CN111150752A

CN111150752A - Application of abrus herb extract in preparing anticancer medicine

Info

Publication number: CN111150752A
Application number: CN202010151221.4A
Authority: CN
Inventors: 黄锁义; 李庭树; 莫仁高; 陈小妮; 雷智冬; 陈石梅; 王金妮
Original assignee: Youjiang Medical University for Nationalities
Current assignee: Youjiang Medical University for Nationalities
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2020-05-15

Abstract

The invention belongs to the field of medicines, and particularly relates to application of an abrus herb extract in preparation of an anticancer medicine. The abrus herb extract is abrus herb petroleum ether extract, abrus herb ethyl acetate extract, abrus herb n-butyl alcohol extract and/or abrus herb water extract. The abrus cantoniensis hance extract provided by the invention can promote bax protein and inhibit bcl-2 expression to enable tumor cells to undergo apoptosis, and has important significance for preparing anticancer drugs, especially drugs for gastric cancer, liver cancer and breast cancer.

Description

Application of abrus herb extract in preparing anticancer medicine

[ technical field ] A method for producing a semiconductor device

The invention relates to the field of medicines, in particular to application of an abrus herb extract in preparation of an anticancer medicine.

[ background of the invention ]

Cancer is the second largest killer of human health, and at present, drug resistance often occurs in the clinical chemotherapy drug application process, cancer cells are often killed, and the tumor is often relapsed and metastasized to cause death. One of the important reasons is that the apoptosis inhibiting gene (Bcl2 gene) in the part of cancer cells is highly expressed, so that the tumor cells are anti-apoptotic and do not die, and the tumor relapse and metastasis frequently occur. Therefore, the effect of inhibiting the expression of the tumor Bcl2 gene is one of the important ways of improving the curative effect of the medicine, completely killing cancer cells and preventing the recurrence and the metastasis of the tumor.

The natural products of the Chinese herbal medicines contain wide bioactive substances, so how to search for anti-tumor medicines and anti-tumor auxiliary medicines with small toxic and side effects and unique effects from the Chinese herbal medicines develops the advantages of the traditional Chinese medicines in China and develops a new way for treating tumors.

Abrus cantoniensis (Abrus cantoniensis) also known as herba Hedyotidis Diffusae, radix seu herba Cayratiae Oligocarpae, herba Rheum palmatum, and herba Pileae Scriptae, which are Leguminosae Leguminosae, and are mainly distributed in Guangdong, Guangxi, etc. The abrus herb can be used as a medicine except for seeds of the abrus herb, has the effects of promoting diuresis, removing jaundice, clearing heat, removing toxicity, soothing liver, relieving pain and the like, is a plant used as both medicine and food, and is used for cooking soup in wet spring and summer to serve as food therapy, prepare herbal tea and the like. The pharmacological actions of abrus cantoniensis are manifold, and the abrus cantoniensis relates to various biological activities of reducing fat, protecting liver, resisting bacteria, resisting inflammation, easing pain, enhancing immunity, resisting oxidation, promoting wound healing, treating maternal-fetal ABO blood group incompatibility and the like.

In the early experimental research, the inventor finds that the abrus herb has a certain anti-tumor effect, and the abrus herb is used as a traditional folk Chinese herbal medicine and has a wide prospect in the aspect of tumor treatment.

At present, no report that abrus cantoniensis hance extract can promote bax protein and inhibit bcl-2 expression to make tumor cell produce apoptosis exists.

[ summary of the invention ]

The invention aims to: aiming at the defects of the prior art, the application of the abrus herb extract in preparing anticancer drugs is provided.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the application of the abrus herb extract in preparing the anti-cancer drugs is that the abrus herb extract is an abrus herb petroleum ether extract, an abrus herb ethyl acetate extract, an abrus herb n-butyl alcohol extract and/or an abrus herb water extract.

The preparation method of the abrus herb extract comprises the following steps:

(1) heating and reflux-extracting herba abri whole plant dry powder with 95% ethanol, filtering, and concentrating the filtrate to obtain herba abri ethanol crude extract;

(2) dissolving the crude ethanol extract in the step (1) in water to form a suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol respectively, recovering the extraction solvent to obtain petroleum ether part extract, ethyl acetate part extract, n-butanol part extract and water layer part extract, which are correspondingly marked as abrus herb petroleum ether extract, abrus herb ethyl acetate extract, abrus herb n-butanol extract and abrus herb water extract.

The anti-cancer drug provided by the invention is an anti-gastric cancer drug, an anti-liver cancer drug and/or an anti-breast cancer drug.

The inventor researches and discovers that the abrus cantoniensis hance extract can promote bax protein and inhibit bcl-2 expression to enable tumor cells to undergo apoptosis.

The anti-cancer medicine comprises an effective amount of abrus herb extract and a pharmaceutically acceptable carrier or auxiliary material. The skilled in the art can optionally add carrier or adjuvant to prepare herba abri extract into various preparations by conventional pharmacy, such as granule, capsule, and tablet by adding starch or dextrin; adding syrup to make into syrup, or directly lyophilizing to make into lyophilized preparation.

Preferably, the anti-cancer drug is a lyophilized formulation.

In the present invention, the 95% ethanol refers to ethanol with a volume fraction of 95%.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) the invention discusses the anti-tumor effect of the nude mice by influencing the tumor inhibition rate and the immune index of the nude mice subcutaneous transplantation tumor. In-vivo efficacy experiments show that the abrus cantoniensis hance ethyl acetate, the n-butyl alcohol and the water extract all have an anti-tumor effect, wherein the anti-tumor effect of the ethyl acetate extract is strongest, the anti-tumor rate of the n-butyl alcohol extract is second, the water extract is worst, and the anti-tumor rate of a high-dose group of the ethyl acetate extract can reach more than 40 percent, and the anti-tumor rate is obviously increased along with the increase of the dose, so that a dose-effect relationship is presented, the liver index can be reduced, but the spleen and thymus index are not obviously. N-butanol and water extracts had no effect on liver, spleen and thymus indices. The abrus herb extract has the function of inhibiting the proliferation of tumor cells in vitro, the main effective components of the abrus herb extract are concentrated in the parts of ethyl acetate, n-butanol and water, and the extraction part of the ethyl acetate is strongest.

(2) In-vivo antitumor activity research shows that the abrus cantoniensis ethyl acetate extract has the strongest tumor inhibition effect on subcutaneous transplanted tumors of BEL-7404 nude mice, and can promote bax protein and inhibit bcl-2 expression to enable tumor cells to undergo apoptosis. In vitro anti-tumor activity studies show that the abrus herb ethyl acetate extract can block BEL-7404 cell cycle in G0/G1 phase and induce tumor cell apoptosis.

(3) The invention discloses the material basis of the in-vivo and in-vitro antitumor effect of the abrus cantoniensis hance, and lays the foundation for further separating antitumor monomer compounds and developing medicinal materials into antitumor innovative medicaments.

[ description of the drawings ]

FIG. 1 is a graph of the effect of abrus herb extract on nude mouse transplantable tumors;

FIG. 2 is a graph (400X) showing the effect of abrus herb extract on the tissue morphology of subcutaneous transplantation tumor of BEL-7404 nude mice;

FIG. 3 is a graph (400X) showing the effect of abrus herb extract on the tissue morphology of subcutaneous transplanted tumors in SGC-7901 nude mice;

FIG. 4 is a graph (400X) showing the effect of abrus herb extract on the tissue morphology of MCF-7 nude mice subcutaneous transplantation tumor;

FIG. 5 is a graph of bax expression in BEL-7404 tumor tissue (400X);

FIG. 6 is a graph (400X) showing the expression of bcl-2 in BEL-7404 tumor tissue;

wherein A is model group, B is 5-FU, C is ethyl acetate extract group, D is n-butanol extract group, and E is water extract group.

FIG. 7 is a graph showing the effect of abrus herb extract on the morphology of 3 tumor cell lines. Wherein, A is human gastric cancer cell SGC-7901; b-human hepatoma cells BEL-7404; c-human breast cancer cell MCF-7; a-control group; b-Petroleum ether extract (0.6 mg. mL-1); c-ethyl acetate extract (0.6 mg. mL-1); d-n-butanol extract (0.6 mg. mL-1); e-aqueous extract (0.6 mg. mL-1).

FIG. 8 is a graph (x 200) showing the effect of ethyl acetate extract of abrus herb on the apoptosis morphology of BEL-7404 by Hoechst 33258 fluorescence staining method; wherein B is human hepatoma cell BEL-7404; 0-0 mg/ml-1; 1-0.2 mg/ml-1; 2-0.4 mg/ml-1; 3-0.6 mg. multidot.mL-1.

FIG. 9 is a graph showing the effect of ethyl acetate extract of abrus herb on BEL-7404 liver cancer cell cycle distribution.

FIG. 10 is a graph showing the effect of ethyl acetate extract of abrus herb on the apoptosis rate of BEL-7404 cells.

[ detailed description ] embodiments

Example 1 preparation of abrus herb extract and study of in vivo antitumor activity.

1 materials and methods

1.1 materials

1.1.1 medicinal herb abrus cantoniensis is purchased from Bai color city, Guangxi (identified as the whole herb of abrus cantoniensis of the genus abrus, Leguminosae, by professor of folk medicine, Ledebouriella canaliculus, Ministry of folk medicine, Youjiang medical academy).

1.1.2 cells and experimental animal cell lines human gastric cancer cell line SGC-7901, human hepatoma cell line BEL-7404 and human breast cancer cell line MCF-7 cell lines were purchased from Kunming cell banks of Chinese academy of sciences. The cells were cultured in DMEM medium containing 10% fetal bovine serum and cultured in an incubator at 37 ℃ and saturated humidity and 5% CO 2. SPF-grade BALB/c nude mice 4-5 weeks old were purchased from Changsha Tianqin, Hunan. The certification number is as follows: no.20181012, raised in the center of laboratory animals of the national medical college of the Yangjiang river.

1.1.3 main reagents and apparatus DMEM high sugar medium: sammer Feishel technologies, Inc.; top grade fetal bovine serum: AusGeneX; sterile PBS, CCK-8 reagent: boster, r; dimethyl sulfoxide (DMSO): sigma company; 0.25% EDTA-pancreatin: beijing Solaibao corporation; rabbit anti-human Bcl-2, Bax polyclonal antibody (ABclonal); other reagents such as ethanol, ethyl acetate, n-butanol and the like are analytically pure. RE-3000A rotary evaporator: shanghai Yangrong Biochemical Instrument factory; circulating water type multipurpose vacuum pump: SHB-B88 model, zheng zhou great wall science and trade ltd; and (3) vacuum drying oven: model DZF-6020, Guangzhou Cishi instruments Inc.; CO2 incubator: shanghai li kang HF 90; a multifunctional microplate reader: german bouttotri Star LB 941; an electronic balance: model FA1204B, shanghai tianmei balance instruments ltd; ALPHA1-2/LD-Puls freeze dryer; CHRIST, germany.

1.2 methods

1.2.1 preparation of Abrus cantoniensis extract

Heating and refluxing herba abri dry powder with 95% ethanol, filtering, and concentrating the filtrate to obtain herba abri ethanol crude extract. Dissolving the crude ethanol extract in water to form suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, recovering the extraction solvent to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water layer extract, which are respectively marked as herba abri petroleum ether extract, herba abri ethyl acetate extract, herba abri n-butanol extract and herba abri water extract. And finally, preparing the extract into freeze-dried powder, accurately weighing 4 different parts of the abrus cantoniensis hance, performing ultraviolet sterilization, dissolving with pure water, preparing a liquid medicine with a corresponding concentration, and subpackaging and storing. Storing in a refrigerator at 4 deg.C, standing at room temperature before use, and recovering to room temperature.

1.2.2 in vivo antitumor Activity detection in nude mouse transplantation tumor model

⑴ SGC-7901, BEL-7404 and MCF-7 nude mice are transplanted subcutaneously, SGC-7901, BEL-7404 and MCF-7 cells in logarithmic growth phase are taken, 0.25% of pancreatin is used for digestion to form cell suspension, DMEM is used for configuring the cell concentration to be 1 × 109/ml and the cell suspension is mixed with Matrigel matrix glue in equal proportion, 0.2ml is inoculated to the right armpit of the nude mice subcutaneously, after 3 weeks, when the tumor volume is 50mm3, the molding success rate is 100%, each tumor cell model is divided into 11 groups according to a random principle, namely a blank group is physiological saline, a positive group is 5-FU 0.2 g.kg-1, an ethyl acetate extract group (0.1,0.4,0.8 g.kg-1), a n-butanol extract group (0.1,0.4,0.8 g.kg-1), a water extract group (0.1,0.4,0.8 g.1, 6 g.8 g.1 g.2 g.a/g, the weight of the tumor is measured after the tumor is transplanted, the tumor is removed, the tumor is treated by taking the weight of the tumor is added to be transplanted once per day, the tumor is added to the tumor is treated, the tumor is added to the.

⑵ nude mice, after sacrifice of tumor growth inhibition rate, the whole transplanted tumor was detached, the tumor mass was weighed, and the tumor growth inhibition rate was calculated, the average tumor weight of each group of mice was used as the therapeutic index, and the tumor inhibition rate (%) was calculated by the following formula (model group average tumor mass-administration group average tumor mass)/administration group average tumor mass × 100% liver, spleen, and thymus index, and organ index was organ mass (mg)/body mass (g).

⑶ HE staining

The detached transplantable tumor was fixed in 4% paraformaldehyde solution, dehydrated, embedded in paraffin, and cut into 4 μm-thick sections. Before dyeing, the slices are placed in an incubator at 60 ℃ for heating overnight, dewaxed, and finally subjected to HE dyeing and observed under a microscope.

⑷ immunohistochemical detection of expression of bax and bal-2 proteins

Firstly, slicing and dewaxing, and sealing with 3% hydrogen peroxide prepared in situ for 15 min; washing with PBS for 5min for 3 times; sealing 10% normal goat serum for 30 min; after blocking, no wash was performed and primary antibody was added for incubation (bax and bal-2 were 1: 200). Overnight at 4 ℃; equilibrating at room temperature for 30min, washing with PBS 3 times, 5min each time; incubating with a second antibody at 37 ℃ for 1 h; washing with PBS for 5min for 3 times; DAB color development; after color development is finished, the mixture is placed in distilled water, dehydrated by ethanol with gradient concentration, transparent xylene and sealed by neutral gum.

1.3 statistical data processing

Statistical methods were analyzed using SPSS16.0 software and the experimental data are expressed as mean ± standard deviation. The comparison among the groups is single-factor analysis of variance, the data mean is represented by X +/-s, and the statistical significance is achieved when P is less than 0.05.

2 results

2.1 Effect of abrus herb extract on tumor inhibition rate, liver, spleen and thymus index of nude mice subcutaneous transplantation tumor

The influence of the compound can be combined with

graphs

1, 2 and 3 and a graph 1, compared with a model group, the positive group can obviously inhibit tumor weight, the inhibition rate of BEL-7404, SGC-7901 and MCF-7 nude mice subcutaneous transplantation tumor reaches 58.67%, 53.42% and 56.14%, and the compound also shows obvious inhibition activity on immune organ indexes; the abrus cantoniensis hance ethyl acetate, n-butanol and water extract groups have the effect of inhibiting tumor weight, and the inhibition rates of the ethyl acetate extract group on BEL-7404, SGC-7901 and MCF-7 nude mouse subcutaneous transplantation tumors are 46.67%, 38.36% and 29.82% respectively; n-butanol extract group: 33.33%, 16.43%, 22.81%; water extract group: 13.33 percent, 9.58 percent and 7.02 percent, and the extracts of the abrus cantoniensis hance ethyl acetate and the n-butyl alcohol show obvious inhibitory activity on liver organ indexes, but have no obvious inhibitory activity on spleen and thymus indexes, and the water extract has no obvious influence on immune organ indexes.

TABLE 1 Effect of Abrus cantoniensis Hance ethyl acetate, n-butanol and water extract on BEL-7404 transplanted tumor volume, tumor inhibition rate and liver, spleen and thymus index ((S))

n＝6)

T Table 1.Effects of ethyl acetate,n-butanol and water extract ontumor volume,tumor inhibition rate and indexes of liver,spleen and thymus ofBEL-7404transplantation(

n＝6)

Note: comparison with model group of P <0.05, P <0.01

TABLE 2 Abrus cantoniensis Hance ethyl acetate, n-butanol and water extract on SGC-7901 transplanted tumor volume, tumor inhibition rate and liver, spleen and thymus indexInfluence of (A), (B)

n＝6)

Table 2.Effects of ethyl acetate,n-butanol and water extract on tumorvolume,tumor inhibition rate and indexes of liver,spleen and thymus of SGC-7901transplantation(

n＝6)

Note: comparison with model group of P <0.05, P <0.01

TABLE 3 influence of Abrus cantoniensis Hance ethyl acetate, n-butanol and water extract on MCF-7 transplanted tumor volume, tumor inhibition rate and liver, spleen and thymus index ((S))

n＝6)

Table 3.Effects of ethyl acetate,n-butanol and water extract on tumorvolume,tumor inhibition rate and indexes of liver,spleen and thymus of MCF-7transplantation(x±s,n＝6)

Note: comparison with model group of P <0.05, P <0.01

2.2 HE staining results HE staining was observed under a 200-fold light microscope. As can be seen from FIGS. 2-4, after HE staining of tumor tissue sections, tumor cells in the model group are uniform, densely arranged, well grown, deeply stained nuclei, circular or oval, varying in size, and normal in tumor cell morphology (FIGS. 2, 3, 4-A); the 5-FU positive group had loosely arranged tumor cells, large areas of cell nucleus shrinkage, tumor cell rupture, lysed necrotic regions and red-stained cheese-like necrotic regions (FIGS. 2, 3, 4-B); the tumor cells in the test group showed different degrees of diffuse distribution, red staining, cheese-like necrosis in the form of pieces, blurring, etc. (FIGS. 2, 3, 4-C, D, E). The ethyl acetate extract group showed cell nuclear compaction, tumor cell rupture, and the most severe zone of lysis necrosis.

2.3 the positive reaction of the abrus herb ethyl acetate extract on the expression of Bax and bcl-2 proteins in the subcutaneous transplanted tumor tissue of BEL-7404 mice is that brown yellow or yellow particles appear on cell membranes, cytoplasm and nuclear membranes, FIG. 5 shows the expression condition of Bax, the lighter brown positive expression is shown in a model group, the brown yellow positive expression is most obvious in a 5-FU positive group, and the brown yellow positive expression is more obvious in an ethyl acetate extract group along with the increase of the administration dosage.

As shown in the expression situation of bcl-2 in FIG. 6, the expression rate of brown yellow is the highest in the model group, the expression rate of brown yellow is the lowest in the 5-FU positive group, and the expression rate of brown yellow is reduced in the abrus herb ethyl acetate extract group with increasing dosage. Because the experiment is in the early exploration stage, only two typical important apoptosis genes, namely bax and bcl-2, are selected for exploration, in the later project research, the sample size needs to be increased, more apoptosis genes, such as caspase and the like, are deeply researched, the expression of the apoptosis genes is detected through immunohistochemistry and WB (wideband) and is statistically compared, so that the further deep research is carried out.

Discussion of 3

The abrus cantoniensis hance is a traditional Chinese medicine commonly used for clearing liver and gallbladder damp-heat in folk, the compound capsule is widely applied clinically, and the anti-tumor research is reported, but the corresponding monomer compound is not specifically researched, so that the in-vivo anti-tumor experiment is carried out on the basis of the research result of the abrus cantoniensis hance in vitro anti-tumor extract in the early stage of a subject group. At present, the mechanism of the antitumor drug mainly acts on tumor cells directly and improves the immune function of the organism, based on the two mechanisms, the early-stage subject group adopts the in-vitro tumor growth inhibition effect to discuss the effect of abrus cantoniensis on the direct killing of the tumor cells (CCK-8 method), and the research discusses the antitumor effect of abrus cantoniensis through the influence on the tumor inhibition rate and the immune index of subcutaneous transplanted tumors of nude mice. In-vivo efficacy experiments show that the abrus cantoniensis hance ethyl acetate, the n-butyl alcohol and the water extract all have an anti-tumor effect, wherein the anti-tumor effect of the ethyl acetate extract is strongest, the anti-tumor rate of a high-dose group of the ethyl acetate extract can reach more than 40% due to the second n-butyl alcohol extract and the worst water extract, and the anti-tumor rate is obviously increased along with the increase of the dose, so that a dose-effect relationship is presented. The abrus herb ethyl acetate and n-butanol extracts can reduce the liver index, but have no obvious influence on the spleen and thymus index. The aqueous extract had no effect on liver, spleen and thymus indices. The research finds that the abrus herb ethyl acetate extract has the strongest tumor inhibition effect on BEL-7404 nude mouse subcutaneous transplantation tumor, and can promote bax protein, inhibit bcl-2 expression and improve the immune function of an organism to ensure that tumor cells are subjected to apoptosis. The research reveals the material basis of the antitumor effect in vivo of the abrus cantoniensis hance, and lays the foundation for further separating antitumor monomer compounds and developing medicinal materials into antitumor innovative medicaments.

Example 2 preparation of Abrus cantoniensis Hance extract and study of in vitro antitumor Activity

2.1 materials and instruments

Abrus cantoniensis is purchased from Bai-color city, Guangxi (identified as the whole grass of abrus cantoniensis, a plant of the genus abrus, Leguminosae, by professor of ethnic medicine, faculty of medicine, Youjiang ethnic medicine, Youjin medicine, Chaozhou channel light); the cell strains, namely the human gastric cancer cell strain SGC-7901, the human hepatoma cell strain BEL-7404 and the human breast cancer cell strain MCF-7, are purchased from Kunming cell banks of Chinese academy of sciences; RPMI-1640 medium, DMEM high-sugar medium: sammer Feishel technologies, Inc.; top grade fetal bovine serum: AusGeneX; sterile PBS, CCK-8 reagent: boster, r; dimethyl sulfoxide (DMSO): sigma company; 0.25% EDTA-pancreatin: beijing Solaibao corporation; hoeshst33258 staining solution: a tridymite organism; the PI staining kit comprises other reagents such as ethanol, petroleum ether, ethyl acetate, n-butanol and the like of the biological (Shanghai) company Limited.

RE-3000A rotary evaporator: shanghai Yangrong Biochemical Instrument factory; circulating water type multipurpose vacuum pump: SHB-B88 model, zheng zhou great wall science and trade ltd; and (3) vacuum drying oven: model DZF-6020, Guangzhou Cishi instruments Inc.; CO2 incubator: shanghai li kang HF 90; a multifunctional microplate reader: german bouttotri Star LB 941; an electronic balance: model FA1204B, shanghai tianmei balance instruments ltd; ALPHA1-2/LD-Puls freeze dryer; CHRIST, Germany; inverted fluorescence microscopy is a product of Nikon corporation. Flow cytometry: BD company.

2.2 preparation of Abrus cantoniensis extract

Heating and refluxing herba abri dry powder with 95% ethanol, filtering, and concentrating to obtain paste to obtain herba abri ethanol crude extract. Dissolving the crude ethanol extract in water to form suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, recovering the extraction solvent to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water layer extract, which are respectively marked as herba abri petroleum ether extract, herba abri ethyl acetate extract, herba abri n-butanol extract and herba abri water extract. And finally, preparing the extract powder into freeze-dried powder, accurately weighing 4 different parts of the abrus cantoniensis hance, dissolving the 4 different parts of the abrus cantoniensis hance with a 1% dimethyl sulfoxide (DMSO) solution to prepare an extract of mother liquor with corresponding concentration, filtering the extract by 0.22 mu m, and subpackaging and storing the extract. The culture medium is stored in a refrigerator at 4 ℃ and diluted to the required concentration in a complete culture medium when in use.

2.3 in vitro antitumor Effect of Abrus cantoniensis Hance extract

2.3.1 cell culture

BEL-7404 cells and SGC-7901 cells were cultured in 1640 medium, and MCF-7 cells were cultured in DMEM medium (containing 10% fetal calf serum, penicillin and streptomycin each 100U. mL-1) at 37 ℃ in a 5% CO2 saturated humidity incubator. The growth state of the cells is observed under a microscope, the culture medium is replaced once a day, and the cells are passaged once every two days.

2.3.2 CCK-8 detection

Logarithmically grown BEL-7404 cells, SGC-7901 cells and MCF-7 cells were inoculated into a 96-well plate at a density of 5000 cells per well and cultured in an incubator. Filtering 4 kinds of extraction parts of abrus cantoniensis hance by a 0.22 mu m filter membrane, diluting with dimethyl sulfoxide (DMSO) in a gradient manner, adding 10 mu L of cells into each well to enable the final concentration of the cells in each well to be 0.1, 0.2, 0.4, 0.6 and 0.8 mg.mL < -1 >, setting 5 multiple wells for each concentration group, adding 10ul cck < -8 > solution into each well after adding medicine and culturing for 24h, continuing culturing for 1h, taking out and detecting the absorbance at 450 nm.

2.3.3 morphological observations

BEL-7404 cells, SGC-7901 cells and MCF-7 cells are inoculated in a 6-well plate at the cell density of 1 ten thousand per well, 200ul of petroleum ether with the concentration of 0.6 mg.mL < -1 >, ethyl acetate, n-butanol and water are added after 24 hours of extraction, and the morphological change of the cells is observed under an inverted microscope after the extraction part is treated for 24 hours.

2.3.4 Hoechst 33258 staining for detecting BEL-7404 apoptosis

BEL-7404 cells in logarithmic growth phase were seeded in 6-well plates, and 20X 105 cells were added per well. Setting blank control group and administration group with different concentrations, adding ethyl acetate extract with concentration of 0.2, 0.4, 0.6 mg/mL-1 after cell adherence. After the drug acts for 24 hours, the culture medium is sucked out, washed for 2 times by PBS, 1ml of 4% formaldehyde fixing solution is added into each hole, the mixture is fixed for 30min in a refrigerator at 4 ℃, the PBS is taken out and washed for 3 times, 500ml of Hoechst 33258 dye solution is added, and the mixture is incubated for 30min in an incubator at 37 ℃ in the dark. Washed 3 times with PBS and randomly photographed under a fluorescent microscope with excitation wavelength of 350nm and emission wavelength of 460 nm.

2.3.5 flow cytometry for detecting the influence of the extraction part of the abrus herb ethyl acetate on the BEL-7404 cell cycle

Inoculating cells in logarithmic growth phase into a 6-well plate at a density of 1 × 105 cells per well, adding 200ul of ethyl acetate extracts (0.2, 0.4 and 0.6 mg/mL < -1 >) with different concentrations after the cells are attached to the wall, continuously culturing for 24h, collecting the cells, fixing the cells with 70% precooled ethanol, keeping the temperature away from the sun overnight at 4 ℃, washing the cells twice with precooled PBS, adding propidium iodide, keeping the cells away from the sun, dyeing for 30min, detecting by an up-flow cytometer, and analyzing the DNA content and the cycle change of each group of samples.

2.3.6 Annexin V-FITC/PI for detecting influence of Abrus cantoniensis hance ethyl acetate extract on BEL-7404 tumor cell apoptosis rate

Inoculating cells in logarithmic growth phase into a 6-well plate at a density of 1 × 106 cells per well, after the cells are attached to the wall, adding 200ul of ethyl acetate extracts (0.2, 0.4, 0.6 mg. mL < -1 >) with different concentrations, continuously culturing for 24h, digesting and centrifuging by using 0.25% pancreatin, adding 1 × Binding Buffer to prepare cell suspension with a concentration of 1 × 106. mL < -1 >, sucking 100ul of cell suspension into an ep tube, respectively adding 5ul of FITC and PI, uniformly mixing and incubating at normal temperature and dark for 15min, adding 400ul of 1 × Binding Buffer, and performing on-machine detection within one hour.

2.3.6 data processing

All data are measured data, expressed as Mean ± standard deviation (Mean ± s.d.). The multiple groups of mean comparisons are performed using one-way analysis of variance. Statistical analysis Using GraphPad Prism software, P <0.05 was considered statistically different.

3. Results of the experiment

3.1 Effect of abrus herb extract on proliferation of four cells

In order to determine the main part of the antitumor activity of the four abrus herb extracts, the CCK-8 method is adopted to detect the antitumor activity of the four abrus herb extracts on BEL-7404 cells, SGC-7901 gastric cancer cells and MCF-7 breast cancer cells. Compared with a control group, after 24 hours of action, the ethyl acetate part, the n-butanol part and the water part have obvious inhibitory action on BEL-7404 cells, SGC-7901 gastric cancer cells and MCF-7 breast cancer cells and are in concentration dependence. It is apparent from table 4 that the anti-tumor activities of the ethyl acetate fraction, the n-butanol fraction and the water fraction are strong, suggesting that the anti-tumor activity of abrus herb may be concentrated on the ethyl acetate fraction, the n-butanol fraction and the water fraction. Through fitting calculation, the IC50 values of the petroleum ether part after being acted on BEL-7404 cells, SGC-7901 gastric cancer cells and MCF-7 breast cancer cells are 77.88, 39.0 and 61.1 mg/mL-1, and the IC50 values of the ethyl acetate part are 0.41, 0.47 and 0.40 mg/mL-1. The n-butanol fraction had IC50 values of 0.66, 0.68, and 0.46 mg/mL-1. The IC50 values of the water portion were 0.64, 0.75 and 0.81 mg/mL-1.

Table 4 influence of extracts from various parts of abrus cantoniensis hance on SCG-7901, BEL-7404, and MCF-7 cell proliferation (inhibition rate,

n＝6)

Table 1 Effects ofextracts from different parts of Abruscantoniensis on proliferation of SCG-7901,BEL-7404and MCF-7cells(Inhibitionrate,

n＝6)

note: and 0 mg. mL^-1Group ratio of P<0.01，*P<0.05

3.2 Effect of abrus herb extract on tumor cell morphology

The morphology of each group of cells is observed under an inverted microscope, so that BEL-7404, SGC-7901 and MCF-7 cells in a control group grow in an adherent manner, and the cells are uniform in size, clear in cytoplasm, good in refractivity and vigorous in proliferation. As shown in figure 7, after the 3 kinds of tumor cells are treated by the ethyl acetate part, the n-butanol part and the water part with the concentration of 60 mg/mL < -1 > for 24h, the shapes of the cells are all changed remarkably, and the shapes of the cells are all changed remarkably, which shows that the cell density is reduced remarkably, the cell contraction becomes round, the cell spacing is enlarged, and the anchorage effect is weakened. The petroleum ether part and the negative control group have no obvious influence on the density of the 3 cell strains, and the petroleum part and the 3 cell morphologies of the negative control group have almost no change.

3.3 promoting action of different parts of abrus herb on apoptosis of tumor cells

The observation of the staining result of the Hoechst 33258 fluorescent staining method under an inverted microscope shows that the negative control group has full cells, rich cytoplasm, round or oval nucleus and uniform chromatin fluorescent staining. The positive control group and the drug-adding group obviously see that the number of cells is reduced, the cell nucleus volume is reduced, the cell nucleus is shriveled, dense and concentrated staining fluorescence is seen, the chromosome DNA is in concentrated staining block-shaped and granular distribution, the obvious apoptosis characteristic is prompted, and the number of apoptotic cells is increased along with the increase of the drug concentration, which is shown in figure 8.

3.4 Effect of different concentrations of Ethyl acetate extract of abrus herb on BEL-7404 cell cycle distribution

The flow cytometry is adopted to detect the cell cycle division conditions of each group, and the results show that (table 5 and figure 9) the extraction part of the abrus cantoniensis hance ethyl acetate can increase the G1 phase proportion of liver cancer BEL-7404 cells, reduce the S phase and G2/M phase proportion, can induce the liver cancer BEL-7404 cells to undergo apoptosis and form a dose-dependent relationship, and has statistical significance (P is less than 0.05 and P is less than 0.01) compared with a normal control group.

TABLE 5 Effect of the extract fraction of Abrus cantoniensis Hance with ethyl acetate on the cell cycle fraction of BEL-7404 liver cancer%

Table 2 Effects of ethyl acetate extract from abrus cantoniensis onthe cycle distribution of BEL-7404hepatocellular carcinoma cells％

Comparison with control group P <0.01

3.5 Effect of Elder ethyl acetate fraction on the apoptosis Rate of BEL-7404 cells

The apoptosis of each group of cells was detected by flow cytometry, and the results showed that (see table 6 and fig. 10): the average apoptosis rate of the cells is calculated by taking early apoptosis and late apoptosis of the cells and the ethyl acetate extract of abrus cantoniensis at each concentration to act on BEL-7404 cells for 24 hours, the apoptosis rate increases along with the increase of the drug concentration, and the statistical significance is achieved compared with a normal control group (P is less than 0.05 and P is less than 0.01).

TABLE 6 Effect of Elder herb Ethyl acetate fraction on BEL-7404 apoptosis Rate

Table 3Effect of ethyl acetate extract from abrus cantoniensis onthe apoptosis rate of BEL-7404cells

3.4 discussion

In recent years, antitumor active ingredients extracted from Chinese herbs have been receiving more and more attention. The botanical drug has the characteristics of more obvious curative effect, less adverse reaction and the like, and the ratio of the botanical drug in the drug for treating cancer is increasing. China has abundant germplasm resources of medicinal plants, screens and separates anticancer active ingredients of traditional Chinese medicines, clarifies the anticancer action and mechanism, and has very important significance for the research and development of anticancer drugs and the treatment of cancers.

The research uses human tumor cells as a screening model, adopts a CCK-8 method and in vitro anti-tumor activity as an index, and screens 4 different extraction parts obtained by separating an abrus herb alcohol extract by a system solvent method for the first time, and the experimental result shows that the ethyl acetate, n-butyl alcohol and water extraction parts of the abrus herb are all the active parts with anti-tumor effect, and the result of the CCK-8 experiment is also verified by the observation result of the morphology of microscopic cells. Based on the above, the ethyl acetate extract with the strongest in vitro anti-tumor effect is selected to analyze and research the apoptosis and the flow cycle of BEL-7404 cells after the BEL-7404 cells act on the ethyl acetate extract, and the results of the Hoechst 33258 fluorescence staining method show that the BEL-7404 cells present typical apoptosis phenomena of cell nucleus condensation, chromatin condensation and breakage to form compact blue fluorescent bright spots with different sizes; flow results show that after the abrus herb ethyl acetate BEL-7404 cells with different concentrations, the cell cycle is obviously affected, the cell percentage in the early stage and the rest stage (G0/G1) of DNA synthesis is obviously increased, and along with the increase of the concentration of the abrus herb ethyl acetate extraction part, the cell percentage in the G0/G1 stage is obviously increased, and the cell percentage in the DNA synthesis stage (S stage), the synthesis later stage and the mitosis stage (G2/M stage) is reduced; the Annexin V-FITC/PI detection result shows that the abrus herb ethyl acetate extract can induce BEL-7404 cell apoptosis, and the apoptosis rate is in direct proportion to the abrus herb ethyl acetate extract concentration.

In conclusion, the abrus cantoniensis hance has the effect of inhibiting tumor cell proliferation in vitro, the main effective components of the abrus cantoniensis hance are concentrated in ethyl acetate, n-butanol and water, and the extraction part of the ethyl acetate is strongest. The abrus herb ethyl acetate extract can block BEL-7404 cell cycle in G0/G1 stage and induce tumor cell apoptosis. The experiment provides experimental basis and theoretical basis for the abrus herb to be used for clinically treating cancers, but the medicine effect substance basis and the molecular action mechanism of the abrus herb for resisting tumors are yet to be further researched by in vivo experiments.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims

1. Application of herba abri extract in preparing anticancer medicine is provided.

2. The use of abrus herb extract in the preparation of an anticancer drug as claimed in claim 1, wherein the abrus herb extract is abrus herb petroleum ether extract, abrus herb ethyl acetate extract, abrus herb n-butanol extract and/or abrus herb water extract.

3. The application of the abrus herb extract in the preparation of anticancer drugs as claimed in claim 2, characterized in that the preparation method of the abrus herb extract is as follows:

4. The use of abrus herb extract in the preparation of an anticancer drug as claimed in claim 1, wherein the anticancer drug is an anti-gastric cancer drug, an anti-liver cancer drug and/or an anti-breast cancer drug.

5. The use of abrus herb extract in the preparation of an anticancer drug as claimed in claim 1, wherein the abrus herb extract causes tumor cell apoptosis by promoting bax protein and inhibiting bcl-2 expression.

6. The application of the abrus herb extract in the preparation of anticancer drugs as claimed in claim 1, wherein the anticancer drugs comprise an effective amount of abrus herb extract and a pharmaceutically acceptable carrier or adjuvant.

7. The use of an abrus herb extract in the preparation of an anticancer drug as claimed in claim 1, wherein the anticancer drug is a lyophilized preparation.