group and the traditional Chinese medicine composition extract group, the apoptosis rate of the traditional Chinese medicine composition extract group and the X-ray irradiation group is obviously increased, and the difference has statistical significance (figure 10).
For MDA-MB-468 cells, compared with the control group, the apoptosis rate of the Chinese medicinal composition extract groups with 0.5mg/mL and 1.0mg/mL increased from 4.16% to 10.66% and 15.94%. Treating MDA-MB-468 cells with 0.5mg/mL and 1.0mg/mL Chinese medicinal composition extract for 24 hr, irradiating with 8Gry intensity, culturing in changed solution for 48 hr, and detecting apoptosis with flow cytometer. Compared with the group only irradiated by 8Gry, the apoptosis rate of the traditional Chinese medicine composition extract pretreatment group is increased from 5.41% to 25.21% and 28.51%. Compared with the Control group and the traditional Chinese medicine composition extract group, the apoptosis rate of the traditional Chinese medicine composition extract combined with the X-ray irradiation group is obviously higher, and the difference has statistical significance (figure 11). After the traditional Chinese medicine composition extract is combined with X-ray irradiation, the apoptosis rate of MDA-MB-231 and MDA-MB-468 cells is obviously improved, which shows that the traditional Chinese medicine composition extract can enhance the sensitivity of breast cancer to radiotherapy.
Claims (10)
1. A traditional Chinese medicine composition is characterized by comprising the following components in parts by weight: 1-5 parts of radix curcumae, 1-5 parts of hairyvein agrimony, 1-5 parts of trogopterus dung, 1-5 parts of alum, 1-5 parts of saltpeter, 0.5-3 parts of prepared dried lacquer, 3-7 parts of bran-fried fructus aurantii and 0.5-4 parts of nux vomica powder; preferably: 2-4 parts of curcuma aromatica, 2-4 parts of agrimony, 2-4 parts of trogopterus dung, 2-4 parts of alum, 2-4 parts of saltpeter, 0.5-2 parts of prepared dried lacquer, 4-6 parts of bran-fried fructus aurantii and 1-3 parts of nux vomica powder; the best is as follows: 3 parts of curcuma aromatica, 3 parts of agrimony, 2.5 parts of trogopterus dung, 3 parts of alum, 3 parts of saltpeter, 1 part of prepared dried lacquer, 5 parts of bran-fried fructus aurantii and 2 parts of nux vomica powder.
2. The extract of the traditional Chinese medicine composition of claim 1, wherein the preparation method of the extract of the traditional Chinese medicine composition comprises the following steps:
(1) pulverizing the dried lacquer, trogopterus dung, alum and saltpeter according to the formula amount, sieving by a 80-mesh sieve, adding water, performing ultrasonic extraction for 1-3 times, mixing the extracting solutions, and concentrating to obtain thick paste a; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract b;
(2) percolating radix Curcumae and fructus Aurantii parched with bran with 70% ethanol, collecting percolate and residue, and concentrating percolate to obtain extract c;
(3) weighing herba et Gemma Agrimoniae according to the formula, mixing with the residues collected in step (2), decocting with water for 1-3 times, mixing decoctions, and concentrating to obtain soft extract d;
(4) weighing semen Strychni Pulveratum according to formula, extracting with water under reflux for 1-3 times, mixing extractive solutions, and concentrating to obtain soft extract e; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract f;
(5) and (4) combining the thick paste a-f obtained in the steps (1) to (4), re-dissolving the thick paste with water, and volatilizing the mixture to obtain the traditional Chinese medicine composition extract.
3. The extract of the Chinese medicinal composition of claim 2, wherein the ultrasonic extraction time in each of the steps (1) to (4) is 15-30min, and the amount of the extraction solvent is preferably 4-6 times (more preferably 5 times) the mass of the medicinal material.
4. A method for preparing the extract of the chinese medicinal composition according to any one of claims 2 to 3, characterized by comprising the steps of:
(1) pulverizing the prepared dried lacquer, trogopterus dung, alum and saltpeter according to the formula amount, sieving by a 80-mesh sieve, adding water, ultrasonically extracting for 1-3 times, mixing the extracting solutions, and concentrating to obtain thick paste a; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract b;
(2) percolating radix Curcumae and fructus Aurantii parched with bran with 70% ethanol, collecting percolate and residue, and concentrating percolate to obtain extract c;
(3) weighing herba et Gemma Agrimoniae according to the formula, mixing with the residues collected in step (2), decocting with water for 1-3 times, mixing decoctions, and concentrating to obtain soft extract d;
(4) weighing semen Strychni Pulveratum according to formula, extracting with water under reflux for 1-3 times, mixing extractive solutions, and concentrating to obtain soft extract e; ultrasonically extracting the residue with chloroform for 1-3 times, mixing the extractive solutions, and concentrating to obtain soft extract f;
(5) and (4) combining the thick paste a-f obtained in the steps (1) to (4), re-dissolving the thick paste with water, and volatilizing the mixture to obtain the traditional Chinese medicine composition extract.
5. Use of the Chinese medicinal composition of claim 1 and/or the extract of the Chinese medicinal composition of any one of claims 2 to 3 in the preparation of a breast cancer radiotherapy sensitizing medicament.
6. A medicament for inhibiting lung metastasis of breast cancer, which comprises the Chinese medicinal composition of claim 1 and/or the extract of the Chinese medicinal composition of any one of claims 2 to 3 as an active ingredient.
7. The medicament of claim 6, further comprising a pharmaceutically acceptable excipient.
8. Pharmaceutical according to any one of claims 6 to 7, characterized in that the pharmaceutical is in a dosage form selected from the group consisting of solid, liquid or semisolid formulations.
9. Use of the composition of claim 1 and/or the extract of the composition of any one of claims 2 to 3 for the preparation of a medicament for sensitizing X-ray induced apoptosis; in particular to the application in preparing the medicine with the sensitization effect on the apoptosis of X-ray induced MDA-MB-231 and MDA-MB-468 cells.
10. Use of the Chinese medicinal composition of claim 1 and/or the Chinese medicinal composition extract of any one of claims 2-3 in the preparation of a medicament for inhibiting lung metastasis of breast cancer.
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| CN101612357A (en) * | 2009-02-13 | 2009-12-30 | 西安正大制药有限公司 | A kind of Chinese medicine composition for the treatment of tumor and preparation method thereof |
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Application publication date: 20220621 |