Pharmaceutical composition for adjuvant therapy of breast cancer and application

Abstract

The invention provides a pharmaceutical composition for adjuvant therapy of breast cancer and application thereof.The pharmaceutical composition comprises morinda citrifolia fermented juice freeze-dried powder, the morinda citrifolia fermented juice freeze-dried powder is prepared through a vacuum freeze-drying technology, and the effective dose of the morinda citrifolia fermented juice freeze-dried powder in adjuvant therapy of breast cancer is 3-12 mg / mL. The combined use of the morinda citrifolia fermented juice and gemcitabine can significantly inhibit the growth of breast cancer cells and vascular hyperplasia, has potential effects of improving chemotherapeutic drugs and reducing side effects of the chemotherapeutic drugs, is helpful to clarify the action mechanism of the morinda citrifolia fermented juice in treating breast cancer and the efficacy of combined chemotherapeutic drugs, and has broad application prospects. The medicinal value of the morinda citrifolia is actually converted into an anti-cancer drug or an auxiliary drug.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a pharmaceutical composition for adjuvant treatment of breast cancer and its application. Background Art

[0002] Breast cancer is the most common cancer in women, accounting for about 30% of new cancers in women each year. Triple-negative breast cancer (TNBC) is a subtype of breast cancer, which refers to breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) genes. Treatments for breast cancer include surgery, radiotherapy, chemotherapy, endocrine therapy and molecular targeted therapy, but chemotherapy remains the main treatment for early and late-stage TNBC patients. Compared with patients with other subtypes of breast cancer, TNBC patients usually have a poorer prognosis, mainly due to their aggressive phenotype and the lack of approved targeted therapies. Although chemotherapy is effective, it has significant toxic side effects and poor quality of life for patients. Therefore, the development of effective and relatively safe anti-tumor drugs is the focus of research on the treatment and prevention of breast cancer.

[0003] Patent application number CN115192662A discloses a Chinese medicine composition containing noni fruit as a medicine for treating tumors. The main medicines are ginseng, longan, white chaga, ganoderma lucidum, astragalus, poria, fried white peony root, yam, and at least two of Hedyotis diffusa, Caulis dasyphyllae, and Scutellaria barbata. The medicine includes millet, noni fruit, yam, ginger, enzyme bacteria, and adult live scorpions. Although it has a definite therapeutic effect on various tumors, noni fruit is not the main ingredient for treating tumors, and the anti-tumor effect of noni fruit cannot be clearly determined; Patent application number CN1711097A discloses a method for the anti-angiogenesis effect of noni, and the processed noni preparation blocks the formation of new blood vessels in the tumor area by inhibiting tubule elongation and endothelial cell migration. However, this method does not show the anti-angiogenesis effect of noni fruit fermented juice on breast cancer, and lacks a systematic evaluation of the anti-breast cancer effect of noni fruit fermented juice. All studies use noni fruit extract juice, and do not study the anti-cancer and anti-angiogenesis effects of noni fruit fermented juice. Natural products are often used to synergize with chemotherapy drugs to improve anti-tumor effects and reduce the side effects of chemotherapy drugs to improve the quality of life of patients. Currently, there is no research on the combination of noni fermented juice and chemotherapy drugs for the treatment of breast cancer. Summary of the invention

[0004] In view of this, and in view of the deficiencies in the prior art, the present invention proposes a pharmaceutical composition and application for the adjuvant treatment of breast cancer, with the aim of providing a natural and safe adjuvant treatment drug for breast cancer to reduce the side effects of chemotherapy; the second purpose is to clarify the mechanism of action of noni fruit fermented juice in inhibiting breast cancer cell activity and angiogenesis through multiple targets; the third purpose is to develop a combination therapy of noni fruit fermented juice and gemcitabine to improve the efficacy of chemotherapy and reduce drug resistance.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a pharmaceutical composition for adjuvant treatment of breast cancer. The pharmaceutical composition comprises freeze-dried powder of fermented noni fruit juice. The freeze-dried powder of fermented noni fruit juice is prepared by vacuum freeze-drying technology. The effective dosage of the freeze-dried powder of fermented noni fruit juice in the treatment of breast cancer is 3-12 mg / mL.

[0007] On the basis of this technical solution, it is further preferred that the effective dosage of the freeze-dried powder of noni fermented juice in the treatment of breast cancer specifically includes: the half inhibition concentration of human breast cancer cells MCF-7 is 15.69 mg / mL, and the half inhibition concentration of human breast cancer cells MDA-MB-231 is 16.09 mg / mL.

[0008] On the basis of this technical solution, further preferably, the pharmaceutical composition also includes gemcitabine, and the mass ratio of the freeze-dried powder of Noni fruit fermented juice to gemcitabine is 1:0.5 to 1:5.

[0009] On the basis of this technical solution, further preferably, the method for preparing the freeze-dried powder of Noni fruit fermented juice comprises the following steps:

[0010] The fermented noni fruit juice is taken, filtered and then packaged; the packaged fermented noni fruit juice is weighed, and pre-frozen at a low temperature of -80°C to partially or completely solidify the fermented noni fruit juice, and then vacuum freeze-dried; the freeze-dried solid is weighed again to calculate the recovery rate of soluble solids; the processed freeze-dried noni fruit juice powder is sealed and packaged, and stored at -20°C for use.

[0011] On the basis of this technical solution, further preferably, the soluble solid yield of the freeze-dried powder of Noni fermented juice is 3.0% to 4.0% (w / w).

[0012] In a second aspect, the present invention further provides use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any one of the first aspects in the treatment of breast cancer.

[0013] On the basis of this technical solution, it is further preferred that the concentration of the freeze-dried powder of Noni fermented juice for combined administration is 1-8 mg / mL, and the dosage of gemcitabine is 10%-50% of the clinically recommended dosage.

[0014] In a third aspect, the present invention further provides use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any one of the first aspects in inhibiting tumor angiogenesis in breast cancer.

[0015] On the basis of this technical solution, it is further preferred that the freeze-dried powder of noni fermented juice is achieved by down-regulating CD31 protein expression and inhibiting the tube-forming and invasion capabilities of human umbilical vein endothelial cells.

[0016] On the basis of this technical solution, it is further preferred that the administration dosage of the freeze-dried powder of noni fermented juice is 400-800 mg / kg, which significantly reduces the proportion of Ki67 and CD31 positive cells in tumor tissues.

[0017] The pharmaceutical composition and application for adjuvant treatment of breast cancer provided by the present invention have the following beneficial effects compared with the prior art:

[0018] Noni fruit fermented juice is extracted and processed from natural plants. It is safe to eat and can reduce the side effects of chemical synthetic drugs. It has multiple anti-cancer effects, including inhibiting cancer cell proliferation, inducing apoptosis, affecting cell cycle, inhibiting cancer cell migration and invasion, and inhibiting tumor angiogenesis. In addition, the anti-cancer effect of Noni fruit fermented juice has a multi-target mechanism of action, which helps to reduce the resistance of breast cancer cells to single treatment methods, and can assist in improving the therapeutic effect of the chemotherapy drug gemcitabine, providing new candidate compounds for the development of new anti-tumor drugs; BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The inhibitory effect of the fermented noni juice on breast cancer cell activity described in Example 3 of the present invention;

[0021] Figure 2 The fermented noni juice described in Example 3 of the present invention induces apoptosis of human breast cancer cells (MCF-7 and MDA-MB-231);

[0022] Figure 3The effect of the fermented noni juice on the cell cycle of human breast cancer cells described in Example 4 of the present invention;

[0023] Figure 4 The effect of the fermented noni juice on the proliferation ability of human breast cancer cells described in Example 5 of the present invention;

[0024] Figure 5 The effect of the fermented noni juice on the migration ability of human breast cancer cells described in Example 6 of the present invention;

[0025] Figure 6 The effect of the fermented noni juice on the invasive ability of human breast cancer cells described in Example 7 of the present invention;

[0026] Figure 7 The effect of the fermented noni fruit juice on the growth of breast cancer tumors in vivo described in Example 8 of the present invention;

[0027] Figure 8 The effect of the fermented noni juice on breast cancer tumor proliferation in vivo described in Example 9 of the present invention;

[0028] Fig. 9 The effect of the fermented noni juice described in Example 10 of the present invention on the angiogenesis of breast cancer tumors in vivo;

[0029] Fig.10 The effect of the fermented noni juice described in Example 11 of the present invention on the tube-forming ability of vascular endothelial cells in vitro;

[0030] Fig.11 The effect of the fermented noni fruit juice described in Example 12 of the present invention on the invasive ability of vascular endothelial cells in vitro;

[0031] Fig.12 This is a combined index analysis of the activity of the noni fruit fermented juice and gemcitabine on breast cancer cell MDA-MB-231 cells as described in Example 13 of the present invention;

[0032] Fig.13 The effect of the combination of Noni fruit fermented juice and gemcitabine on apoptosis of breast cancer cells described in Example 14 of the present invention;

[0033] Fig.14 This is the effect of the combination of Noni fruit fermented juice and gemcitabine on the function of vascular endothelial cells in Example 15 of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Noni is a unique medicinal plant among tropical fruit trees, mainly grown in South Pacific islands such as Fiji. Noni is rich in phenolic acids, terpenes and other ingredients. Experimental studies have shown that Noni has a variety of biological activities, such as anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory.

[0036] The present invention provides a pharmaceutical composition and application for the adjuvant treatment of breast cancer. By combining fermented Noni fruit juice and gemcitabine, the anti-breast cancer effect of fermented Noni fruit juice is systematically evaluated. From the proteomics and genomics levels, it is preliminarily found that fermented Noni fruit juice can regulate the signal pathway related to tumor angiogenesis and has an anti-angiogenesis effect on breast cancer. The combination of fermented Noni fruit juice and gemcitabine can significantly inhibit the growth and angiogenesis of breast cancer cells, and has the potential to improve the effect of chemotherapy drugs and reduce their side effects. The present invention helps to clarify the mechanism of action of fermented Noni fruit juice in treating breast cancer, and the efficacy of combined chemotherapy drugs, and actually converts the medicinal value of Noni fruit into an anti-cancer drug or adjuvant drug.

[0037] In a preferred embodiment, a pharmaceutical composition for adjuvant treatment of breast cancer includes freeze-dried powder of fermented noni fruit juice, wherein the freeze-dried powder of fermented noni fruit juice is prepared by vacuum freeze-drying technology, and the effective dose of the freeze-dried powder of fermented noni fruit juice in the treatment of breast cancer is 3 to 12 mg / mL.

[0038] Specifically, the effective dosage of the freeze-dried powder of Noni fermented juice in the treatment of breast cancer includes:

[0039] The half inhibitory concentration for human breast cancer cells MCF-7 is 15.69 mg / mL, and the half inhibitory concentration for human breast cancer cells MDA-MB-231 is 16.09 mg / mL.

[0040] Specifically, the pharmaceutical composition also includes gemcitabine, and the mass ratio of the freeze-dried powder of Noni fruit fermented juice to gemcitabine is 1:0.5 to 1:5.

[0041] Specifically, the method for preparing the freeze-dried powder of Noni fermented juice comprises the following steps:

[0042] The fermented noni fruit juice is taken, filtered and then packaged; the packaged fermented noni fruit juice is weighed, and pre-frozen at a low temperature of -80°C to partially or completely solidify the fermented noni fruit juice, and then vacuum freeze-dried; the freeze-dried solid is weighed again to calculate the recovery rate of soluble solids; the processed freeze-dried noni fruit juice powder is sealed and packaged, and stored at -20°C for use.

[0043] Specifically, the soluble solid yield of the freeze-dried powder of Noni fermented juice is 3.0% to 4.0% (w / w).

[0044] In a preferred embodiment, the use of the pharmaceutical composition for adjuvant treatment of breast cancer in the treatment of breast cancer is also provided.

[0045] Specifically, the concentration of the noni fruit fermented juice freeze-dried powder for combined administration is 1-8 mg / mL, and the dosage of gemcitabine is 10%-50% of the clinically recommended dosage.

[0046] In a preferred embodiment, the use of the pharmaceutical composition for adjuvant treatment of breast cancer in inhibiting angiogenesis in breast cancer tumors is also provided.

[0047] Specifically, the freeze-dried powder of noni fermented juice is achieved by down-regulating the expression of CD31 protein and inhibiting the tube-forming and invasion capabilities of human umbilical vein endothelial cells.

[0048] Specifically, the administration dosage of the freeze-dried powder of the fermented noni fruit juice is 400-800 mg / kg, which significantly reduces the proportion of Ki67 and CD31 positive cells in tumor tissues.

[0049] Example 1

[0050] A method for treating breast cancer using a pharmaceutical composition for adjuvant treatment of breast cancer comprises the following steps:

[0051] S1. Preparing freeze-dried powder of noni fruit fermented juice: using vacuum freeze-drying technology to concentrate the freeze-dried noni fruit fermented juice, and sealing and packaging the processed freeze-dried powder of noni fruit fermented juice, and storing it at -20°C for use.

[0052] S2. The inhibitory effect of fermented noni fruit juice on breast cancer cell activity: The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method was used to determine the inhibitory effect of fermented noni fruit juice on the cell activity of human breast cancer cells (MCF-7 and MDA-MB-231).

[0053] S3. Effect of fermented noni fruit juice on apoptosis of breast cancer cells: Annexin V-FITC / PI apoptosis detection kit and flow cytometry were used to detect the promoting effect of different concentrations of fermented noni fruit juice on cell apoptosis.

[0054] S4. Effect of fermented noni fruit juice on the cell cycle of breast cancer cells: Flow cytometry was performed using propidium iodide (PI) DNA staining to analyze the effects of different concentrations of fermented noni fruit juice on the cell cycle.

[0055] S5. Effect of fermented noni fruit juice on breast cancer cell proliferation: The cell proliferation protein marker Ki67 was stained and labeled, and flow cytometry was performed to analyze the cell proliferation. Cells treated with fermented noni fruit juice were cultured in monoclonal culture to detect the cell proliferation ability.

[0056] S6. Effect of fermented noni juice on the migration ability of breast cancer cells: The cell scratch test was used to analyze the migration of cells after 24 hours of treatment with fermented noni juice.

[0057] S7. Effect of fermented noni juice on the invasion ability of breast cancer cells: Matrigel was spread on transwell to detect the invasion ability of cells.

[0058] S8. Effect of fermented noni juice on breast cancer tumor growth in vivo: A nude mouse subcutaneous tumor model of MDA-MB-231 cells was established. The treated group was gavaged with three doses of fermented noni juice (200, 400, and 800 mg / kg bw) per day, and the blank control group was gavaged with normal saline. After 21 days of the experiment, the mice were killed to obtain tumor tissues.

[0059] S9. Effect of fermented noni juice on breast cancer tumor proliferation in vivo: Immunofluorescence staining of the proliferation marker protein Ki67 in tumor tissue sections was used to study the cancer cell proliferation ratio of tumor tissues in different treatment groups.

[0060] S10. Effect of fermented noni juice on tumor angiogenesis in vivo: Immunofluorescence staining of tumor tissue sections was used to detect the expression of vascular endothelial cell marker protein CD31 in tumor tissues of different treatment groups.

[0061] S11. Effect of fermented noni juice on endothelial cell function in vitro: Matrigel was placed on transwell plates to detect the invasion ability of human umbilical vein endothelial cells (HUVEC). Matrigel was placed on 96-well plates, HUVEC cells were inoculated at a certain density and treated with medium or conditioned medium containing different concentrations of fermented noni juice for 4-8 hours, and the tube formation was observed to evaluate the angiogenesis ability.

[0062] S12. The combined effect of noni fruit fermented juice and gemcitabine: The study used the drug combination dose-effect analysis software "CompuSyn" developed by the Chou-Talaly median pharmacodynamics mathematical model to analyze the combined anti-breast cancer effect of noni fruit fermented juice and gemcitabine.

[0063] S13. Effect of the combination of noni fruit fermented juice and gemcitabine on apoptosis of breast cancer cells: Annexin V-FITC / PI apoptosis detection kit and flow cytometry were used to detect the promoting effect of low concentrations of noni fruit fermented juice and / or gemcitabine on cell apoptosis.

[0064] S14. Effect of the combination of noni fruit fermented juice and gemcitabine on vascular endothelial cell function: Matrigel was spread on the transwell to detect the invasion ability of human umbilical vein endothelial cells HUVEC. Matrigel was spread on a 96-well plate, a certain density of HUVEC cells were inoculated and treated with low concentrations of noni fruit fermented juice or / and gemcitabine for 4-8 hours, and the tube formation was observed to evaluate the angiogenesis ability.

[0065] In a preferred embodiment, more specifically, Example 2

[0066] The method for preparing freeze-dried powder of fermented noni fruit juice comprises the following steps:

[0067] S1. Take the fermented noni fruit juice, filter out the solid residue with filter paper and package it in a clean bench. The packaged juice is weighed and pre-frozen at -80°C to partially or completely solidify it, and then vacuum freeze-dried. The freeze-dried solid is weighed again to calculate the recovery rate of soluble solids. The processed freeze-dried noni fruit juice powder is sealed and packaged, and stored at -20°C for use.

[0068] Table 1 Soluble solids yield of freeze-dried concentrated noni fermented juice

[0069] batch 1 2 3 4 5 6 7 8 9 10 Yield (%) 3.667 3.613 3.542 3.597 3.493 3.642 3.405 3.647 3.628 3.535 batch 11 12 13 14 15 16 17 18 19 20 Yield (%) 3.621 3.565 3.952 3.833 3.889 3.920 3.886 3.553 3.699 3.858

[0070] Results: As shown in Table 1, a total of 20 batches of extraction and concentration were carried out, and the soluble solid yields obtained were shown in Table 1. The average soluble solid ratio of the noni fruit fermented juice was 3.678±0.154%.

[0071] In a preferred embodiment, more specifically, Example 3

[0072] The study on the effect of fermented noni juice on breast cancer cell activity has the following steps:

[0073] S2, get the Noni fruit fermented juice freeze-dried powder, with DMEM culture fluid, the freeze-dried powder is made into a certain concentration of fruit pulp, and gradient dilution is carried out, with 0.22 μm microporous filter suction filtration sterilization, and 4 ℃ are stored for standby use. Human breast cancer cells (MCF-7 and MDA-MB-231) in logarithmic growth phase are inoculated on 96-well culture plates by 5000 per well, 37 ℃, 5% CO2 incubator, cultivate 24h, after cell adhesion, the experimental group adds the Noni fruit fermented juice of different concentrations to process, the experimental control group adds the DMEM culture fluid without Noni fruit fermented juice, the blank control group does not contain cells, and each concentration repeats 6 multiple holes, is placed in 37 ℃, 5% CO2 incubator and hatches 24h. After cultivation, every well adds the MTT (5mg / mL) of 10 μL, is placed in 37 ℃, 5% CO2 incubator and hatches 4h. The supernatant was removed, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the cells were shaken on a horizontal shaker for 10 s. The absorbance was measured at a wavelength of 490 nm using an enzyme reader.

[0074] The cell activity inhibition effects of experimental drugs and control drugs on human breast cancer cells (MCF-7 and MDA-MB-231) were calculated according to the following formula.

[0075] Cell activity (%) = [(absorbance value of drug action group - absorbance value of blank control group) / (absorbance value of cell control group - absorbance value of blank control group)] * 100%.

[0076] Results: The experimental results are as follows Figure 1 The results show that after 24 hours of treatment, the IC50 (half inhibitory concentration) of the fermented noni juice of the present invention on human breast cancer cells MCF-7 is 15.69 mg / mL. Figure 2 It shows that the IC50 (half inhibitory concentration) of noni fruit fermented juice on human breast cancer cells MDA-MB-231 is 16.09 mg / mL.

[0077] In a preferred embodiment, more specifically, Example 4

[0078] The study of the effect of fermented noni juice on breast cancer cell apoptosis has the following steps:

[0079] S3. Human breast cancer cells (MCF-7 and MDA-MB-231) in logarithmic growth phase were cultured at 2.5×10 5The cells were inoculated in 6-well plates. The experimental groups were added with low, medium and high concentrations (3, 6, 12 mg / mL) of noni fruit fermented juice, and the blank control group was added with culture medium without noni fruit fermented juice. After 24 hours of drug treatment, all cells (adherent cells and cells floating in the culture medium) were collected and washed once with PBS. 100 μL of buffer was added to each group and blown evenly. 5 μL FITC Annexin V and 5 μL PI were added to each tube, stained at room temperature in the dark for 15 minutes, 100 μL of buffer was added, blown evenly, and collected in the flow cytometer loading tube. The samples were collected and analyzed by flow cytometry for fluorescence intensity data.

[0080] Results: Figure 2 The results showed that noni fruit fermented juice could significantly induce apoptosis of human breast cancer cells (MCF-7 and MDA-MB-231), and as the concentration of noni fruit fermented juice increased, the proportion of apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) gradually increased.

[0081] In a preferred embodiment, more specifically, Example 5

[0082] The study of the effect of fermented Noni juice on the cell cycle of breast cancer cells has the following steps:

[0083] S4. Human breast cancer cells (MCF-7 and MDA-MB-231) with different logarithmic growth phases were divided into low, medium, high concentration groups and a blank control group. After different treatments, all cells (adherent and suspended cells) were collected in a 15mL centrifuge tube and washed once with PBS. Each group was resuspended in 1mL pre-cooled PBS and blown evenly. Slowly add 5mL 70% ice-cold ethanol for fixation and place at 4℃ overnight. Wash with pre-cooled PBS before staining, centrifuge at 3000rpm for 5min at 4℃, remove the fixative, add 50μL RNaseA and propidium iodide PI (propdium iodide) and 0.3% Triton X-100, and stain at room temperature in the dark for 45min. After staining, add 300μL PBS to wash once, collect cells in a flow cytometer loading tube, and perform cell cycle analysis using a flow cytometer to obtain the percentage of each cell cycle stage (G1, G2, S and M phases).

[0084] Results: Figure 3 The results showed that fermented noni juice could effectively inhibit the normal transition of cell cycle by reducing G2 / M proliferating cells and increasing G0 / G1 arrested cells, causing MCF-7 cells to accumulate in G1 phase, preventing cell mitosis and inhibiting cell proliferation, thereby achieving anti-human breast cancer cell activity.

[0085] In a preferred embodiment, more specifically, Example 6

[0086] The study of the effect of fermented noni juice on breast cancer cell proliferation has the following steps:

[0087] S5. Human breast cancer cells (MCF-7 and MDA-MB-231) in different logarithmic growth phases were divided into low, medium, high concentration groups (3, 6, 12 mg / mL) and a blank control group. After different treatments, all adherent cells were collected in a 15 mL centrifuge tube, washed once with PBS, added with Fixation / Permeabilization Solution and placed on ice for 30-60 min to fix and permeabilize the cells, added with Perm / Wash Buffer to wash once, centrifuged and discarded the supernatant, added Ki67 antibody to stain in the dark for 15-30 min, washed with Perm / Wash Buffer after staining, collected the cells in a flow cytometer loading tube, and analyzed the cell cycle using a flow cytometer.

[0088] Human breast cancer cells (MCF-7 and MDA-MB-231) with different logarithmic growth phases were taken, 1000 cells were inoculated in each well of a 6-well plate, and divided into low, medium, high concentration groups and a blank control group for treatment with different concentrations of noni fruit fermented juice for 24 hours. After the treatment, all experimental groups were replaced with DMEM culture medium without noni fruit fermented juice for culture, and cultured in an incubator for 1-2 weeks. When clones visible to the naked eye appeared in the experimental group, the culture was terminated. The supernatant was discarded, washed twice with PBS, 4% paraformaldehyde was added to fix the cells for 15 minutes, the fixative was removed and washed once with PBS, and an appropriate amount of crystal violet staining solution was added for staining for 1 hour. After the staining was completed, the staining solution was slowly washed away with running water, dried at room temperature, and each group was photographed with a digital camera, and the number of cloned cells was counted.

[0089] Results: Figure 4 As shown in ab, high concentrations of noni fermented juice can effectively inhibit the proliferation of MDA-MB-231 cells, but the inhibitory effect on the proliferation of MCF-7 cells is not obvious. Figure 4 As shown in cd, medium concentration of Noni fruit fermented juice can reduce the cell clone number of MDA-MB-231, and high concentration of Noni fruit fermented juice can reduce the cell clone number of MDA-MB-231 and MCF-7.

[0090] In a preferred embodiment, more specifically, Example 7

[0091] The study of the effect of fermented noni juice on the migration of breast cancer cells has the following steps:

[0092] S6. Different human breast cancer cells (MCF-7 and MDA-MB-231) were inoculated in 6-well plates at a density of 100%, and the cell monolayer was scraped vertically with a 1 mL or 200 μL gun tip. The culture supernatant was discarded, and the cells were washed 2-3 times with PBS, and the scraped cells were removed. Serum-free medium containing different concentrations of noni fruit fermented juice and serum-free medium without noni fruit fermented juice was added to each well, and the cells were cultured at 37°C and 5% CO2 for 24 hours. The cells were imaged and photographed under a microscope at 0 hours and 24 hours to observe the changes in cell migration.

[0093] Results: Figure 5 As shown, Noni fruit fermented juice can significantly inhibit the migration ability of human breast cancer cells MCF-7 and MDA-MB-231.

[0094] In a preferred embodiment, more specifically, Example 8

[0095] The study of the effect of fermented noni juice on the invasion of breast cancer cells has the following steps:

[0096] S7. Human breast cancer cells (MCF-7 and MDA-MB-231) at different logarithmic growth phases were suspended in serum-free DMEM medium and 2.5×10 4 The cells were placed in the upper chamber of the transwell of a 24-well plate. The upper chamber was coated with 3% Matrigel, and 800 μL of medium containing low, medium, and high concentrations of noni fermented juice and medium without noni fermented juice were added to the lower chamber. After culturing for 24 hours at 37°C and 5% CO2, the upper chamber medium was discarded, and 4% PFA was added to fix the cells at room temperature for 2 hours. After fixation, the upper and lower chambers were rinsed with PBS, and crystal violet was added to stain the cells on the surface of the upper chamber. After staining, the transwell was slowly rinsed with tap water, dried at 37°C, and imaged and photographed with a microscope, and the number of cells was calculated by ImageJ software.

[0097] Results: Figure 6 As shown in the data, low, medium and high concentrations of noni fruit fermented juice can significantly inhibit the invasion ability of human breast cancer cells MCF-7 and MDA-MB-231, and as the concentration of noni fruit fermented juice increases, the inhibitory effect on the invasion ability becomes more obvious.

[0098] In a preferred embodiment, more specifically, Example 9

[0099] Effects of Noni Fermented Juice on Breast Cancer Tumor Growth in Vivo:

[0100] S8. Human breast cancer MDA-MB-231 cells were inoculated subcutaneously in the axilla of 30 female nude mice. The treated groups were gavaged with 200, 400, and 800 mg / kg bw of noni fruit fermented juice every day, and the blank control group was gavaged with normal saline. After 21 days of the experiment, the mice were killed to obtain tumor tissues.

[0101] Results: Figure 7 As shown, after three weeks of treatment, daily gavage of high-dose Noni fruit fermented juice (Noni-H, 800 mg / kg bw) can significantly inhibit the growth of breast cancer tumors, and there is no significant decrease in the body weight of mice.

[0102] In a preferred embodiment, more specifically, Example 10

[0103] Effects of fermented noni juice on breast cancer tumor proliferation in vivo:

[0104] S9. Fix the tumor tissue with 4% paraformaldehyde solution for 48 hours, soak in 50%, 70%, 85%, 95%, and 100% ethanol for 1 hour for dehydration, then put it in xylene for 45 minutes for permeabilization, and put it in paraffin overnight for waxing. Then embed the waxed tissue in paraffin, cut into 5μm thick tissue sections, and dry them in a 37℃ oven overnight. Before staining, the tissue sections were fixed in a 55℃ oven, soaked in xylene for 5 minutes for dewaxing, and then soaked in 100%, 95%, 70%, 50%, and 30% ethanol for 1 minute for dehydration, placed in a high-temperature antigen repair solution, heated to 95℃ for 20 minutes, cooled to 65℃ at room temperature, and washed with PBST. The slices were soaked in 1% H2O2 to inhibit the activity of endogenous catalase, then washed with PBST, and then soaked in 10% goat serm containing 0.3% Trition-100X for 2 hours. After the blocking, the slices were washed with PBST, and the diluted Ki67 primary antibody working solution was added, and the slices were incubated in a humidified box at 4°C overnight. The samples were placed at room temperature for 15 minutes, the primary antibody working solution was removed, washed with PBST, and the fluorescent secondary antibody working solution was added for 1 hour, and then washed with PBST after incubation. Diluted DAPI working solution was added to the sample to stain the nucleus, incubated for 10 minutes, the DAPI working solution was removed and washed with PBST. Anti-fluorescence attenuation mounting agent was added, and the coverslip was covered to seal the slices, and the images were observed and collected under a fluorescence microscope.

[0105] Results: Figure 8 As shown, high doses of noni fermented juice can significantly inhibit the proliferation of breast cancer cells in vivo.

[0106] In a preferred embodiment, more specifically, Example 11

[0107] Effects of Noni Fermented Juice on Tumor Angiogenesis in Vivo:

[0108] S10. Take paraffin sections of tumor tissue and perform the above-mentioned dehydration and antigen retrieval process, add vascular endothelial cell marker protein CD31 primary antibody working solution and incubate overnight, then add fluorescent secondary antibody and DAPI working solution, seal the sections, and place them under a fluorescence microscope for observation and image acquisition.

[0109] Results: Fig. 9 As shown, high doses of noni fermented juice can significantly inhibit the proliferation of breast cancer tumor blood vessels in vivo.

[0110] In a preferred embodiment, more specifically, Example 12

[0111] Effects of Noni Fermented Juice on Vascular Endothelial Cell Function in Vitro:

[0112] S11. Place the 96-well plate on ice, slowly and evenly add 100 μL of 50% Matrigel, and place at 37°C for 30 min. Add 100 μL of cell suspension containing or without different concentrations of noni fermented juice to each well of the Matrigel surface. Add 2×10 4 cells / well, and cultured at 37°C, 5% CO2 for 4-8 hours. Remove the culture medium and wash with PBS, add 4% PFA for fixation, and observe and photograph the tube formation under a microscope.

[0113] Results: Fig.10 and Fig.11 As shown, the fermented noni juice significantly inhibited the tube formation and invasion ability of vascular endothelial cells HUVEC, and contained fewer factors that promoted the tube formation and invasion ability.

[0114] In a preferred embodiment, more specifically, Example 13

[0115] Combined effects of Noni fruit fermented juice and gemcitabine:

[0116] S12. The drug combination effect analysis software "CompuSyn" developed by the Chou-Talaly median pharmacodynamics mathematical model was used to analyze the combined effect of noni fruit fermented juice and gemcitabine. The drug combination index was calculated in CompuSyn software, and its general formula is:

[0117]

[0118] The combination index formula of the two drugs is:

[0119] In the formula, D is the single dose, D xis the combined dose, and CI is the combined index. The model also involves multiple parameters such as inhibition rate Fa, survival rate Fu, and dose reduction index DRI. The dose-effect relationship data of noni fruit fermented juice and gemcitabine were obtained through MTT experiment, and the equivalent dose comparison and equivalent dose comparison were processed to qualitatively and quantitatively describe the drug combination effect.

[0120] Results: Fig.12 As shown, low concentrations of Noni fruit fermented juice (1, 2 and 4 mg / mL) had a synergistic effect with gradually increasing doses of gemcitabine, indicating that the combination of Noni fruit fermented juice and gemcitabine has a potential anti-breast cancer tumor effect.

[0121] In a preferred embodiment, more specifically, Example 14

[0122] Effect of Noni fruit fermented juice combined with gemcitabine on apoptosis of breast cancer cells:

[0123] S13, human breast cancer cells MDA-MB-231 in logarithmic growth phase were collected and cultured at 2.5×10 5 100 μL of FITC Annexin V and 5 μL of PI were added to each tube, and the cells were stained at room temperature in the dark for 15 minutes, and 100 μL of buffer was added, which was blown evenly and collected in the sample tube of the flow cytometer. The samples were collected and analyzed for fluorescence intensity data by flow cytometry.

[0124] Results: Fig.13 The results showed that low concentrations of noni fruit fermented juice or gemcitabine alone could not significantly induce apoptosis of human breast cancer cells MDA-MB-231, but the combination of the two drugs could significantly induce apoptosis of human breast cancer cells MDA-MB-231.

[0125] In a preferred embodiment, more specifically, Example 15

[0126] Effects of noni fruit fermented juice and gemcitabine combined with medication on vascular endothelial cell function:

[0127] S14. Place the 96-well plate on ice, slowly and evenly add 100 μL of 50% Matrigel, and place at 37°C for 30 min. Add 100 μL of cell suspension containing no or low concentration of noni fruit fermented juice and / or gemcitabine to each well of the Matrigel surface. Add 2×10 4cells / well, and cultured at 37°C, 5% CO2 for 4-8 hours. Remove the culture medium and wash with PBS, add 4% PFA for fixation, and observe and photograph the tube formation under a microscope.

[0128] Results: Fig.14 The results showed that low-concentration gemcitabine alone could not significantly inhibit the tube-forming ability of vascular endothelial cells, but combined with low-concentration fermented noni juice could effectively inhibit the tube-forming and invasion ability of vascular endothelial cells, indicating that the combination of low-concentration fermented noni juice and gemcitabine may exert its anti-breast cancer effect by inhibiting angiogenesis, and low-concentration fermented noni juice can enhance the anti-cancer effect of gemcitabine.

[0129] In summary, the present invention provides a pharmaceutical composition and application for adjuvant treatment of breast cancer. Noni fruit fermented juice is extracted and processed from natural plants, has high food safety, and reduces the side effects that may be caused by chemical synthetic drugs. Noni fruit fermented juice has multiple anti-cancer effects, including cancer cell proliferation, inducing apoptosis, affecting the cell cycle, inhibiting cancer cell migration and invasion, and can inhibit tumor angiogenesis. The anti-cancer effect of Noni fruit fermented juice has a multi-target mechanism of action, which helps to reduce the resistance of breast cancer cells to a single treatment method, and can also assist in improving the therapeutic effect of the chemotherapy drug gemcitabine, and can provide new candidate compounds for the development of new anti-tumor drugs.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition for adjuvant treatment of breast cancer, characterized in that: The pharmaceutical composition comprises freeze-dried powder of fermented noni fruit juice, which is prepared by vacuum freeze-drying technology. The effective dosage of the freeze-dried powder of fermented noni fruit juice in adjuvant treatment of breast cancer is 3-12 mg / mL.

2. The pharmaceutical composition for adjuvant treatment of breast cancer according to claim 1, characterized in that: The dosage of the freeze-dried powder of noni fermented juice in the adjuvant treatment of breast cancer specifically includes: the half inhibition concentration for human breast cancer cells MCF-7 is 15.69 mg / mL, and the half inhibition concentration for human breast cancer cells MDA-MB-231 is 16.09 mg / mL.

3. The pharmaceutical composition for adjuvant treatment of breast cancer according to claim 2, characterized in that: The pharmaceutical composition also includes gemcitabine, and the mass ratio of the freeze-dried powder of Noni fruit fermented juice to gemcitabine is 1:0.5 to 1:

5.

4. The pharmaceutical composition for adjuvant treatment of breast cancer according to claim 3, characterized in that: The method for preparing the freeze-dried powder of Noni fermented juice comprises the following steps: The fermented noni fruit juice is taken, filtered and then packaged; the packaged fermented noni fruit juice is weighed, and pre-frozen at a low temperature of -80°C to partially or completely solidify the fermented noni fruit juice, and then vacuum freeze-dried; the freeze-dried solid is weighed again to calculate the recovery rate of soluble solids; the processed freeze-dried noni fruit juice powder is sealed and packaged, and stored at -20°C for use.

5. The pharmaceutical composition for adjuvant treatment of breast cancer according to claim 4, characterized in that: The soluble solid yield of the freeze-dried powder of Noni fermented juice is 3.0% to 4.0% (w / w).

6. Use of the pharmaceutical composition for adjuvant treatment of breast cancer as claimed in any one of claims 1 to 5 in the treatment of breast cancer.

7. Use of the pharmaceutical composition for adjuvant treatment of breast cancer according to claim 6 in treating breast cancer, characterized in that: The concentration of the noni fruit fermented juice freeze-dried powder for combined administration is 1-8 mg / mL, and the dosage of gemcitabine is 10%-50% of the clinically recommended dosage.

8. Use of the pharmaceutical composition for adjuvant treatment of breast cancer according to any one of claims 1 to 5 in inhibiting angiogenesis in breast cancer tumors.

9. Use of the pharmaceutical composition for adjuvant treatment of breast cancer as claimed in claim 8 in inhibiting angiogenesis of breast cancer tumors, characterized in that: The freeze-dried powder of noni fermented juice is achieved by down-regulating the expression of CD31 protein and inhibiting the tube-forming and invasion capabilities of human umbilical vein endothelial cells.

10. Use of the pharmaceutical composition for adjuvant treatment of breast cancer according to claim 9 in inhibiting angiogenesis of breast cancer tumors, characterized in that: The dosage of the freeze-dried powder of the fermented noni fruit juice is 400-800 mg / kg, which significantly reduces the proportion of Ki67 and CD31 positive cells in tumor tissues.

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