CN113368150B

CN113368150B - Application of sophora japonica aqueous extract in preparing medicine for preventing or treating triple negative breast cancer

Info

Publication number: CN113368150B
Application number: CN202110185337.4A
Authority: CN
Inventors: 刘洪远; 奇日迈励图; 王允亮
Original assignee: Beijing Yunhui Medicine Technology Co ltd
Current assignee: Beijing Yunhui Medicine Technology Co ltd
Priority date: 2021-02-10
Filing date: 2021-02-10
Publication date: 2022-10-14
Anticipated expiration: 2041-02-10
Also published as: CN113368150A

Abstract

Experiments prove that the inhibition rate of the concentration of the sophora japonica aqueous extract on MDA231 cells under the normal cell non-toxic condition can reach 55.89% and 63.53% to the maximum, and the sophora japonica aqueous extract has an obvious inhibition effect on triple-negative breast cancer cell lines. The sophora japonica decocted and the water extract have no inhibition effect on MCF7 cells and HER2 positive breast cancer cell lines under the condition that normal cells are nontoxic.

Description

Application of sophora japonica aqueous extract in preparing medicine for preventing or treating triple-negative breast cancer

Technical Field

The invention relates to the technical field of medicines, and in particular relates to application of a sophora japonica aqueous extract in preparation of a medicine for preventing or treating triple negative breast cancer.

Background

The breast cancer is the most common tumor of women in China, accounts for 15 percent of the total incidence rate of cancer of all women, the incidence rate is 2.5 percent per year, the death rate is continuously increased by 3 percent per year, the new number and the death number respectively account for 12.2 percent and 9.6 percent of the whole world per year, the breast cancer is the most serious disease in women aged 45-59 years, and the breast cancer is the most serious disease threatening the health of the women in China. Breast cancers are classified into different subtypes according to the expression conditions of Estrogen Receptor (ER), progesterone Receptor (PR), human epidermal growth factor receptor 2 (HER 2), and the like. The triple negative breast cancer with negative indexes of ER, PR and HER2 accounts for about 15% of female breast cancer, and is the subtype with the highest recurrence rate and death rate in breast cancer. This is because triple negative breast cancer lacks the expression of hormone receptors and HER2, and is insensitive to endocrine and targeted therapies. At present, the curative effect is mainly to reduce the relapse of the disease by adopting a chemotherapy method, but the curative effect reaches the bottleneck.

In recent years, chinese scholars verify that the traditional Chinese medicine has the effect of killing cancer cells through experimental research. The method for treating breast cancer by using the traditional Chinese medicine comprises the measures of resisting cancer, disinfecting, removing food retention, strengthening body resistance, tonifying deficiency and the like. The traditional Chinese medicine for treating breast cancer has the following effects: 1. the traditional Chinese medicine induces apoptosis of breast cancer cells; 2. the traditional Chinese medicine can inhibit the growth, metastasis and recurrence of breast cancer tumor cells; 3. the traditional Chinese medicine can improve the immunity of the organism after breast cancer chemotherapy.

The formula of the traditional Chinese medicine, the variety, the production area, the pretreatment, the processing and the like of the medicinal materials can generate great influence on the drug effect of the traditional Chinese medicine, thereby influencing the treatment effect on the breast cancer. The existing traditional Chinese medicine formulas for treating breast cancer are all combined compound traditional Chinese medicines, the number of the medicines is generally more than 5, the processing treatment is rough, the extraction and processing technologies are complicated, and meanwhile, the active ingredients of the medicinal materials are lost or deteriorated. In conclusion, the formula which has fewer tastes and is effective in treating the negative breast cancer is screened, so that the treatment effect can be achieved, and the preparation process of the traditional Chinese medicine can be simplified.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the application of the sophora japonica aqueous extract in preparing the medicine for preventing or treating triple negative breast cancer.

The invention provides application of a sophora japonica aqueous extract in preparing a medicament for preventing or treating triple negative breast cancer.

In the application, the preparation method of the sophora flower aqueous extract comprises the following steps: adding water 1-3 times the weight of flos Sophorae Immaturus into the flos Sophorae Immaturus, decocting, collecting decoction, and concentrating.

Optionally, water is added in an amount of 2 times the weight of the drug.

Optionally, in the decocting step, the decocting time is 40 minutes, and the decocting times are 2 times.

In the application, the preparation method of the sophora flower aqueous extract comprises the following steps: adding water 15-20 times the weight of flos Sophorae Immaturus into the flos Sophorae Immaturus, reflux extracting for 2.5-3 hr, collecting concentrated solution, and drying.

Optionally, adding water in an amount which is 18 times the weight of the medicament into the sophora flower, extracting for 2.8 hours under reflux, collecting the concentrated solution, and drying.

The technical scheme of the invention has the following advantages:

1. experiments prove that the inhibition rate of the concentration of the sophora japonica aqueous extract on MDA231 cells under the normal cell non-toxic condition can reach 55.89% and 63.53% to the maximum extent, the sophora japonica aqueous extract has an obvious inhibition effect on triple negative breast cancer cell lines, and the sophora japonica decoction and aqueous extract have no inhibition effect on MCF7 cells and HER2 positive breast cancer cell lines under the normal cell non-toxic condition.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of tumor formation and tumor suppression in nude mice in Experimental example 1 of the present invention.

Detailed Description

The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1

The embodiment provides a preparation method of a sophora japonica aqueous extract, which comprises the following steps: 30g of sophora flower is added with water which accounts for 2 times of the total weight of the medicine for decoction and extraction, the decoction time is 40 minutes, the decoction times are 2 times, and the decoction is collected and concentrated to clear paste for later use.

Example 2

The embodiment provides a preparation method of a sophora japonica aqueous extract, which comprises the following steps: taking 100g of sophora flower, adding 1800ml of water, carrying out reflux extraction for 2.8 hours, collecting concentrated solution, placing the concentrated solution in a water bath kettle to be evaporated to dryness, and placing the concentrated solution in a vacuum drying oven to be dried for three days to obtain an aqueous extract for later use.

Experimental example 1

1. Experiment main reagent

TABLE 1 main reagents for the experiment

Name of reagent	Manufacturer and goods number
		RPMI 1640 Medium	GIBCO Cat.No.11875-093
ECM	ScienCell Cat.No.1001
		Fetal Bovine Serum (FBS)	GIBCO Cat.No.12664-025
MTT detection kit	GIBCO Cat.No.V13154
		DMSO	Sigma Cat.No.D4540
0.25% pancreatin	Ruercang biological Cat. No. REK3012
		10mM PBS (cell culture, sterile)	Ruercang biological Cat. No. REK3013
Nude mouse	Beijing Huafukang

2. Experiment main instrument

TABLE 2 Main Experimental instruments

Name of the instrument	Instrument type	Manufacturer of the product
			Carbon dioxide incubator	MCO-15AC type	SANYO
Inverted microscope	XDS-2B type	Chongqing photoelectricity
			Super clean bench	SW-CJ-1D type	Jiangsu Tongjing medicine
Centrifugal machine	Centrifuge 5415D	Eppendorf
			Enzyme-linked immunosorbent assay (ELISA) instrument	MultiSkan3	Thermo
Laminar flow cabinet		Su Hang

3. Experimental method

1. Cell culture and Collection

1.1 cell Resuscitation

Rapidly dissolving the frozen cells in water bath at 42 ℃ within 1min, and placing the cells in a T-25 culture flask for culture;

1.2 cell routine maintenance

1.2.1 discarding the cell primary growth culture medium, and cleaning for 2 times by using a serum-free culture medium;

1.2.2 adding 0.25% pancreatin, 1ml/T-25, and incubating at 37 deg.C for 1-3min based on covering the bottom of the cell container;

1.2.3 adding 5ml growth medium, resuspending cells, counting cells, centrifuging at 1000rpm for 5min;

1.2.4 discarding the supernatant, and freezing the cell sediment for drug screening or cell collection;

1.2.5 various cell lines correspond to growth media:

TABLE 3 growth medium for various cell lines

English name	Name of Chinese	Characteristics of growth	Culture conditions
				HUVEC	Human umbilical vein endothelial cells	Adherent growth	ECM containing 10% FBS
MDA231	Human breast cancer cell, three yin	Growth by adherence	1640 of 10% FBS
				MCF7	Human breast cancer cells	Growth by adherence	1640 of 10% FBS

2. Non-toxic concentration screening of drug on normal cells

2.1, the HUVEC cell stock culture was discarded, the cells were digested with 0.25% trypsin at 37 ℃ for 2min, and then suspended in growth medium with cell density adjusted to 5X 10 ⁴ Per ml;

2.2, adding the mixture into a 96-hole cell culture plate, and continuously culturing for 24 hours at a rate of 100 ul/hole;

2.3, replacing serum-free culture and synchronizing for 24 hours;

2.4, acting the cells with different concentrations of the drug (20%, 10%, 5%, 2.5% and 1.25% of the obtained liquid drug concentration of example 1 (diluted with 20%, 10%, 5%, 2.5% and 1.25% of the mass percentage of the extract contained in the cell growth medium for the extract obtained using example 1), 50, 25, 12.5, 6.25ug/ml of the aqueous extract obtained using example 2 (dissolved with the cell growth medium for the extract obtained using example 2 to a concentration of 50, 25, 12.5, 6.25ug/ml of the extract)) for 48h;

discarding the original culture medium after 2.5 and 48 hours, adding 100ul culture medium and 10ul MTT, reacting for 4 hours at 37 ℃;

2.6, discard the medium, add 100ul of 10% SDS to lyse the cells, read the absorbance at 570nm, and calculate the cell inhibition = (control OD value-experimental OD value)/control OD value × 100%; screening out the nontoxic concentration of the A to normal cells;

3. detection of growth inhibition effect of drug on breast cancer cells under normal cell non-toxic concentration

3.1 discarding all breast cancer cell stock culture solution, digesting cells with 0.25% pancreatin at 37 deg.C for 2min, adding growth medium to resuspend cells, adjusting cell density to 5 × 10 ⁶ Per ml;

3.2 adding the mixture into a 96-hole cell culture plate, culturing for 24 hours continuously at a rate of 100 ul/hole;

3.3 serum-free culture is replaced, and synchronization is carried out for 24 hours;

3.4 with the method 3, the medicine with non-toxic concentration obtained by screening acts on the breast cancer cells for 48 hours;

3.5 abandoning the original culture medium, adding 100ul culture medium and 10ul MTT, reacting for 4h at 37 ℃;

3.6 discard the medium, add 100ul 10% SDS to lyse the cells, read the absorbance at 570nm, and calculate the cell inhibition = (control OD value-experimental OD value)/control OD value × 100%; the growth inhibition rate of breast cancer cells was observed, 5 wells at a time.

4. Cell collection and inoculation

4.1 discarding the MDA231 cell primary growth culture medium, and washing with serum-free culture medium for 2 times;

4.2 adding 0.25% pancreatin, 1ml/T-25, and incubating at 37 deg.C for 1-3min based on covering the bottom of the cell container;

4.3 adding 5ml growth medium, resuspending the cells, counting the cells, centrifuging at 1000rpm for 5min;

4.4 discard the supernatant, wash the cells twice with 10mM PBS, centrifuge 5min at 1000rpm each time;

4.5 cell Density adjusted to 5X 10 with 10mM PBS ⁷ Per ml;

4.6 by 5X 10 ⁶ The amount of each mouse is 0.1 ml/mouse, and the mouse is inoculated to the armpit of a BCLB/C nude mouse;

4.7 macroscopic tumors 7-10 days after inoculation, were randomized into four groups: the treatment group of the traditional Chinese medicine decoction (example 1) is subjected to intragastric administration according to the concentration of 1.0ml per patient per day; the treatment group of the traditional Chinese medicine water extract (example 2) is prepared into an aqueous solution according to 100ug/ml, and is irrigated with 1.0 ml/stomach per day; the control group was perfused with sterile PBS at 1.0 ml/mouse/day;

4.8 after 18 days of continuous gavage, two groups of axillary tumor tissues are contrastingly observed for tumors and sizes, and the inhibition effect of the traditional Chinese medicine on breast cancer cells is confirmed.

3. Results of the experiment

1. Results of non-toxic concentration probing of normal cells (HUVEC) (in the following table, the corresponding concentration of decoction refers to the preparation of the extract prepared in example 1, and the corresponding concentration of aqueous extract of Sophora japonica refers to the preparation of the aqueous extract of Sophora japonica prepared in example 2)

TABLE 4 results of non-toxic concentrations of Normal cells (HUVEC)

The maximum nontoxic concentration is respectively 5% of decoction and 12.5ug/ml of water extract

Results of the MDA-231 breast cancer cell inhibition assay (in the following table, the decoction at the corresponding concentration is prepared by using the extract prepared in example 1, and the sophora flower aqueous extract at the corresponding concentration is prepared by using the sophora flower aqueous extract prepared in example 2)

TABLE 5 results of MDA-231 test for the inhibitory Effect of Breast cancer cells

Test results of breast cancer cell inhibitory effect of mcf7 (in the following table, the decoction with the corresponding concentration means the extract prepared by using the extract prepared in example 1, and the sophora japonica aqueous extract with the corresponding concentration means the extract prepared by using the sophora japonica aqueous extract prepared in example 2)

TABLE 6 results of detecting the inhibitory effect of mcf7 breast cancer cells

From the above, the inhibition rate of the concentration of the sophora flower decoction and the water extract on MDA231 cells under the normal cell non-toxic condition can reach 55.89% and 63.53% at most, and the sophora flower decoction and the water extract have obvious inhibition effect on triple negative breast cancer cell lines.

The sophora japonica decocted and the water extract have no inhibition effect on MCF7 cells and HER2 positive breast cancer cell lines under the condition that normal cells are nontoxic.

4. Tumor formation and tumor suppression results in nude mice

The tumor formation and tumor inhibition results of nude mice are shown in fig. 1, from top to bottom, in lines 1 to 4, MDA231 transplanted tumor group, solvent control group, decoction (example 1) treatment group, and water extract (example 2) treatment group, respectively, as can be seen from fig. 1, sophora japonica decoction and water extract have significant MDA231 tumor inhibition effects.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. The application of the sophora japonica aqueous extract in preparing the medicine for preventing or treating triple negative breast cancer comprises the following steps: adding water 1-3 times the weight of flos Sophorae Immaturus into the flos Sophorae Immaturus, decocting, collecting decoction, and concentrating.

2. Use according to claim 1, wherein 2 times the weight of the drug is added.

3. The use according to claim 1 or 2, wherein in the step of decocting, the time of decoction is 40 minutes and the number of times of decoction is 2.

4. The application of the sophora japonica aqueous extract in preparing the medicine for preventing or treating triple negative breast cancer comprises the following steps: adding water 15-20 times the weight of flos Sophorae Immaturus into the flos Sophorae Immaturus, reflux extracting for 2.5-3 hr, collecting concentrated solution, and drying.

5. The use as claimed in claim 4, wherein flos Sophorae Immaturus is added with water 18 times of the weight of the medicine, and the extract is extracted under reflux for 2.8 hr, and the concentrated solution is collected and dried.