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 application relates to the field of medicine, and in particular to a pharmaceutical composition for adjuvant therapy of breast cancer and application thereof. BACKGROUND

[0002] Breast cancer is the most common cancer in women, accounting for about 30% of new cases of female cancer each year. Triple-negative breast cancer (TNBC) is one of the subtypes of breast cancer, referring to breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR) expression and lacks epidermal growth factor receptor 2 (HER2) gene expression. The treatment methods for breast cancer include surgical treatment, radiotherapy, chemotherapy, endocrine therapy and molecular targeted therapy, etc., but chemotherapy is still the main treatment for early and advanced TNBC patients. Compared with patients with other subtypes of breast cancer, the prognosis of TNBC patients is usually poor, mainly because of its invasive phenotype and lack of approved targeted treatment methods. Although chemotherapy has significant effects, it has great side effects and poor quality of life for patients, so the development of effective and relatively safe antitumor drugs is the focus of research on the treatment and prevention of breast cancer. The patent with application number CN115192662A discloses a traditional Chinese medicine composition containing noni fruit as a medicine for treating tumors, the main drugs being ginseng, longan, white birch mushroom, ganoderma lucidum, astragalus, poria cocos, fried white peony root, Chinese yam, and at least two of whiteflower snake tongue grass, wuboteng, and half branch lotus, and the medicine including millet, noni fruit, Chinese yam, ginger, enzyme bacteria and adult scorpion, although it has exact effects on various tumors, but noni fruit is not the main component for treating tumors, and the antitumor efficacy of noni fruit cannot be determined; the patent with application number CN1711097A discloses a method for anti-angiogenesis of noni, 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 efficacy of noni fruit fermented juice on breast cancer, and lacks systematic evaluation of the anti-breast cancer effect of noni fruit fermented juice. Both studies use noni fruit extract juice, and there is no study on the anti-cancer and anti-angiogenesis effect of noni fruit fermented juice. Natural products are often used to improve the antitumor effect of chemotherapy drugs and reduce the side effects of chemotherapy drugs to improve the quality of life of patients, and there is currently no study on the combination of noni fruit fermented juice and chemotherapy drugs for the treatment of breast cancer. SUMMARY

[0003] In view of this, and in response to the shortcomings of the existing technology, this invention proposes a pharmaceutical composition and its application for adjuvant treatment of breast cancer. The objectives are: firstly, to provide a natural and safe adjuvant therapy for breast cancer and reduce the side effects of chemotherapy; secondly, to elucidate the mechanism of action of noni fruit fermented juice in inhibiting breast cancer cell activity and angiogenesis through multiple targets; and thirdly, to develop a combination therapy regimen of noni fruit fermented juice and gemcitabine to improve the efficacy of chemotherapy and reduce drug resistance.

[0004] The technical solution of this invention is implemented as follows: This invention provides a pharmaceutical composition for adjuvant treatment of breast cancer, the pharmaceutical composition comprising freeze-dried powder of fermented noni fruit juice, the freeze-dried powder of fermented noni fruit juice being 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 being 3-12 mg / mL. Based on this technical solution, and further preferably, the effective dose of the freeze-dried noni fruit fermented juice powder in the treatment of breast cancer specifically includes: a half-maximal inhibitory concentration (IC50) of 15.69 mg / mL for human breast cancer cells MCF-7 and a half-maximal inhibitory concentration (IC50) of 16.09 mg / mL for human breast cancer cells MDA-MB-231. Based on this technical solution, and more preferably, the pharmaceutical composition further includes gemcitabine, and the mass ratio of freeze-dried noni fruit fermentation juice powder to gemcitabine is 1:0.5 to 1:5.

[0005] Based on this technical solution, and further preferably, the preparation method of the freeze-dried powder of noni fruit fermented juice includes the following steps: Take fermented noni fruit juice, filter it, and then bottle it. Weigh the bottled fermented noni fruit juice and test it at -80°C. C. Pre-freeze at low temperature to partially or completely solidify the fermented noni fruit juice, then perform vacuum freeze-drying; weigh the freeze-dried solid again to calculate the recovery rate of soluble solids; seal the processed freeze-dried noni fruit juice powder and store it at -20°C. Condition C is pending use. Based on this technical solution, and more preferably, the soluble solids yield of the freeze-dried noni fruit fermentation juice powder is 3.0%~4.0% (w / w). In a second aspect, the present invention also provides the use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any of the first aspects in the treatment of breast cancer. Based on this technical solution, and more preferably, the concentration of the freeze-dried powder of the fermented noni fruit juice used in combination is 1~8 mg / mL, and the dose of gemcitabine is 10%~50% of the clinically recommended dose. Thirdly, the present invention also provides the use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any of the first aspects in inhibiting angiogenesis in breast cancer tumors. Based on this technical solution, and further preferably, the freeze-dried powder of noni fruit fermentation juice achieves its effect by downregulating CD31 protein expression and inhibiting the tube-forming and invasive abilities of human umbilical vein endothelial cells. Based on this technical solution, and further preferably, the dosage of the freeze-dried powder of fermented noni fruit juice is 400~800 mg / kg, which significantly reduces the proportion of Ki67 and CD31 positive cells in tumor tissue. The pharmaceutical composition and its application provided by this invention for adjuvant treatment of breast cancer have the following advantages over the prior art: Noni fruit fermented juice is extracted and processed from natural plants, making it relatively safe for consumption and reducing the side effects of chemically synthesized drugs. It possesses multiple anti-cancer effects, including inhibiting cancer cell proliferation, inducing apoptosis, affecting the cell cycle, inhibiting cancer cell migration and invasion, and suppressing tumor angiogenesis. Furthermore, the anti-cancer effects of noni fruit fermented juice have a multi-target mechanism of action, helping to reduce the resistance of breast cancer cells to single-treatment methods and potentially improving the efficacy of the chemotherapy drug gemcitabine, thus providing new candidate compounds for the development of novel anti-tumor drugs. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0006] Figure 1 This demonstrates the inhibitory effect of fermented noni fruit juice on breast cancer cell activity as described in Example 3 of the present invention. Figure 2 The noveli fruit fermentation juice described in Example 3 of this invention induces apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231). Figure 3 The effect of fermented noni fruit juice on the cell cycle of human breast cancer cells as described in Example 4 of this invention; Figure 4 The effect of Noni fruit fermentation juice as described in Example 5 of this invention on the proliferation ability of human breast cancer cells; Figure 5 The effect of Noni fruit fermentation juice as described in Example 6 of this invention on the migration ability of human breast cancer cells; Figure 6 The effect of Noni fruit fermented juice as described in Example 7 of this invention on the invasive ability of human breast cancer cells; Figure 7 The effect of Noni fruit fermented juice as described in Example 8 of this invention on the growth of breast cancer tumors in vivo; Figure 8 This refers to the effect of Noni fruit fermentation juice as described in Example 9 of the present invention on the proliferation of breast cancer tumors in vivo. Figure 9 The effect of fermented noni fruit juice as described in Example 10 of this invention on angiogenesis in breast cancer tumors in vivo; Figure 10 The effect of noni fruit fermentation juice as described in Example 11 of this invention on the tube-forming ability of vascular endothelial cells in vitro; Figure 11 This describes the effect of Noni fruit fermentation juice as described in Example 12 of the present invention on the invasive ability of vascular endothelial cells in vitro. Figure 12 This study analyzes the combined index of noni fruit fermentation juice and gemcitabine on the activity of MDA-MB-231 breast cancer cells as described in Example 13 of this invention. Figure 13 The effect of the combined use of fermented noni fruit juice and gemcitabine as described in Example 14 of this invention on apoptosis of breast cancer cells; Figure 14 This invention relates to Example 15, which describes the effect of combined use of fermented noni fruit juice and gemcitabine on vascular endothelial cell function. Detailed Implementation

[0007] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0008] Noni fruit is a unique medicinal plant among tropical fruit trees, mainly growing in the South Pacific islands, such as Fiji. Noni fruit is rich in phenolic acids, terpenes, and other components, and experimental studies have demonstrated that it possesses various biological activities, such as anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory effects. This invention provides a pharmaceutical composition and its application as an adjunct therapy for breast cancer. By combining noni fruit fermentation juice with gemcitabine, the anti-breast cancer effect of noni fruit fermentation juice was systematically evaluated. Preliminary findings at the proteomics and genomics levels indicate that noni fruit fermentation juice can regulate signaling pathways associated with tumor angiogenesis and has an anti-angiogenic effect on breast cancer. The combined use of noni fruit fermentation juice and gemcitabine significantly inhibits the growth and angiogenesis of breast cancer cells, potentially enhancing the efficacy of chemotherapy drugs and reducing their side effects. This invention helps elucidate the mechanism of action of noni fruit fermentation juice in treating breast cancer and the efficacy of its combination with chemotherapy drugs, effectively transforming the medicinal value of noni fruit into an anti-cancer drug or adjuvant drug. In a preferred embodiment, a pharmaceutical composition for adjuvant treatment of breast cancer includes lyophilized noni fruit fermented juice powder, which is prepared by vacuum freeze-drying technology, and the effective dose of the lyophilized noni fruit fermented juice powder in the treatment of breast cancer is 3-12 mg / mL. Specifically, the effective dosage of the freeze-dried powder of fermented noni fruit juice in the treatment of breast cancer includes: The half-maximal inhibitory concentration (IC50) against human breast cancer cells MCF-7 was 15.69 mg / mL, and the half-maximal inhibitory concentration (IC50) against human breast cancer cells MDA-MB-231 was 16.09 mg / mL. Specifically, the pharmaceutical composition further includes gemcitabine, and the mass ratio of noni fruit fermented juice freeze-dried powder to gemcitabine is 1:0.5 to 1:5. Specifically, the preparation method of the freeze-dried powder of fermented noni fruit juice includes the following steps: Take fermented noni fruit juice, filter it, and then bottle it. Weigh the bottled fermented noni fruit juice and test it at -80°C. C. Pre-freeze at low temperature to partially or completely solidify the fermented noni fruit juice, then perform vacuum freeze-drying; weigh the freeze-dried solid again to calculate the recovery rate of soluble solids; seal the processed freeze-dried noni fruit juice powder and store it at -20°C. Condition C is pending use.

[0009] Specifically, the soluble solids yield of the freeze-dried noni fruit fermentation juice powder is 3.0%~4.0% (w / w).

[0010] In a preferred embodiment, the pharmaceutical composition described above as an adjunct therapy for breast cancer is also provided for use in the treatment of breast cancer. Specifically, the concentration of the freeze-dried powder of fermented noni fruit juice used in combination is 1-8 mg / mL, and the dose of gemcitabine is 10%-50% of the clinically recommended dose. In a preferred embodiment, the pharmaceutical composition for adjuvant treatment of breast cancer is also provided for the use of inhibiting angiogenesis in breast cancer tumors. Specifically, the freeze-dried powder of fermented noni fruit juice achieves its effect by downregulating CD31 protein expression and inhibiting the tube-forming and invasive abilities of human umbilical vein endothelial cells. Specifically, the dosage of the freeze-dried powder of fermented noni fruit juice is 400-800 mg / kg, which significantly reduces the proportion of Ki67 and CD31 positive cells in tumor tissue. Example 1 The use of a pharmaceutical composition as an adjunct therapy for breast cancer in the treatment of breast cancer includes the following steps: S1. Preparation of freeze-dried noni fruit fermented juice powder: The freeze-dried noni fruit fermented juice is concentrated using vacuum freeze-drying technology. The processed freeze-dried noni fruit fermented juice powder is then sealed and packaged, and stored at -20°C. Condition C is pending use. S2. Inhibitory effect of Noni fruit fermentation juice on breast cancer cell activity: The inhibitory effect of Noni fruit fermentation juice on human breast cancer cells (MCF-7 and MDA-MB-231) was determined by the MTT assay (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide). S3. Effect of Noni Fruit Fermentation Juice on Apoptosis of Breast Cancer Cells: The promoting effect of different concentrations of Noni fruit fermentation juice on cell apoptosis was detected using the Annexin V-FITC / PI cell apoptosis detection kit and flow cytometry. S4. Effects of Noni Fermentation Juice on the Cell Cycle of Breast Cancer Cells: Flow cytometry was used to analyze the effects of different concentrations of Noni fermentation juice on the cell cycle using propidium iodide (PI) DNA staining. S5. Effect of Noni Fruit Fermentation Juice on Breast Cancer Cell Proliferation: Cell proliferation was analyzed by staining and labeling with the cell proliferation protein marker Ki67 and performing flow cytometry. Cells treated with Noni fruit fermentation juice were cultured in single-clone form to assess their proliferative capacity. S6. Effect of Noni Fruit Fermentation Juice on the Migration Ability of Breast Cancer Cells: The cell scratch assay was used to analyze the migration of cells after 24 hours of treatment with Noni fruit fermentation juice. S7. Effect of Noni fruit fermentation juice on the invasive ability of breast cancer cells: Matrigel was applied to a transwell to detect the invasive ability of cells. S8. Effects of Noni Fruit Fermentation Juice on Breast Cancer Tumor Growth in Vivo: A subcutaneous tumor model of MDA-MB-231 cells was established in nude mice. The treatment groups were administered three doses of Noni fruit fermentation juice daily by gavage: 200, 400, and 800 mg / kg bw. The blank control group was administered physiological saline by gavage. After 21 days of experimentation, the mice were sacrificed to obtain tumor tissue. S9. Effects of Noni Fruit Fermentation 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 proportion of cancer cell proliferation in tumor tissues of different treatment groups. S10, Effects of Noni Fruit Fermentation Juice on Tumor Angiogenesis in Vivo: Immunofluorescence staining of tumor tissue sections was used to detect the expression of CD31, a marker protein of vascular endothelial cells, in tumor tissues of different treatment groups. S11. Effects of Noni Fruit Fermentation Juice on In Vitro Vascular Endothelial Cell Function: Matrigel was seeded in Transwell plates to detect the invasive ability of human umbilical vein endothelial cells (HUVECs). Matrigel was seeded in 96-well plates, and HUVECs were inoculated at a certain density and treated with medium or conditioned medium containing different concentrations of Noni fruit fermentation juice for 4-8 hours. Tube formation was observed to assess angiogenesis capacity.

[0011] S12. Combined effect of noni fruit fermentation juice and gemcitabine: The combined anti-breast cancer effect of noni fruit fermentation juice and gemcitabine was analyzed using the dose-effect analysis software "CompuSyn" developed by the Chou-Talaly median pharmacodynamic model. S13. Effects of S13 noni fruit fermentation juice and gemcitabine combined on apoptosis of breast cancer cells: The promoting effect of low concentrations of noni fruit fermentation juice and / or gemcitabine on apoptosis was detected by Annexin V-FITC / PI apoptosis detection kit and flow cytometry. S14. Effects of Noni Fermentation Juice and Gemcitabine Combination on Vascular Endothelial Cell Function: Matrigel was seeded in Transwell plates to detect the invasive ability of human umbilical vein endothelial cells (HUVECs). Matrigel was seeded in 96-well plates, HUVECs were inoculated at a certain density, and treated with a low concentration of noni fermentation juice and / or gemcitabine for 4-8 hours. Tube formation was observed to assess angiogenesis capacity. In a preferred embodiment, more specifically, Example 2 The preparation of freeze-dried powder from fermented noni fruit juice includes the following steps: S1. Take the fermented noni fruit juice, filter out solid residue with filter paper, and dispense it in a laminar flow hood. Weigh the dispensed juice and sterilize it at -80°C. Pre-freezing was performed at a low temperature (C) to partially or completely solidify the fruit, followed by vacuum freeze-drying. The freeze-dried solid was weighed again to calculate the recovery rate of soluble solids. The processed freeze-dried noni fruit juice powder was then sealed and stored at -20°C. Condition C is pending use.

[0012] Table 1. Yield of soluble solids from freeze-dried concentration of Noni fruit fermentation juice

[0013] Results: As shown in Table 1, a total of 20 batches of extraction and concentration were carried out, and the yield of soluble solids obtained is shown in Table 1. The average soluble solids content of the noni fruit fermentation juice was 3.678 ± 0.154%.

[0014] In a preferred embodiment, more specifically, Example 3 The study on the effect of fermented noni fruit juice on breast cancer cell activity involved the following steps: S2. Take the freeze-dried powder of noni fruit fermentation juice, prepare a fruit pulp of a certain concentration with DMEM culture medium, and perform serial dilution. Sterilize by filtration through a 0.22 µm microporous filter, and then... Store at C for later use. Human breast cancer cells (MCF-7 and MDA-MB-231) in logarithmic growth phase were seeded at 5000 cells per well in 96-well culture plates and incubated at 37°C. C. Cells were cultured in a 5% CO2 incubator for 24 hours. After cell attachment, the experimental groups were treated with different concentrations of noni fruit fermentation juice, the experimental control group was treated with DMEM medium without noni fruit fermentation juice, and the blank control group contained no cells. Each concentration was repeated in 6 replicates and placed at 37°C. C. Incubate in a 5% CO2 incubator for 24 hours. After incubation, add 10 µL of MTT (5 mg / mL) to each well and incubate at 37°C. C. Incubate in a 5% CO2 incubator for 4 hours. Remove the supernatant, add 100 µL of dimethyl sulfoxide (DMSO) to each well, shake horizontally for 10 seconds, and measure the absorbance at 490 nm using a microplate reader.

[0015] The inhibitory effects of the experimental and control drugs on the cell activity of human breast cancer cells (MCF-7 and MDA-MB-231) were calculated using the following formula.

[0016] Cell viability (%) = [(absorbance value of drug treatment group - absorbance value of blank control group) / (absorbance value of cell control group - absorbance value of blank control group)] * 100%. Results: The experimental results are as follows Figure 1The results showed that after 24 hours of treatment, the IC50 (half-maximal inhibitory concentration) of the fermented noni fruit juice of this invention against human breast cancer cells MCF-7 was 15.69 mg / mL. Experimental Results Figure 2 The results showed that the IC50 (half-maximal inhibitory concentration) of fermented noni fruit juice against human breast cancer cells MDA-MB-231 was 16.09 mg / mL. In a preferred embodiment, more specifically, Example 4 The study on the effect of noni fruit fermentation juice on breast cancer cell apoptosis involves the following steps: S3. Human breast cancer cells (MCF-7 and MDA-MB-231) in logarithmic growth phase were harvested at a concentration of 2.5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. Experimental groups were treated with low, medium, and high concentrations (3, 6, and 12 mg / mL) of noni fruit fermentation juice, while the control group received culture medium without noni fruit fermentation juice. After 24 hours of 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 the mixture was thoroughly mixed. 5 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. 100 µL of buffer was added, and the mixture was thoroughly mixed. The samples were collected in flow cytometry tubes, and fluorescence intensity data were collected and analyzed. Result: As Figure 2 The results showed that fermented noni fruit juice could significantly induce apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231), and the proportion of apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) gradually increased with the increase of noni fruit fermented juice concentration. In a preferred embodiment, more specifically, Example 5 The study on the effects of fermented noni fruit juice on the cell cycle of breast cancer cells involved the following steps: S4. Human breast cancer cells (MCF-7 and MDA-MB-231) at different logarithmic growth phases were divided into low, medium, and high concentration groups and a blank control group. After different treatments, all cells (adherent and suspension cells) were collected in 15 mL centrifuge tubes and washed once with PBS. Each group was resuspended in 1 mL of pre-chilled PBS and thoroughly mixed. 5 mL of 70% ice-cold ethanol was slowly added for fixation, and the cells were incubated for 4 days. Incubate overnight at 4°C. Wash with pre-cooled PBS before staining. Centrifuge at 3000 rpm for 5 min at C, remove fixative, add 50 µL RNase A, propidium iodide (PI), and 0.3% Triton X-100, and stain at room temperature in the dark for 45 min. After staining, wash once with 300 µL PBS, collect cells in flow cytometry tubes, and perform cell cycle analysis to obtain the percentage of each cell cycle phase (G1, G2, S1, and M phases). Result: As Figure 3 The results showed that noni fruit fermentation juice effectively inhibited the normal cell cycle transition by reducing G2 / M proliferating cells and increasing G0 / G1 arrested cells, causing MCF-7 cells to accumulate in the G1 phase, preventing cell mitosis, inhibiting cell proliferation, and thus achieving anti-human breast cancer cell activity.

[0017] In a preferred embodiment, more specifically, Example 6 The study on the effects of fermented noni fruit juice on breast cancer cell proliferation involved the following steps: S5. Human breast cancer cells (MCF-7 and MDA-MB-231) in different logarithmic growth phases were divided into low, medium, and high concentration groups (3, 6, and 12 mg / mL) and a blank control group. After different treatments, all adherent cells were collected in 15 mL centrifuge tubes, washed once with PBS, and fixed and permeabilized with Fixation / Permeabilization Solution on ice for 30-60 min. After washing once with Perm / Wash Buffer, the cells were centrifuged and the supernatant was discarded. Ki67 antibody was added for staining in the dark for 15-30 min. After staining, the cells were washed with Perm / Wash Buffer and collected in flow cytometry tubes for cell cycle analysis. Human breast cancer cells (MCF-7 and MDA-MB-231) at different logarithmic growth phases were seeded at 1000 cells per well in 6-well plates, divided into low, medium, and high concentration groups and a blank control group. Each group was treated with different concentrations of noni fruit fermentation juice for 24 hours. After treatment, all experimental groups were replaced with DMEM medium without noni fruit fermentation juice and cultured in an incubator for 1-2 weeks. Culture was terminated when visible clones appeared in the experimental groups. The supernatant was discarded, and the cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde for 15 min, the fixative was removed, and the cells were washed once with PBS. Crystal violet staining solution was added for 1 h. After staining, the staining solution was slowly washed away with running water, and the cells were air-dried at room temperature. Each group was photographed with a digital camera, and the number of cloned cells was counted. Result: As Figure 4As shown in ab, high concentrations of noni fruit fermentation juice effectively inhibited the proliferation of MDA-MB-231 cells, but its inhibitory effect on the proliferation of MCF-7 cells was not significant. Figure 4 As shown in cd, medium concentrations of noni fruit fermentation juice can reduce the number of MDA-MB-231 cell clones, while high concentrations of noni fruit fermentation juice can reduce the number of both MDA-MB-231 and MCF-7 cell clones. In a preferred embodiment, more specifically, Example 7 The study on the effect of fermented noni fruit juice on the migration ability of breast cancer cells involved the following steps: S6. Different human breast cancer cells (MCF-7 and MDA-MB-231) were seeded at 100% density in 6-well plates. Gaps were created in the cell monolayer using a 1 mL or 200 µL pipette tip. The supernatant was discarded, and the cells were washed 2-3 times with PBS to remove the scraped cells. Serum-free medium containing different concentrations of noni fruit fermentation juice and no noni fruit fermentation juice was added to each well, and the plates were incubated at 37°C. C. Incubate under 5% CO2 for 24 hours, and take images under a microscope at 0h and 24h to observe changes in cell migration. Result: As Figure 5 As shown, fermented noni fruit juice can significantly inhibit the migration ability of human breast cancer cells MCF-7 and MDA-MB-231. In a preferred embodiment, more specifically, Example 8 The study on the ability of fermented noni fruit juice to inhibit the invasion of breast cancer cells involved the following steps: S7. Human breast cancer cells (MCF-7 and MDA-MB-231) in different logarithmic growth phases were suspended in serum-free DMEM medium, and 2.5 × 10⁻⁶ cells were added to each well. 4 Cells were cultured in the upper chamber of a 24-well transwell plate, coated with 3% Matrigel. The lower chamber contained 800 µL of medium containing low, medium, and high concentrations of noni fruit fermentation juice, as well as medium without noni fruit fermentation juice. The culture was kept at 37°C. C. After culturing for 24 hours under 5% CO2 conditions, discard the upper chamber medium and fix the cells with 4% PFA at room temperature for 2 hours. After fixation, rinse both chambers with PBS, and stain the cells on the surface of the upper chamber with crystal violet. After staining, slowly rinse the transwell with tap water and incubate at 37°C. The cells were dried at C20°C, and then imaged using a microscope. The cell count was calculated using ImageJ software. Result: As Figure 6As shown, low, medium and high concentrations of noni fruit fermentation juice can significantly inhibit the invasive ability of human breast cancer cells MCF-7 and MDA-MB-231, and the inhibitory effect on invasive ability is more obvious as the concentration of noni fruit fermentation juice increases. In a preferred embodiment, more specifically, Example 9 The effect of fermented noni fruit juice on the growth of breast cancer tumors in the body: S8. Human breast cancer cells MDA-MB-231 were subcutaneously inoculated into the axillary region of 30 female nude mice. The treatment groups were administered noni fruit fermented juice by gavage at doses of 200, 400, and 800 mg / kg bw daily, respectively, while the blank control group was administered physiological saline by gavage. After 21 days of the experiment, the mice were sacrificed to obtain tumor tissue. Result: As Figure 7 As shown, after three weeks of treatment, daily gavage administration of high-dose Noni fermented juice (Noni-H, 800 mg / kg bw) significantly inhibited the growth of breast cancer tumors, and the mice did not experience a significant decrease in body weight. In a preferred embodiment, more specifically, Example 10 The effect of fermented noni fruit juice on the proliferation of breast cancer tumors in vivo: S9. Tumor tissue was fixed with 4% paraformaldehyde solution for 48 hours, then dehydrated by soaking in 50%, 70%, 85%, 95%, and 100% ethanol for 1 hour each, followed by permeation in xylene for 45 minutes, and then paraffin infiltration overnight. The paraffin-infiltrated tissue was then embedded in paraffin and sectioned into 5 µm thick tissue sections, which were then incubated at 37°C. Dry overnight in a C oven. Place tissue sections at 55°C before staining. The antigen was fixed in a C oven, dewaxed by soaking in xylene for 5 minutes, and then dehydrated by soaking in 100%, 95%, 70%, 50%, and 30% ethanol for 1 minute each. Finally, it was placed in a high-temperature antigen retrieval solution and heated to 95°C. Continue at C for 20 minutes, then cool to 65°C from room temperature. C. Wash with PBST. Immerse the sections in 1% H2O2 to inhibit endogenous catalase activity, then wash with PBST, and then block with 10% goat serum containing 0.3% Tritium-100X for 2 hours. After blocking, wash with PBST, add diluted Ki67 primary antibody working solution, and place the sections in a humidified chamber for 4 hours. Incubate overnight at C. Warm the sample to room temperature for 15 min, remove the primary antibody working solution, wash with PBST, add fluorescent secondary antibody working solution and incubate for 1 h, then wash with PBST. Add diluted DAPI working solution to the sample for nucleus staining, incubate for 10 min, remove the DAPI working solution and wash with PBST. Add anti-fluorescence attenuation mounting medium, cover with a coverslip, and observe and acquire images under a fluorescence microscope.

[0018] Result: As Figure 8 As shown, high doses of fermented noni fruit juice can significantly inhibit the proliferation of breast cancer cells in the body. In a preferred embodiment, more specifically, Example 11 The effect of fermented noni fruit juice on tumor angiogenesis in vivo: S10. Take paraffin sections of tumor tissue and perform the above dehydration and antigen retrieval process. Add CD31 primary antibody working solution and incubate overnight. Then add fluorescent secondary antibody and DAPI working solution, mount the slide, and observe and acquire images under a fluorescence microscope. Result: As Figure 9 As shown, high doses of fermented noni fruit juice can significantly inhibit the proliferation of blood vessels in breast cancer tumors in the body. In a preferred embodiment, more specifically, Example 12 Effects of Noni fruit fermentation juice on in vitro vascular endothelial cell function: S11. Place the 96-well plate on ice and slowly and evenly add 100 µL of 50% Matrigel. Incubate at 37°C. At C, for 30 min. Add 100 µL of cell suspension (containing or without different concentrations of noni fruit fermentation juice) to the Matrigel surface in each well, and add 2 × 10⁻⁶ cells per well. 4 One cell / well, placed at 37°C C. Incubate under 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. Result: As Figure 10 and Figure 11 As shown, fermented noni fruit juice significantly inhibits the tube-forming and invasive abilities of vascular endothelial cells (HUVECs) and contains fewer factors that promote tube-forming and invasive abilities. In a preferred embodiment, more specifically, Example 13 The combined effects of noni fruit fermentation juice and gemcitabine: S12. This study used the CompuSyn software, developed using the Chou-Talaly median pharmacodynamic model, to analyze the combined effects of noni fruit fermentation juice and gemcitabine. The drug combination index was calculated in CompuSyn software, with the general formula as follows: .

[0019] The formula for the combined index of the two drugs is:

[0020] In the formula, D is the single-use dose, D x The combined dose is represented by CI, and the combination index is used for comparison. The model also involves multiple parameters such as inhibition rate (Fa), survival rate (Fu), and dose reduction index (DRI). Dose-response relationship data of noni fruit fermentation juice and gemcitabine were obtained through MTT assays, and equivalent dose-response and equivalent dose comparisons were performed to qualitatively and quantitatively describe the combined drug effects. Result: As Figure 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, suggesting that the combination of noni fruit fermented juice and gemcitabine has potential anti-breast cancer tumor activity. In a preferred embodiment, more specifically, Example 14 The effect of combined use of fermented noni fruit juice and gemcitabine on apoptosis in breast cancer cells: S13. Take human breast cancer cells MDA-MB-231 in the logarithmic growth phase and use 2.5 × 10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates. The experimental groups were treated with a low concentration of noni fruit fermentation juice and / or gemcitabine, while the control group received blank culture medium. After 24 hours of 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 the mixture was thoroughly mixed. 5 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. 100 µL of buffer was added, and the mixture was thoroughly mixed and collected in flow cytometry tubes. Fluorescence intensity data were collected and analyzed using flow cytometry. Result: As Figure 13 The results showed that low concentrations of noni fruit fermentation juice or gemcitabine alone could not significantly induce apoptosis in human breast cancer cells MDA-MB-231, but the combination of the two drugs could significantly induce apoptosis in human breast cancer cells MDA-MB-231. In a preferred embodiment, more specifically, Example 15 Effects of Noni fruit fermentation juice combined with gemcitabine on vascular endothelial cell function: S14. Place the 96-well plate on ice and slowly and evenly add 100 µL of 50% Matrigel. Incubate at 37°C. At C for 30 min. Add 100 µL of cell suspension containing noni fruit fermentation juice and / or gemcitabine to the surface of each Matrigel, and add 2 × 10⁻⁶ cells per well. 4 Cells / well, placed at 37°C C. Incubate under 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. Result: As Figure 14 The study showed that low concentrations of gemcitabine alone could not significantly inhibit the tube-forming ability of vascular endothelial cells, but the combination with low concentrations of noni fruit fermented juice could effectively inhibit the tube-forming and invasive abilities of vascular endothelial cells. This suggests that the combined use of low concentrations of noni fruit fermented juice and gemcitabine may exert its anti-breast cancer effect by inhibiting angiogenesis, and that low concentrations of noni fruit fermented juice can enhance the anti-cancer effect of gemcitabine.

[0021] In summary, this invention provides a pharmaceutical composition and its application for adjuvant therapy of breast cancer. Noni fruit fermented juice is extracted and processed from a natural plant, exhibiting high food safety and reducing potential side effects from chemically synthesized drugs. Noni fruit fermented juice possesses multifaceted anti-cancer effects, including inhibiting cancer cell proliferation, inducing apoptosis, affecting the cell cycle, suppressing cancer cell migration and invasion, and inhibiting tumor angiogenesis. The anti-cancer effects of noni fruit fermented juice have a multi-target mechanism of action, helping to reduce the resistance of breast cancer cells to single-treatment methods, while also enhancing the therapeutic effect of the chemotherapy drug gemcitabine, and providing new candidate compounds for the development of novel anti-tumor drugs.

[0022] 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 within the protection scope of the present invention.

Claims

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

2. The pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 1, characterized in that, The specific dosage of the freeze-dried noni fruit fermented juice powder in adjuvant therapy for breast cancer includes: a half-maximal inhibitory concentration (IC50) of 15.69 mg / mL for human breast cancer cells MCF-7 and a half-maximal inhibitory concentration (IC50) of 16.09 mg / mL for human breast cancer cells MDA-MB-231.

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

5.

4. The pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 3, characterized in that, The method for preparing the freeze-dried powder of fermented noni fruit juice includes the following steps: Take fermented noni fruit juice, filter it, and then bottle it. Weigh the bottled fermented noni fruit juice and test it at -80°C. C. Pre-freeze at low temperature to partially or completely solidify the fermented noni fruit juice, then perform vacuum freeze-drying; weigh the freeze-dried solid again to calculate the recovery rate of soluble solids; seal the processed freeze-dried noni fruit juice powder and store it at -20°C. Condition C is pending use.

5. The pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 4, characterized in that, The soluble solids yield of the freeze-dried noni fruit fermentation juice powder is 3.0%~4.0% (w / w).

6. The use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any one of claims 1-5 in the treatment of breast cancer.

7. The application of the pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 6 in the treatment of breast cancer, characterized in that, The concentration of the freeze-dried powder of fermented noni fruit juice used in combination is 1-8 mg / mL, and the dose of gemcitabine is 10%-50% of the clinically recommended dose.

8. The use of the pharmaceutical composition for adjuvant treatment of breast cancer as described in any one of claims 1-5 in inhibiting angiogenesis in breast cancer tumors.

9. The application of the pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 8 in inhibiting angiogenesis in breast cancer tumors, characterized in that, The freeze-dried powder of fermented noni fruit juice achieves its effect by downregulating CD31 protein expression and inhibiting the tube-forming and invasive abilities of human umbilical vein endothelial cells.

10. The application of the pharmaceutical composition for adjuvant treatment of breast cancer as described in claim 9 in inhibiting angiogenesis in breast cancer tumors, characterized in that, The dosage of the freeze-dried powder of fermented noni fruit juice was 400-800 mg / kg, which significantly reduced the proportion of Ki67 and CD31 positive cells in tumor tissue.

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