CN112263592A

CN112263592A - Application of pueraria flower health-promoting prescription-containing serum containing medicines in cancer cells

Info

Publication number: CN112263592A
Application number: CN202011209872.0A
Authority: CN
Inventors: 李军; 杨柱; 唐东昕; 何金秀
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2021-01-26

Abstract

According to the invention, the pueraria flower health-improving prescription drug-containing serum is prepared, the influence of the drug-containing serum with different concentrations on the invasion and transfer of HepG2 liver cancer cells and the influence of the drug-containing serum with different concentrations on the proliferation and apoptosis of HepG2 liver cancer cells are observed, the effects of inhibiting the proliferation and promoting the apoptosis of the HepG2 liver cancer cells and the like of the pueraria flower health-improving prescription drug-containing serum are proved, and theory and data support are provided for the clinical anti-liver cancer transfer application of the prescription.

Description

Application of pueraria flower health-promoting prescription-containing serum containing medicines in cancer cells

Technical Field

The invention relates to the field of medicine invention, in particular to application of pueraria flower health-promoting medicine-containing serum in cancer cells.

Background

The primary liver cancer is a malignant tumor with high incidence in China, and the new patients in China are reported to account for about half of the total number of the patients all over the world every year and are in a trend of rising year by year^[1 ^2]. However, liver cancer is hidden, so that most patients have advanced to the middle and late stages when the liver cancer is found, the treatment effect is poor, and complications are many, so that the prognosis is extremely poor. However, the occurrence and development of liver cancer is a multi-stage and multi-process progression, and a long pre-liver cancer lesion period is experienced in the process from the beginning of hepatitis and liver cirrhosis to the final diagnosis of malignant evolution of liver cancer^[3 ^4]. Therefore, the 'three-early' early discovery, early diagnosis and early treatment are the key for preventing and treating liver cancer, and the prevention and blocking of the pre-liver cancer lesion is particularly important, which is the embodiment of the 'preventive treatment' idea in the traditional Chinese medicine. At present, the treatment aiming at the liver cancer is mainly performed by operation, but metastasis and invasion are main reasons of postoperative relapse and death of liver cancer patients, and the liver cancer is low in sensitivity to radiotherapy and chemotherapy, so that the search for a proper and effective treatment method and a proper and effective medicament are very practical^[5-8]. Research shows that epithelial-mesenchymal transition (EMT) plays an important role in the process of tumor infiltration and metastasis, and epithelial cells lose the interaction among cells, lose the polarity of the cells and reduce the adhesion capacity, so that the invasion and metastasis capacity is obtained^[9 10]，

Therefore, inhibiting the EMT process of liver cancer cells is especially important in the prevention and treatment of liver cancer. The pueraria flower health promoting formula consists of pueraria flower, round cardamom, villous amomum fruit and other medicines, has the functions of eliminating dampness of wine, regulating qi and strengthening spleen, and treats the spleen injury caused by wine accumulation under the principle of combining the functions of dispersing and permeating and promoting health. The preliminary study of the subject finds that the formula has important effects of reducing liver damage, reducing hepatocyte destruction, preventing steatosis of hepatocytes, promoting lipid metabolism in liver, inhibiting growth of tumor tissue, and the like^[11-14]。

Therefore, the invention provides theory and data support for clinical anti-liver cancer metastasis application of the prescription by preparing the pueraria flower health-beneficial prescription drug-containing serum, observing the influence of the drug-containing serum with different concentrations on the invasion and metastasis of HepG2 liver cancer cells and epithelial-mesenchymal transition and observing the influence of the drug-containing serum with different concentrations on the proliferation and apoptosis of HepG2 liver cancer cells.

Disclosure of Invention

The invention aims to provide application of pueraria flower health-promoting medicine-containing serum in preparation of a medicine or preparation for treating cancer cells, in particular HepG2 liver cancer cells.

The invention relates to an application of pueraria flower health-promoting prescription containing medicament serum in preparing a medicament for treating cancer cells;

preferably, the pueraria flower health-beneficial formula-containing serum disclosed by the invention is applied to preparation of a medicine for treating liver cancer cells.

Further preferably, the pueraria flower health-care medicine-containing serum disclosed by the invention is applied to preparation of a medicine for treating HepG2 liver cancer cells.

Further preferably, the pueraria flower health-beneficial medicine-containing serum disclosed by the invention is applied to preparation of a medicine for treating HepG2 liver cancer cell migration.

Further preferably, the pueraria flower health-beneficial formula is applied to preparation of a medicine for treating drug-containing serum in HepG2 liver cancer cell invasion.

Further preferably, the pueraria flower health-beneficial medicine-containing serum disclosed by the invention is applied to preparation of medicines for treating protein expression of HepG2 liver cancer cells E-cadherin and N-cadherin.

Further preferably, the pueraria flower health-promoting prescription-containing serum disclosed by the invention is applied to preparation of a medicine for treating and inhibiting the proliferation capacity of HepG2 liver cancer cells.

Further preferably, the pueraria flower health-promoting medicine-containing serum disclosed by the invention is applied to preparation of a medicine for promoting HepG2 liver cancer cell apoptosis.

Further preferably, the pueraria flower health-promoting medicine-containing serum disclosed by the invention is applied to preparation of a preparation for treating liver cancer cells.

The preparation is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable auxiliary materials.

The preparation provided by the invention is capsule, granule, powder, pill, tablet, injection preparation and freeze-dried powder injection.

The pharmaceutically acceptable auxiliary materials are not limited, and can be one or more of common auxiliary materials in the field, such as a filling agent, a disintegrating agent, an adhesive, an antioxidant, a lubricant, a surfactant or a flavoring agent.

Has the advantages that:

the research proves that the invention has the following beneficial effects:

1. the invention detects the influence of the pueraria flower healthy-improving prescription drug-containing serum on the protein expression of E-cadherin and N-cadherin of HepG2 liver cancer cells through Western Blot, and proves that the pueraria flower healthy-improving prescription drug-containing serum can inhibit the epithelial-mesenchymal transition process by enhancing the protein expression of E-cadherin and reducing the expression of N-cadherin, thereby inhibiting the invasion and metastasis of tumors, playing the role of resisting the tumors and providing powerful experimental data and support for the clinical application of the pueraria flower healthy-improving prescription.

2. The migration invasion capacity of the pueraria flower health-promoting prescription-containing serum on HepG2 liver cancer cells is detected by Transwell, and the pueraria flower health-promoting prescription-containing serum can effectively inhibit the migration capacity of the HepG2 cells in high, medium and low dose groups. Compared with a blank control group, the cell migration rate in each administration group is obviously reduced (P is less than 0.05), and the concentration is dependent.

3. According to the invention, the CCK8 method is used for detecting the proliferation influence of the pueraria flower health-promoting prescription drug-containing serum on HepG2 liver cancer cells, and after the pueraria flower health-promoting prescription drug-containing serum is intervened for 24 hours, the influence of 5% and 10% concentration groups on cell proliferation is small, and compared with a control group, the difference is not statistically significant (p is more than 0.05). After 48h and 72h of intervention in each administration group, the proliferation rates of the 10% and 20% concentration groups were 72.08%, 69.80% and 68.04%, 62.91%, respectively, and the effect on the proliferation rate of cells was statistically significant (P < 0.05) compared with the control group.

4. The influence of the pueraria lobata health-improving prescription drug-containing serum on the apoptosis of HepG2 liver cancer cells is detected by a flow cytometry Annexin V-FITC/PI double staining method, and the influence of the pueraria lobata health-improving prescription drug-containing serum on the apoptosis of the HepG2 liver cancer cells is proved that the pueraria lobata health-improving prescription drug-containing serum can promote the apoptosis of the HepG2 cells to a certain degree, and compared with a blank control group, each administration group obviously promotes the apoptosis of the cells (P is less than 0.01) and is concentration-dependent.

Drawings

FIG. 1 shows the effect of healthy drug-containing serum in Pueraria lobata on HepG2 cell migration (0.1% crystal violet X200, A. blank control group; B. low dose serum group; C. medium dose serum group; D. high dose serum group).

FIG. 2 shows the effect of healthy drug-containing serum of Pueraria lobata on the invasion of HepG2 cells (0.1% crystal violet X100, A. blank control group; B. low dose serum group; C. medium dose serum group; D. high dose serum group).

FIG. 3 Western Blot method for detecting the influence of health-beneficial prescription of flos Puerariae Lobatae on E-cad and N-cad in hepatoma cell lines.

FIG. 4 health benefits of Pueraria lobata (Willd.) Ohwi serum on the apoptosis of HepG 2.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

Example 1

The application of the healthy-improving prescription of the pueraria flower drug-containing serum in migration and invasion of HepG2 liver cancer cells and the application of E-cadherin and N-cadherin protein expression.

1 materials and methods

1.1 Experimental materials

1.1.1 Experimental animals and cells

SD rats, half male and female, SPF grade, weight 180-: SCXK (Xiang) 2014-. The human liver cancer HepG2 cell strain is purchased from Shanghai cell bank of Chinese academy of sciences, is numbered SCSP-510, is cultured, passaged and frozen strictly according to the conventional specification, and is subjected to experiment when in logarithmic growth phase.

1.1.2 Experimental drugs

The health promoting formula of pueraria flower comprises 0.9 g of green tangerine orange peel of lotus flower, 1.5 g of costus root, 4.5 g of orange peel, 4.5 g of ginseng, 4.5 g of polyporus, 4.5 g of white poria, 6 g of medicated leaven, 6 g of rhizoma alismatis, 6 g of dried ginger, 6 g of bighead atractylodes rhizome, 15 g of cardamom, 15 g of pueraria flower, 15 g of fructus amomi and the like, is purchased from Guiyang Tongrentang Tang, and is prepared into water decoction (crude drug concentrations are 1g/ml, 2g/ml and 4g/ml respectively) by the pharmacy department of the first subsidiary hospital of Guizhou pharmaceutical university according to the original formula proportion for later use.

1.1.3 Primary reagents and instruments

Fetal bovine serum (Gibco, USA, lot number: 2036224C); e-cadherin and N-cadherin (Abcam company, the numbers are ab40772 and ab18203 respectively); a primer (BD Biosciences, USA, model 9119018); transwell chamber (Corning corporation, USA, lot number 08619005); flow cytometry (BD corporation, USA, model: BD FACS Canto); an immunoblotting assay system (Bio-Rad, USA, model: BIO-RAD Chemidoc Touch); an inverted fluorescence microscope (Olympus, Japan, model: Olympus IX 71).

1.2 Experimental methods

1.2.1 Experimental groups

The liver cancer cell strain HepG2 is taken as a research object, and after the SD rat is adaptively fed for two days, the liver cancer cell strain is randomly divided into a control group, a low dose group of the Pueraria flower healthy and healthy formula, a medium dose group of the Pueraria flower healthy and healthy formula and a high dose group of the Pueraria flower healthy and healthy formula.

1.2.2 preparation of health promoting prescription containing serum from flos Puerariae Lobatae

The contrast group is subjected to normal saline intragastric administration, the experimental group is subjected to intragastric administration of pueraria lobata health-improving formula water decoction with different concentrations (crude drug concentrations are 1g/ml, 2g/ml and 4g/ml respectively), the dosage is 1ml/100g, 2 times per day, continuous intragastric administration is carried out for 5 days, 2 hours after the last administration, 1% sodium pentobarbital is used for anesthetizing rats, blood is obtained by adopting a femoral artery blood-taking method, supernatant is obtained after centrifugation and is placed in a 56-degree water bath kettle for inactivation for 30 minutes, filtration and sterilization are carried out, and the obtained supernatant is subpackaged by an ultraclean bench and stored in a-80 refrigerator for later use.

1.2.3 Transwell test of migration and invasion abilities of Pueraria lobata health-promoting prescription-containing serum to HepG2 liver cancer cells

All culture reagents and Transwell chamber were incubated at 37 ℃ until the cells were grown to logarithmic growth phase, the cells were digested, washed once with PBS followed by serum-free medium, the cells were suspended in serum-free medium, and the cell concentration was adjusted by counting to 2X 10⁵And/ml. 800ul of medium containing 20% serum was added to the lower chamber, 200ul of cell suspension and drug were added to the upper chamber, and after further incubation in the cell incubator for 24h, the Transwell chamber was removed, transferred to a well to which approximately 800ul of methanol had previously been added, and fixed at room temperature for 15 min. After staining, the cells in the chamber were wiped off with a sterile cotton swab, the bottom membrane of the upper chamber was removed with forceps, air dried, transferred to a slide glass, mounted with neutral gum, and observed under the mirror.

Cell migration inhibition rate (number of control group migrated cells-number of experimental group migrated cells)/number of control group migrated cells

The matrix substrate was dissolved at 4 ℃ overnight, the matrix was diluted with pre-cooled serum-free medium to a final concentration of 1mg/ml, 100ul of diluted matrix was added vertically to the center of the bottom of the chamber, and incubated at 37 ℃ for 1h to dry into a gel. 200ul of each cell suspension was added to the chamber and 800ul of medium containing 20% serum was added to the lower chamber. After 24h of culture, washing with PBS for 2 times, fixing with methanol for 15min, wiping off cells in the chamber with a sterile cotton swab after staining, taking off the bottom membrane of the upper chamber with a pair of tweezers, air-drying, transferring to a glass slide, sealing with neutral gum, and observing under a mirror. Randomly selecting 5 visual fields for observation, shooting and counting.

The inhibition rate of cell invasion was (number of cells in treatment group/number of cells in blank control group) × 100%.

1.2.4 Western Blot to test the influence of Pueraria health-promoting prescription-containing serum on the expression of E-cadherin and N-cadherin proteins in HepG2 liver cancer cells

Preparing single cell suspension from HepG2 cells in logarithmic phase, counting, planting in a culture bottle, adding different concentrations of pueraria flower health-promoting medicated serum after the cells adhere to the wall, and continuously culturing in an incubator for 24 h; discarding a culture medium, washing by precooled pbs, adding 0.6ml of cell lysate containing PMSF, cracking for 10min on ice, repeatedly blowing and sucking by using a 1ml pipette gun, transferring into an ep tube at 4 ℃ and 12000r/min, centrifuging for 15min, sucking supernatant to obtain total cell protein, quantifying by using a BCA quantitative kit, performing SDS-PAGE electrophoresis, transferring to a membrane, sealing a PVDF membrane by using special sealing liquid, performing primary anti-incubation, washing the membrane, performing secondary antibody incubation, washing the membrane, finally performing ECL development, and exposing in a chemiluminescence imaging analyzer; and (3) analyzing results by adopting Image analysis software, measuring the gray value of the strip, calculating the ratio of the target strip and the internal reference of each group, and comparing the difference between the groups.

1.3 statistical analysis

Statistical analysis was performed using SPSS21.0 software, and the data were measured as mean. + -. standard deviation

Showing that the variance analysis is adopted for the comparison among groupsHomogeneous is analyzed by adopting one-factor variance, pairwise comparison among groups is carried out by using an LSD method, and P is less than 0.05, which shows that the homogeneous has statistical significance.

2 results of the experiment

2.1 Effect of Pueraria flower anti-health prescription drug-containing serum on HepG2 liver cancer cell migration

Through observation under a mirror, the Pueraria lobata health-beneficial formula containing high, medium and low dose of medicine serum can effectively inhibit the migration capability of HepG2 cells. Compared with a blank control group, the cell migration rate in each administration group is obviously reduced (P is less than 0.05), and the concentration is dependent. The results are shown in FIG. 1.

2.2 Effect of Pueraria flower anti-health prescription drug-containing serum on HepG2 liver cancer cell invasion

Through observation under a mirror, the Pueraria lobata health-beneficial formula containing high, medium and low dose of medicine serum can effectively inhibit the invasion capacity of HepG2 cells. Compared with a blank control group, the cell invasion rate of each administration group is obviously reduced (P is less than 0.05), and the concentration is dependent. The results are shown in FIG. 2.

2.3 Effect of Pueraria flower anti-health prescription drug-containing serum on expression of E-cadherin and N-cadherin proteins of HepG2 liver cancer cells

The Pueraria health promoting formula comprises high, medium and low dose groups containing drug serum, which can effectively up-regulate the expression level of E-cadherin in HepG2 cells, effectively down-regulate the expression level of N-cadherin in the cells, and has obvious difference (P is less than 0.05) compared with a blank control group. The results are shown in Table 1 and FIG. 3.

TABLE 1 influence of the healthy-health-promoting formula of Pueraria lobata serum containing drugs on the expression of E-cadherin and N-cadherin proteins in HepG2 liver cancer cells

Note: p < 0.01 compared to control; compared with 10% of the serogroup containing the medicine, # P < 0.05

3. Summary of the invention

Liver cancer is the most common malignant tumor with extremely poor prognosis, the selection of the treatment method is closely related to factors such as the differentiation degree of the tumor, the size of the tumor, whether vascular invasion exists or not, and the like, for most of early and middle patients, liver transplantation and surgical treatment are relatively effective treatment means which are clinically recognized at present, even if radical resection can be performed on the patient, the clinical metastasis rate of the liver cancer is very high, and invasion and metastasis after the operation are the most main reasons for causing poor prognosis and final death of the patient, so that the regulation and control of liver cancer cell invasion and metastasis and the molecular mechanism for guiding the clinic have important scientific significance. At present, with the continuous development and wide application of traditional Chinese medicine, more and more liver cancer patients are treated by selecting traditional Chinese medicine from multiple levels, multiple links and multiple targets, and the traditional Chinese medicine is unique and plays an important role in various aspects such as inhibiting tumor cell proliferation, regulating the immune function of the body, improving the clinical symptoms of the patients, reducing the damage of radiotherapy and chemotherapy to the body and the like.

Pueraria lobata healthy qi promoting formula is mainly used for treating spleen and stomach function hypofunction caused by excessive drinking and accompanied with vomiting and vomiting in the spleen and stomach theory, and is used as a classic formula combining dispersion and seepage and promoting digestion and tonifying, and the formula is found to be capable of relieving liver damage and protecting liver function in early-stage research^[15]Reversing pre-hepatoma lesions^[16]And can also inhibit tumor growth, improve immune function, but its influence and possible action mechanism on liver cancer cell migration and invasion have not been elucidated.

In recent years, it has been found that liver cancer is treated with^[17]Lung cancer and lung cancer^[18]Among various tumors, the epithelial-mesenchymal transition process induces the invasion and metastasis of tumor cells, and is mainly characterized in that E-cadherin which plays a main role among epithelial cells is reduced, Vimentin and N-cadherin among interstitial cells are increased, and then the contact between the epithelial cells and surrounding cells is reduced after the epithelial cells lose polarity, so that the connection among the cells is damaged, the adhesion among the cells is further reduced, and finally the invasion and metastasis of the tumor cells are caused. Among them, E-cad is one of the key molecules for maintaining epithelial phenotype, and can mediate adhesion between cells, thereby maintaining the stability of cell structure and morphology. If the expression of E-cad is reduced, the adhesion between cells is reduced, so that the cells are exfoliated, and further epithelial-mesenchymal transition is caused, so that the invasion and metastasis of tumor cells occur^[19]. Research shows that abnormal expression of E-cad and liver cancerSince invasion and metastasis of cells are closely related, it is important to select this index as a mechanism subject.

In the experiment, results of Transwell and substrate gel experiments show that compared with a control group, the invasion and metastasis effects of liver cancer cells of each medicine-containing serogroup of the pueraria flower health-beneficial formula are weakened, and the differences have statistical significance (P is less than 0.05 and P is less than 0.01), so that the formula can inhibit migration and invasion capacity of HepG2 cells. Western Blot results show that each drug-containing serogroup of the healthy formula of the pueraria flower can up-regulate the expression of E-cadherin in cells and down-regulate the expression of N-cadherin. The experimental results show that the pueraria flower healthy-producing prescription drug-containing serum can inhibit the epithelial-mesenchymal transition process by enhancing the protein expression of E-cadherin and reducing the expression of N-cadherin, so that the tumor invasion and metastasis are inhibited, the anti-tumor effect is exerted, and powerful experimental data and support are provided for the clinical application of the pueraria flower healthy-producing prescription.

Example 2

Application of Pueraria health promoting medicated serum in proliferation ability of HepG2 liver cancer cell and apoptosis of HepG2 liver cancer cell is provided.

1 materials and methods

1.1 Experimental materials

1.1.1 Experimental animals and cells

SD rats, male and female halves, SPF grade, body weight 180-: SCXK (Xiang) 2014-. The human liver cancer HepG2 cell strain is purchased from Shanghai cell bank of Chinese academy of sciences, is numbered SCSP-510, and is cultured, passaged and frozen strictly according to the conventional cell specification.

1.1.2 Experimental drugs

The health promoting formula of pueraria flower comprises 0.9 g of green tangerine orange peel of lotus flower, 1.5 g of costus root, 4.5 g of orange peel, 4.5 g of ginseng, 4.5 g of polyporus, 4.5 g of white poria, 6 g of medicated leaven, 6 g of rhizoma alismatis, 6 g of dried ginger, 6 g of bighead atractylodes rhizome, 15 g of cardamom, 15 g of pueraria flower, 15 g of fructus amomi and the like, is purchased from the same benevolence hall, and is prepared into water decoction (crude drug concentration is 4g/ml) according to the original formula proportion by the first subsidiary hospital of Guizhou traditional Chinese medicine university.

1.1.3 Primary reagents and instruments

Fetal bovine serum (Gibco, USA, lot number: 2036224C); cell proliferation-toxicity test kit CCK8 (Tortoise chemical Co., Ltd., batch No.: NN 710); annexin V-FITC/PI double-staining apoptosis detection kit (Jiangsu Kayji biotechnology, Inc., batch number: KGA 108); flow cytometry (BD corporation, USA, model: BD FACS Canto); an inverted fluorescence microscope (Olympus, Japan, model: Olympus IX 71).

1.2 Experimental methods

1.2.1 preparation of health promoting blood serum containing flos Puerariae Lobatae

After 20 SD rats are adaptively fed for two days, the SD rats are randomly divided into a blank group and a pueraria flower health promoting formula removing group, 10 rats in each group are respectively fed with physiological saline and a pueraria flower health promoting formula water decoction for intragastric administration, the dosage is 1ml/100g, the administration is carried out 2 times per day for 5 days continuously, after 2 hours of last administration, the rats are anesthetized with 1% pentobarbital sodium, blood is obtained by adopting a femoral artery blood taking method, after centrifugation, a supernatant is obtained and placed in a 56-degree water bath kettle for inactivation for 30 minutes, filtration sterilization is carried out, and the SD rats are subpackaged on an ultra-clean bench and stored in a-80 refrigerator for later use.

1.2.2 cell grouping

Blank, 5% drug-containing serogroup, 10% drug-containing serogroup and 20% drug-containing serogroup.

1.2.3 CCK8 method for detecting the effect of health-promoting blood serum containing flos Puerariae Lobatae on proliferation of HepG2 hepatocarcinoma cell

Preparing single cell suspension from HepG2 liver cancer cells in logarithmic phase, and counting, wherein the cell number is about 1x10⁶Inoculating the mixture to a 96-well plate, adding 100 mu L of cell suspension into each well, after 24h, completely adhering the cells to the wall, adding different concentrations of pueraria flower health-promoting prescription-containing serum into the 96-well plate, arranging 6 multiple wells in each group, placing the groups in an incubator for 24h, 48h and 72h respectively, adding 10 mu L of CCK8 solution into each well, placing the mixture into the incubator for incubation for 1h, detecting the absorbance of each well at the wavelength of 450nm by using an enzyme labeling instrument, calculating the proliferation rate of HepG2 liver cancer cells at different concentrations, and repeating the experiment for 3 times.

Cell proliferation rate (%) - (OD value of experiment group-blank OD value)/(OD value of control group-blank OD value) × 100%

1.2.4 flow cytometry Annexin V-FITC/PI double staining method for detecting influence of pueraria flower health-promoting prescription-containing serum on apoptosis of HepG2 liver cancer cells

Preparing single cell suspension from liver cancer cells in logarithmic growth phase, counting, and performing 2 × 10 cell per well⁵Inoculating each cell in a 6-hole plate, placing in an incubator overnight, after 24h, after the cells adhere to the wall, giving different concentrations of pueraria flower health-giving medicine-containing serum for intervention, after 24h, adding pancreatin without EDTA to digest each group of cells, centrifuging at 4 ℃ for 5 minutes to collect the cells, washing the cells for 2 times by precooled PBS (phosphate buffer solution), and centrifuging at 4 ℃ for 5 minutes each time; adding 1ml of 1 × Binding Buffer to resuspend the cells, adding Annexin V-FITC/PI label respectively, mixing gently, incubating at room temperature in the dark for 15min, and detecting with a flow cytometer. The apoptosis rate (%) × 100% (number of apoptotic cells/total number of observed cells).

1.3 statistical analysis

Statistical analysis was performed using SPSS21.0 software, and the data were measured as mean. + -. standard deviation: (

And +/-s) represents that the comparison among the groups adopts variance analysis, the homogeneity of variance adopts single-factor variance analysis, pairwise comparison among the groups is carried out by an LSD method, and P is less than 0.05, which represents that the statistical significance is achieved.

2 results of the experiment

2.1 Effect of Pueraria flower health promoting prescription containing medicated serum on cell proliferation ability of HepG2

After 24 hours of intervention of drug-containing serum at each concentration of the pueraria lobata health promoting formula, 5% and 10% concentration groups have little influence on cell proliferation, and compared with a control group, the difference has no statistical significance (p is more than 0.05). After 48h and 72h of intervention in each administration group, the proliferation rates of the 10% and 20% concentration groups were 72.08%, 69.80% and 68.04%, 62.91%, respectively, and the effect on the proliferation rate of cells was statistically significant (P < 0.05) compared with the control group. The results are shown in Table 2.

TABLE 2 influence of healthy health-promoting prescription of Pueraria lobata (Willd.) Ohwi medicated serum on cell proliferation ability of HepG2

Note: comparison with the normal cell group: p < 0.05, P < 0.01;

2.2 Effect of Pueraria flower anti-health prescription drug-containing serum on HepG2 apoptosis

Flow cytometry detection results show that drug-containing serum of each concentration of the healthy formula for flos puerariae lobatae can promote HepG2 cells to apoptosis to a certain degree, and compared with a blank control group, each administration group obviously promotes the cells to apoptosis (P is less than 0.01) and is concentration-dependent. The results are shown in Table 3 and FIG. 4.

TABLE 3 influence of healthy health-promoting formulation of Pueraria lobata (Willd.) Ohwi-containing serum on apoptosis of HepG2 cells

Note: comparison with the normal cell group: p < 0.01

3. Summary of the invention

Liver cancer is the most common malignant tumor with poor prognosis, most of patients in early and middle stages still take operation as the main treatment means, but invasive metastasis after operation is the most main reason for death of patients. At present, with the continuous development and wide application of traditional Chinese medicine, more and more liver cancer patients are treated by selecting traditional Chinese medicine from multiple levels, multiple links and multiple targets, and the traditional Chinese medicine is unique and plays an important role in various aspects such as inhibiting tumor cell proliferation, regulating the immune function of the body, improving the clinical symptoms of the patients, reducing the damage of radiotherapy and chemotherapy to the body and the like. The pueraria flower health promoting formula is a classic formula combining powder infiltration and elimination and tonifying, wherein the pueraria flower is used for treating yangming and relieving alcoholism and activating spleen; polyporus, Poria, and Alismatis rhizoma have the effects of inducing diuresis and removing dampness, so that the pathogenic factors of alcohol dampness come out from urine and stool; fructus Amomi, fructus Amomi rotundus, pericarpium Citri Reticulatae viride, pericarpium Citri Tangerinae, radix aucklandiae, and dried rhizoma Zingiberis recens have effects of warming spleen and stomach, activating qi-flowing, and regulating stomach; ginseng radix and Atractylodis rhizoma have effects of invigorating qi and invigorating spleen; medicated leaven has effects of relieving hangover and digestion promoting. The medicines are used together to play the roles of dispelling dampness of wine, warming middle-jiao and invigorating spleen. Subject groups found in earlier studies that the formula can not only alleviate liver damage and protect liver function^[20]Reversing pre-hepatoma lesions^[21]，

Meanwhile, the effects of inhibiting tumor growth, improving immune function and the like can be achieved, but the influence and possible action mechanism on the migration and invasion of liver cancer cells are not clarified.

The research preliminarily discusses the influence of the pueraria flower health-promoting prescription-containing serum on the proliferation and apoptosis of the high-metastasis liver cancer cell HepG 2. The research result shows that after the cell culture is carried out for 24 hours, the influence of 5 percent and 10 percent concentration groups on cell proliferation is small, and compared with a control group, the difference is not statistically significant (p is more than 0.05). After 48h and 72h of intervention in each administration group, the proliferation rates of the 10% and 20% concentration groups were 72.08%, 69.80% and 68.04%, 62.91%, respectively, and the effect on the proliferation rate of cells was statistically significant (P < 0.05) compared with the control group. In an apoptosis experimental study, after 24 hours of intervention, the pueraria flower health-beneficial formula and the drug-containing serum in various concentrations can promote the hepatoma cells HepG2 to apoptosis to a certain extent in various administration groups, and compared with a blank control group, the administration groups obviously promote the apoptosis (P is less than 0.01) and are concentration-dependent. In conclusion, the research finds that the pueraria flower health-promoting medicated serum with different concentrations has obvious effects of inhibiting proliferation and promoting apoptosis of HepG2 liver cancer cells, and the effect is dose-dependent, but the mechanism of the effect of the pueraria flower health-promoting medicated serum through which the pueraria flower health-promoting medicated serum is used is needed to be further researched.

Example 3

Taking the health promoting medicinal serum of flos Puerariae Lobatae as raw material, adding 1/30 starch, and granulating to obtain granule.

Example 4

Taking the medicinal serum of the health promoting prescription of flos puerariae lobatae as a raw material medicine, adding 1/30 starch, mixing, pulverizing, and encapsulating to obtain the capsule.

Example 5

Taking flos Puerariae Lobatae health promoting medicinal serum as raw material, adding dextrin 1/20, mixing, drying, and making into pill.

Example 6

Taking the health promoting medicinal serum of flos Puerariae Lobatae as raw material, adding 1/15 starch, granulating, tabletting, and making into tablet.

Example 7

Taking the health promoting kudzuvine flower medicinal serum as a raw material medicament, adding 8 times of injection water, and sterilizing to obtain the injection.

Example 8

Taking the medicinal serum of the health promoting prescription of flos puerariae lobatae as a raw material medicine, and freeze-drying to obtain freeze-dried powder.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference documents:

[1]Li Y，Xu D，Bao C，et al.MicroRNA-135b,a HSF1 target,promotes tumor invasion and metastasis by rugulating RECK and EVI5 in hepatocellular carcinoma[J].Oncotarget,2015,6(4):2421-2433.

[2] chendongfeng, Sunweishi, Liukai Jun, etc. epidemiological status quo and pathogenesis of nonalcoholic fatty liver disease-related liver cancer [ J ]. China liver disease journal, 2017, 25 (2): 111.

[3]Tommaso L D,Sangiovanni A,Borzio M,et al.Advanced precancerous lesion in theliver[J].Best Practice&Research Clinical Gastroenterology,2013,27(2):269-284.

[4] zhangshuang, wangfeng, mengqingling, etc. epidemiology and hepatitis b virus infection investigation of primary liver cancer patients [ J ] chinese vaccine and immunization, 2018, 24 (2): 133.

[5]Mak GW，Chan MM，Leong VY，et al.Overexpression of a novel activator of PAK4，the CDK5 kinase-Associated protein CDK5RAP3，promotes hepatocellular carcinomametastasis[J].CancerRes，2011，71(8):2949－2958.

[6]Ren N，Wu JC，Dong QZ，et al.Association of specificgenotypes in metastatic suppressor HTPAP with tumor metastasis and clinical prognosis in hepatocellular carcinoma[J].CancerRes，2011，71(9):3278－3286.

[7]Newhook T E,Lapar D J,Lindberg J M,et al.Morbidity and mortality of hepatectomy for benign liver tumors[J].Am J Surg,2016,211(1):102.

[8]Liu B,Long J,Wang W,et al.Predictive factors of treatment outcomes after percutaneous ablation of hepato-cellular carcinoma in the caudate lobe:a retrospective study[J].BMC Cancer,2019,19(1):699.

[9]Singh M,Yelle N,Venugopal C,et al.EMT:Mechanisms and therapeutic implications[J].Pharmacol Ther,2018,182(2):80-94.

[10]Chaffer C L,San Juan B P,Lim E,et al.EMT,cell plasticity and metastasis[J].Cancer Metastasis Rev,2016,35(2):645-654.

[11] 3 anti-alcoholism formulas such as puerarin powder and the like have a protective effect on acute liver injury of a mouse caused by ethanol [ J ] in the university of Liaoning traditional Chinese medicine, 2010 and 12 (4): 7-9.

[12] Shuqi, Wangping, Wangwangjia, etc. 3 sobering-up prescriptions such as Kudzuvine root powder, etc. have influence on the apoptosis of liver cells of mice with acute alcoholic liver injury [ J ] Chinese traditional medicine journal, 2011,26(9): 2046-.

[12] The influence of the pueraria flower anti-aging prescription on the content of immune inflammatory factors in peripheral blood of transplanted liver cancer mice [ J ]. precious national and medical remedies 2015, 26 (12): 2845.

[14] li Jun, Tang Dong Xin, Longfeng xi, etc. Pueraria Hua Jiesheng formula has an influence on the inhibition of tumor tissue growth and the expression of APC protein thereof in mice with transplanted liver cancer [ J ]. J, J of Chinese Experimental prescriptions, 2016,22 (15): 112-117.

[15] Influence of the pueraria flower anti-growth formula on the antioxidation of hepatoma cell orthotopic transplantation tumor mice [ J ]. reported by Beijing university of traditional Chinese medicine, 2015,38 (10): 690-693.

[16] Bin, tangzui, torvu xi, etc. influence of the pueraria flower healthy-development prescription on the expression of the cell cycle regulatory factor of the pre-hepatoma pathological condition of alcoholic HBV transgenic mice [ J ]. press by beijing university of traditional chinese medicine 2015,38 (11): 745-748.

[17]Zoheiry MM,Hasan SA,EI-Ahwany E,et al.Serum markers of epithelial mesenchymal transition as predictors of HCV-induced liver brosis,Cirrhosis and Hepatocellular Carcinoma[J].Electronic physician,2015,7:1626-1637.

[18]Wang LK,Pan SH,Chang YL,et al.MDA-9/Syntenin-Slug transcriptional complex promote epithelial-mesenc-hymal transition and invasion/metastasis in lung adenocarcinoma[J].Oncotarget,2016,7:386-401.

[19]Nakagawa H,Hikiba Y,Hirata Y,et al.Loss of E-cadherin induces sclerosing cholangitis and promotes carcino-genesis[J].Proc Natl Acad Sci USA,2014,111(3):1090-1095.

[20] Bin, tangzui, torvu xi, etc. influence of the pueraria flower healthy-development prescription on the expression of the cell cycle regulatory factor of the pre-hepatoma pathological condition of alcoholic HBV transgenic mice [ J ]. press by beijing university of traditional chinese medicine 2015,38 (11): 745-748.

[21]Zoheiry MM，Hasan SA，EI-Ahwany E，et al.Serum markers of epithelial mesenchymal transition as pred-ictors of HCV-induced liver fibrosis,Cirrhosis and Hepatocellular Carcinoma[J].Electronic physician,2015,7:1626-1637.

Claims

1. An application of pueraria flower health-promoting prescription-containing serum in preparing a medicine for treating cancer cells is characterized in that the pueraria flower health-promoting prescription-containing serum is applied in preparing the medicine for treating liver cancer cells.

2. The use of claim 1, wherein the health-beneficial and health-beneficial pueraria flower serum is used for preparing a medicament for treating HepG2 liver cancer cells.

3. The use of claim 2, wherein the health-beneficial and health-beneficial pueraria flower-containing serum is used for preparing a medicament for treating HepG2 liver cancer cell migration.

4. The use of claim 2, wherein the health-beneficial formula of pueraria lobata flower is used for preparing a medicament for treating drug-containing serum invasion of HepG2 liver cancer cells.

5. The use of the healthy-producing medicinal serum of pueraria lobata as claimed in claim 2, wherein the healthy-producing medicinal serum is used for preparing a medicament for treating protein expression of HepG2 liver cancer cells E-cadherin and N-cadherin.

6. The use of claim 2, wherein the health-beneficial and health-promoting pueraria flower-containing serum is used for preparing a medicament for treating and inhibiting HepG2 liver cancer cell proliferation capacity.

7. The use of claim 2, wherein the health-beneficial and health-promoting serum is used for preparing a medicament for promoting apoptosis of HepG2 liver cancer cells.

8. The use of any one of claims 1-7, wherein the pueraria flower health-promoting and drug-containing serum is used for preparing a preparation for treating liver cancer cells.

9. The use of claim 8, wherein the preparation is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable auxiliary materials.

10. The use of claim 9, wherein the formulation is a capsule, granule, powder, pill, tablet, injectable formulation, lyophilized powder for injection.