A reagent for inhibiting the proliferation of ovarian cancer cells, its preparation method and application

Abstract

This invention belongs to the field of ovarian cancer cell inhibitor technology, specifically relating to a reagent for inhibiting the proliferation of ovarian cancer cells, its preparation method, and its application. The reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and a decoction of *Rhizophora stylosa* leaves, with a mass ratio of 0.4~0.7:5~10:1000. This invention utilizes vitamin B6 and *Rhizophora stylosa* leaf decoction to enhance the inhibitory effect of gemcitabine on ovarian cancer cells, showing significant killing effects on OVCAR3 and ES-2 cells, while exhibiting weak killing effects on normal human ovarian epithelial cells IOSE-80, suggesting that the reagent of this invention has anti-ovarian cancer activity.

Description

Technical Field

[0001] This invention belongs to the field of ovarian cancer cell inhibitor technology, specifically relating to a reagent for inhibiting the proliferation of ovarian cancer cells, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is a malignant tumor originating from the ovarian epithelium, germ cells, or sex cord-stromal tissue. 85%–90% are epithelial ovarian cancers, with the remainder being germ cell tumors, sex cord-stromal tumors, etc. Early-stage ovarian cancer is often asymptomatic; in advanced stages, symptoms may include abdominal distension, pelvic and abdominal masses, ascites, easy satiety after meals, decreased appetite, weight loss, urinary frequency, urgency, or constipation, menstrual irregularities, and abnormal vaginal bleeding. Current treatments for ovarian cancer include chemotherapy drugs such as paclitaxel and platinum-based drugs, but these are mostly used after early-stage ovarian cancer surgery and often lead to drug resistance, resulting in limited efficacy. Therefore, the development of new therapeutic drugs is necessary.

[0003] The pathogenesis of ovarian cancer is multifactorial. For example, human ovarian adenocarcinoma cells OVCAR3 originate from ascites fluid in patients with high-grade serous ovarian carcinoma and exhibit high chromosomal instability and p53 gene mutations. Human ovarian adenocarcinoma cells ES-2 represent clear cell carcinoma of the ovary and often cause mutations in the PI3K / AKT pathway. The PI3K / AKT pathway, also known as the phosphatidylinositol 3-kinase / protein kinase B signaling pathway, is a phosphatidylinositol-related signal transduction pathway mediated by receptor tyrosine kinases. These factors regulate and participate in various physiological functions, and mutations in the corresponding signaling pathways have a significant impact on the body's physiological functions. Studies have shown that ANXA2 is highly expressed in ovarian cancer cells, and its expression level is significantly higher than that in normal ovarian cells. Inhibiting ANXA2 expression can improve the survival rate of normal ovarian cells.

[0004] Therefore, exploring agents that inhibit the proliferation of ovarian cancer cells based on the above factors is key to the treatment of ovarian cancer. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a reagent for inhibiting the proliferation of ovarian cancer cells, its preparation method, and its application.

[0006] The first objective of this invention is to provide a reagent for inhibiting the proliferation of ovarian cancer cells, which is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4~0.7:5~10:1000.

[0007] Preferably, the preparation method of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells, namely, the decoction of *Rhizophora stylosa* leaves, is as follows: add water equivalent to 8 to 10 times the mass of *Rhizophora stylosa* leaves to the medicinal material, boil for 20 to 30 minutes, filter, and collect the filtrate as the decoction of *Rhizophora stylosa* leaves.

[0008] Preferably, the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells involves filtration using medical gauze or a 100-mesh filter.

[0009] Preferably, the mass ratio of gemcitabine, vitamin B6, and decoction of sheep's foot leaf in the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells is 0.6:5:1000.

[0010] The second objective of this invention is to provide a method for preparing a reagent that inhibits the proliferation of ovarian cancer cells, wherein gemcitabine, vitamin B6, and a decoction of sheep's foot leaves are mixed in a mass ratio of 0.4~0.7:5~10:1000 to obtain the reagent that inhibits the proliferation of ovarian cancer cells.

[0011] A third objective of this invention is to provide an application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells, wherein the application refers to inhibiting the proliferation of human ovarian adenocarcinoma cells OVCAR3 and human ovarian adenocarcinoma cells ES-2.

[0012] Preferably, the selective application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells means that the inhibitory effect of the reagent on human ovarian cancer adenocarcinoma cells OVCAR3 and ES-2 is greater than its inhibitory effect on normal human ovarian epithelial cells IOSE-80.

[0013] Preferably, the application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells refers to the preparation of anti-ovarian cancer drugs using the reagent for inhibiting the proliferation of ovarian cancer cells.

[0014] Preferably, in the application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells, the drug uses the reagent for inhibiting the proliferation of ovarian cancer cells as its sole active ingredient.

[0015] Preferably, in the application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells, the drug further includes pharmaceutically permissible excipients.

[0016] Preferably, in the application of the above-mentioned reagent for inhibiting the proliferation of ovarian cancer cells, the pharmaceutically permissible excipients include solvents, cosolvents, preservatives, antioxidants, surfactants, or stabilizers.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention provides a reagent for inhibiting the proliferation of ovarian cancer cells, composed of the following raw materials: gemcitabine, vitamin B6, and a decoction of *Rhizophora stylosa* leaves, in a mass ratio of 0.4~0.7:5~10:1000. Gemcitabine is a commonly used antitumor drug, vitamin B6 is a water-soluble vitamin with high safety, and the decoction of *Rhizophora stylosa* leaves extracts the water-soluble components of *Rhizophora stylosa* leaves. The decoction of *Rhizophora stylosa* leaves, vitamin B6, and gemcitabine are mixed and administered by injection or gavage, which is convenient. This invention utilizes vitamin B6 and the decoction of *Rhizophora stylosa* leaves to enhance the inhibitory effect of gemcitabine on human ovarian adenocarcinoma cells. It shows significant killing effects on human ovarian adenocarcinoma cells OVCAR3 and ES-2, but weak killing effects on IOSE-80, indicating that the reagent of this invention has selective inhibitory effects on different cell types.

[0019] 2. The reagent developed in this invention can inhibit the expression of ANXA2, suggesting its value in treating ovarian cancer.

[0020] 3. The preparation method of the decoction of *Rhizophora stylosa* leaves provided by this invention is as follows: Add water equivalent to 8 to 10 times the mass of *Rhizophora stylosa* leaves, boil for 20 to 30 minutes, filter, and collect the filtrate as the decoction of *Rhizophora stylosa* leaves. This method can extract various water-soluble active ingredients such as hyperoside from *Rhizophora stylosa* leaves, which helps to exert the anti-ovarian cancer efficacy of the decoction of *Rhizophora stylosa* leaves. Attached Figure Description

[0021] Figure 1 This is a statistical result of the cell mortality rate of human ovarian adenocarcinoma cells OVCAR3.

[0022] Figure 2 The statistical results of cell mortality rate for human ovarian adenocarcinoma cells ES-2.

[0023] Figure 3 Statistical results of cell mortality in normal human ovarian epithelial cells IOSE-80.

[0024] Figure 4 The results of Western Blot analysis show the expression levels of ANXA2. Detailed Implementation

[0025] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0026] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0027] The inventive concept of this invention is as follows:

[0028] A reagent for inhibiting the proliferation of ovarian cancer cells is provided, which is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, with a mass ratio of 0.4~0.7:5~10:1000.

[0029] Gemcitabine is a commonly used anti-tumor drug, but its inhibitory effect on ovarian cancer cells is generally limited when used alone. This invention utilizes vitamin B6 and a decoction of *Rhizophora stylosa* leaves to enhance the inhibitory effect of gemcitabine on ovarian cancer cells. Experiments were conducted on different ovarian cancer cells, different single-factor drugs, and different ratios of gemcitabine, vitamin B6, and *Rhizophora stylosa* leaf decoction. The results showed that the combination of gemcitabine, vitamin B6, and *Rhizophora stylosa* leaf decoction significantly increased the inhibitory effect on ovarian cancer cell proliferation compared to gemcitabine alone, suggesting a synergistic effect. Furthermore, the synergistic effect of these three drugs inhibited ANXA2 expression, indicating potential value in treating ovarian cancer. The results of control groups 4 to 9 showed that when the dosage of vitamin B6, the dosage of the decoction of *Rumex acetosa* leaves, or the dosage of gemcitabine were too high or too low, the killing rate of human ovarian cancer adenocarcinoma cells OVCAR3 and ES-2 was significantly lower than that of the control groups in each example. This indicates that the dosage of vitamin B6, the dosage of decoction of *Rumex acetosa* leaves, and gemcitabine directly affects the anti-ovarian cancer effect of the drug, and the ratio of "0.4~0.7:5~10:1000" is the optimal ratio.

[0030] In addition, vitamin B6 is a water-soluble vitamin with high safety. When used alone in mice, it can inhibit the expression of ANXA2, thereby inhibiting the proliferation of ovarian cancer cells.

[0031] The decoction of *Rumex acetosa* leaves extracts the water-soluble components of the leaves. When mixed with vitamin B6 and gemcitabine, it can be conveniently administered via injection or gavage. Furthermore, the decoction contains numerous active ingredients, such as hyperoside. When used alone, the decoction can slightly inhibit the proliferation of ovarian cancer cells.

[0032] Specific embodiments of the present invention are as follows.

[0033] Example 1

[0034] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:5:1000.

[0035] The preparation method is as follows:

[0036] (1) Preparation of decoction of sheep hoof leaves

[0037] Add water equivalent to 8 times the weight of the Rumex acetosa leaves to the herb, boil for 20 minutes, filter through two layers of medical gauze, and collect the filtrate as the Rumex acetosa leaf decoction.

[0038] (2) Mixing

[0039] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.4:5:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0040] Example 2

[0041] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.6:5:1000.

[0042] The preparation method is as follows:

[0043] (1) Preparation of decoction of sheep hoof leaves

[0044] The preparation method is the same as in Example 1.

[0045] (2) Mixing

[0046] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.6:5:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0047] Example 3

[0048] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.7:5:1000.

[0049] The preparation method is as follows:

[0050] (1) Preparation of decoction of sheep hoof leaves

[0051] The preparation method is the same as in Example 1.

[0052] (2) Mixing

[0053] Gemcitabine, vitamin B6, and a decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.7:5:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0054] Example 4

[0055] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:8:1000.

[0056] The preparation method is as follows:

[0057] (1) Preparation of decoction of sheep hoof leaves

[0058] The preparation method is the same as in Example 1.

[0059] (2) Mixing

[0060] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.4:8:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0061] Example 5

[0062] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:10:1000.

[0063] The preparation method is as follows:

[0064] (1) Preparation of decoction of sheep hoof leaves

[0065] The preparation method is the same as in Example 1.

[0066] (2) Mixing

[0067] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed at a mass ratio of 0.4:10:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0068] Control group 1

[0069] One reagent is gemcitabine of the same mass as in Example 1, with the other components of Example 1 replaced by sterile water.

[0070] The preparation method is as follows:

[0071] Gemcitabine and sterile water were thoroughly mixed to obtain the reagent.

[0072] Control group 2

[0073] One reagent is vitamin B6 of the same mass as in Example 1, with the other components of Example 1 replaced by sterile water.

[0074] The preparation method is as follows:

[0075] Vitamin B6 and sterile water are thoroughly mixed to obtain the reagent.

[0076] Control group 3

[0077] One reagent is a decoction of sheep's hoof leaves of the same mass as in Example 1, with the other components of Example 1 replaced by sterile water.

[0078] The preparation method is as follows:

[0079] (1) Preparation of decoction of sheep hoof leaves

[0080] Add water equivalent to 8 times the weight of the Rumex acetosa leaves to the herb, boil for 20 minutes, filter through two layers of medical gauze, and collect the filtrate as the Rumex acetosa leaf decoction.

[0081] (2) Mixing

[0082] A reagent for inhibiting the proliferation of ovarian cancer cells was obtained by thoroughly mixing sterile water with the decoction of sheep's hoof leaves.

[0083] Control group 4

[0084] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.3:5:1000.

[0085] The preparation method is as follows:

[0086] (1) Preparation of decoction of sheep hoof leaves

[0087] The preparation method is the same as in Example 1.

[0088] (2) Mixing

[0089] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.3:5:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0090] Control group 5

[0091] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.8:5:1000.

[0092] The preparation method is as follows:

[0093] (1) Preparation of decoction of sheep hoof leaves

[0094] The preparation method is the same as in Example 1.

[0095] (2) Mixing

[0096] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed at a mass ratio of 0.8:5:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0097] Control group 6

[0098] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:4:1000.

[0099] The preparation method is as follows:

[0100] (1) Preparation of decoction of sheep hoof leaves

[0101] The preparation method is the same as in Example 1.

[0102] (2) Mixing

[0103] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.4:4:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0104] Control group 7

[0105] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:11:1000.

[0106] The preparation method is as follows:

[0107] (1) Preparation of decoction of sheep hoof leaves

[0108] The preparation method is the same as in Example 1.

[0109] (2) Mixing

[0110] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed at a mass ratio of 0.4:11:1000 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0111] control group 8

[0112] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:5:900.

[0113] The preparation method is as follows:

[0114] (1) Preparation of decoction of sheep hoof leaves

[0115] The preparation method is the same as in Example 1.

[0116] (2) Mixing

[0117] Gemcitabine, vitamin B6, and a decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.4:5:900 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0118] Control group 9

[0119] A reagent for inhibiting the proliferation of ovarian cancer cells is composed of the following raw materials: gemcitabine, vitamin B6, and decoction of sheep's foot leaves, in a mass ratio of 0.4:5:1100.

[0120] The preparation method is as follows:

[0121] (1) Preparation of decoction of sheep hoof leaves

[0122] The preparation method is the same as in Example 1.

[0123] (2) Mixing

[0124] Gemcitabine, vitamin B6, and decoction of sheep's foot leaves were thoroughly mixed in a mass ratio of 0.4:5:1100 to obtain a reagent that inhibits the proliferation of ovarian cancer cells.

[0125] The experimental process and results for inhibiting ovarian cancer are as follows:

[0126] (1) Experimental materials

[0127] 12-week-old female BALB / c nude mice: purchased from Beijing Vital River Anima Co., Ltd.

[0128] Human ovarian cancer adenocarcinoma cells OVCAR3: purchased from Shanghai Yubo Biotechnology Co., Ltd.

[0129] Human ovarian adenocarcinoma cells ES-2: purchased from Beijing Ita Biotechnology Co., Ltd.

[0130] Normal human ovarian epithelial cells IOSE-80: purchased from Shanghai Crown Biotechnology Co., Ltd.

[0131] (2) Cell experiments

[0132] All cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS). The culture conditions were: 37°C and 5% CO2. After culture, the concentration was adjusted to 10% with sterile water. 5 Cell sap per mL.

[0133] Add 100 μL of reagent to each milliliter of cell culture and incubate at 37°C with 5% CO2 for 24 hours. Count the number of viable cells and calculate the cell death rate using the following formula.

[0134] Cell death rate = 100% × (1 - number of surviving cells / original number of cells)

[0135] Use an equal volume of sterile water instead of the reagent as a blank control.

[0136] See results Figures 1-3 . Figure 1 It is the cell death rate of human ovarian adenocarcinoma OVCAR3 cells. Figure 2This refers to the cell death rate of human ovarian adenocarcinoma cells ES-2. The results showed that the reagents in Examples 1-5 had significantly higher killing rates against human ovarian adenocarcinoma cells OVCAR3 and ES-2 than gemcitabine in Control Group 1, and also significantly higher than vitamin B6 in Control Group 2 and the decoction of *Rhizophora stylosa* leaves in Control Group 3. Control Group 2 was the case where vitamin B6 was used alone, and Control Group 3 was the case where the decoction of *Rhizophora stylosa* leaves was used alone. When these two were combined with gemcitabine, they constituted the reagent in Example 1 that showed a significant inhibitory effect on tumor cells, indicating that vitamin B6 and the decoction of *Rhizophora stylosa* leaves can enhance the killing effect of gemcitabine on human ovarian adenocarcinoma cells OVCAR3 and ES-2. Figure 3 It is the cell death rate of normal human ovarian epithelial cells IOSE-80, from Figure 3 It can be seen that the killing rate of the reagents in Examples 1-5 against normal human ovarian epithelial cells IOSE-80 is lower than that of gemcitabine against normal human ovarian epithelial cells IOSE-80. Comparison of cell death rate data for the three cell types shows that Examples 1-5 can selectively kill human ovarian cancer adenocarcinoma cells OVCAR3 and ES-2, while exhibiting weak killing activity against normal human ovarian epithelial cells IOSE-80, indicating a low impact on healthy tissues. Control groups 4 and 5 were those with excessively high or low gemcitabine dosages, control groups 6 and 7 were those with excessively high or low vitamin B6 dosages, and control groups 8 and 9 were those with excessively high or low dosages of the decoction of *Rhizophora stylosa* leaves. The results showed that the killing rates of human ovarian cancer adenocarcinoma cells OVCAR3 and ES-2 in control groups 4 to 9 were significantly lower than those in the example groups. This indicates that the dosages of vitamin B6, *Rhizophora stylosa* leaf decoction, and gemcitabine directly affect the anti-ovarian cancer efficacy of the drug, and the optimal ratio is "0.4~0.7:5~10:1000".

[0137] Human ovarian cancer adenocarcinoma cells OVCAR3 originate from ascites fluid in patients with high-grade serous ovarian carcinoma, while human ovarian cancer adenocarcinoma cells ES-2 originate from clear cell carcinoma of the human ovary. Both cells exhibit a relationship of different origins within the same disease in terms of pathological origin, molecular characteristics, and biological behavior, representing a classic model of different pathological subtypes within the same organ. They also show significant differences in genome, phenotype, and treatment response. For example, human ovarian cancer adenocarcinoma cells OVCAR3 represent the most common type of high-grade serous ovarian carcinoma, characterized by high chromosomal instability and p53 gene mutations. Human ovarian cancer adenocarcinoma cells ES-2, on the other hand, represent clear cell carcinoma of the human ovary, often causing mutations in the PI3K / AKT pathway. The PI3K / AKT pathway is a phosphatidylinositol-related signal transduction pathway mediated by receptor tyrosine kinases. Its core components include phosphatidylinositol 3-kinase and protein kinase B. This pathway participates in various physiological functions by regulating insulin signaling, cell survival, and metabolism; therefore, mutations in this signaling pathway have a significant impact on the body's physiological functions. This invention uses the two cell types mentioned above as research subjects, which can reflect the treatment characteristics of ovarian cancer from different perspectives.

[0138] (3) Mouse experiment

[0139] To investigate the role of exosome-borne ANXA2 in ovarian cancer, this invention established an orthotopic ovarian transplantation tumor model, referencing the paper "Zhang J, Liu H, Wu Q, et al. Exosomal ANXA2 facilitates ovarian cancer peritoneal metastasis by activating peritoneal mesothelial cells through binding with TLR2[J]. Cell communication and signaling: CCS, 2025, 22(1):616". Twelve-week-old female BALB / c nude mice were selected for experimental grouping, and the drug dosage for each group was as follows:

[0140] Model group: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of sterile water was injected.

[0141] Group 1: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of the ES-2-NC cell exosomes, 200 μL of the reagent from Example 1 was injected.

[0142] Group 2: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of the exosomes from ES-2-NC cells, 200 μL of the reagent from Example 2 was injected.

[0143] Group 3: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of the exosomes from ES-2-NC cells, 200 μL of the reagent from Example 3 was injected.

[0144] Group 4: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of the exosomes from ES-2-NC cells, 200 μL of the reagent from Example 4 was injected.

[0145] Group 5: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of the exosomes from ES-2-NC cells, 200 μL of the reagent from Example 5 was injected.

[0146] Group 6: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 1 was injected.

[0147] Group 7: Mice received intraperitoneal injections of exosomes derived from ES-2-NC cells at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were administered every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 2 was injected.

[0148] Group 8: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 3 was injected.

[0149] Group 9: Mice received intraperitoneal injections of exosomes derived from ES-2-NC cells at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were administered every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 4 was injected.

[0150] Group 10: Exosomes from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given once every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 5 was injected.

[0151] Group 11: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of the ES-2-NC cell exosomes, 200 μL of the reagent from control group 6 was injected.

[0152] Group 12: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 7 was injected.

[0153] Group 13: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of exosomes from ES-2-NC cells, 200 μL of the reagent from control group 8 was injected.

[0154] Group 14: Exosomes derived from ES-2-NC cells were injected intraperitoneally at a concentration of 50 μg / 200 μL, with a dosage of 50 μg per mouse. Injections were given every 3 days for a total of 4 injections. Four hours after each injection of the ES-2-NC cell exosomes, 200 μL of the reagent from control group 9 was injected.

[0155] Healthy control group: Intraperitoneal injection of an equal volume of sterile water. No exosomes from ES-2-NC cells were injected.

[0156] See results Figure 4 GAPDH is used as an internal reference, from Figure 4 The results show that Examples 1 to 5 can significantly reduce the expression level of ANXA2.

[0157] Furthermore, existing research shows that ANXA2 is highly expressed in ovarian cancer, especially significantly upregulated in tumor-peritoneal cell interactions, promoting tumor cell adhesion, migration, and invasion. Using ANXA2-specific siRNA or neutralizing antibodies can significantly inhibit the migration ability of ovarian cancer cells and reduce their spread within the peritoneum. Other studies have shown that ANXA2 is involved in reversing the epithelial-mesenchymal transition (EMT), a key mechanism for tumor metastasis. Inhibiting ANXA2 can reverse EMT, reduce the invasive phenotype of tumor cells, and thus inhibit distant metastasis of ovarian cancer. Therefore, ANXA2 can serve as an ideal target for the treatment of ovarian cancer. The reagent developed in this invention can inhibit ANXA2 expression, suggesting its potential value in treating ovarian cancer.

[0158] It should be noted that the above embodiments only provide an exemplary method for preparing a decoction of *Rhizophora stylosa* leaves. In other embodiments, the preparation method of the decoction of *Rhizophora stylosa* leaves can also be as follows: add water equivalent to 8 to 10 times the mass of *Rhizophora stylosa* leaves to the medicinal material, boil for 20 to 30 minutes, filter through 2 to 4 layers of medical gauze, and the collected filtrate is the decoction of *Rhizophora stylosa* leaves. Any value from "8 to 10 times" (e.g., 9 to 10 times), any value from "20 to 30 minutes" (e.g., 25 to 30 minutes), and any value from "2 to 4 layers" (e.g., 3 to 4 layers) can be used to obtain a decoction of *Rhizophora stylosa* leaves suitable for inhibiting the proliferation of ovarian cancer cells. Furthermore, this decoction of *Rhizophora stylosa* leaves, when used alone, has a certain effect on inhibiting the proliferation of ovarian cancer cells. Alternatively, the gauze filtration method can be replaced with filtration through a 100-mesh filter.

[0159] It should be noted that when the reagent that inhibits the proliferation of ovarian cancer cells is used as an anti-ovarian cancer drug, the drug has the reagent that inhibits the proliferation of ovarian cancer cells as its sole active ingredient.

[0160] Preferably, the drug further includes pharmaceutically permissible excipients.

[0161] More preferably, the pharmaceutically permissible excipients include solvents, cosolvents, preservatives, antioxidants, surfactants, or stabilizers. The solvent is sterile water or physiological saline, the preservative is sodium benzoate or potassium sorbate, the antioxidant is ascorbic acid, the surfactant is sodium dodecyl sulfate, and the stabilizer is citric acid.

[0162] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0163] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. An agent that inhibits proliferation of ovarian cancer cells, characterized in that, The reagent for inhibiting proliferation of ovarian cancer cells is prepared from the following raw materials in a mass ratio: gemcitabine, vitamin B6, and water decoction of Rumex japonicus Houtt., 0.4-0.7:5-10:1000. The preparation method of the water decoction of Rumex japonicus Houtt. is as follows: water is added to Rumex japonicus Houtt. at 8 times the mass of the Rumex japonicus Houtt., boiled for 20 min, filtered, and the collected filtrate is the water decoction of Rumex japonicus Houtt.

2. The agent for inhibiting proliferation of ovarian cancer cells according to claim 1, wherein The filtering refers to filtering with medical gauze or filtering with a 100-mesh filter screen.

3. The agent for inhibiting proliferation of ovarian cancer cells according to claim 1, wherein The mass ratio of gemcitabine, vitamin B6, and water decoction of Rumex japonicus Houtt. is 0.6:5:1000.

4. The method for preparing the reagent for inhibiting the proliferation of ovarian cancer cells according to claim 1, characterized in that, The reagent for inhibiting proliferation of ovarian cancer cells is prepared from the following raw materials in a mass ratio: gemcitabine, vitamin B6, and water decoction of Rumex japonicus Houtt., 0.4-0.7:5-10:1000.

5. The use of an agent that inhibits proliferation of ovarian cancer cells according to claim 1, wherein The application refers to a medicine for inhibiting proliferation of human ovarian adenocarcinoma cells OVCAR3 and human ovarian adenocarcinoma cells ES-2.

6. The use of an agent that inhibits proliferation of ovarian cancer cells according to claim 5, wherein The medicine takes the reagent for inhibiting proliferation of ovarian cancer cells as the only active ingredient.

7. The use of an agent that inhibits proliferation of ovarian cancer cells according to claim 5, wherein The medicine further comprises pharmaceutically acceptable adjuvants.

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