Use of leaf alcohol in preparation of drugs for urinary system tumors

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to application of leaf alcohol in preparation of urinary system tumor medicine. Through animal experiment verification, the leaf alcohol has a significant improvement effect on tumors caused by human bladder cancer cells, and can be used for treatment of urinary system tumors. The application first determines that the leaf alcohol has an inhibitory effect on proliferation, migration ability and invasion ability of urinary system tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to the application of leaf alcohol in the preparation of drugs for urinary system tumors. Background Technology

[0002] A tumor is a new growth formed by the abnormal proliferation of cells in a local tissue under the influence of various carcinogenic factors, often manifesting as a local mass. Tumor cells have abnormal morphology and metabolic function, grow rapidly, and often exhibit continuous growth.

[0003] Urinary system tumors refer to tumors occurring in any part of the urinary system, including tumors of the kidneys, renal pelvis, ureters, bladder, and urethra. The organs below the renal pelvis are tubular organs, all lined with urothelial tissue, and their internal environment is urine. Carcinogens often cause tumors of the urothelial tissue through urine; therefore, urothelial tumors of the renal pelvis, ureters, bladder, and urethra share common characteristics and can affect multiple organs. Because urine stays in the bladder the longest, bladder cancer is the most common type of urinary tract cancer caused by it. Clear cell renal cell carcinoma is an adenocarcinoma originating from renal tubular epithelial cells. It often involves hemorrhage, necrosis, cystic degeneration, and calcification. It grows within the renal parenchyma, and as it matures, it infiltrates, compresses, and destroys the renal pelvis and calyces, extending beyond the renal capsule to form hemangioma emboli or metastasize to lymph nodes and other organs. Currently, the treatment for bladder cancer or clear cell renal cell carcinoma is primarily surgical, with radiotherapy, chemotherapy, and biotherapy playing adjunctive roles. Drug therapy options are limited, and most are expensive.

[0004] Cis-3-Hexen-1-ol, also known as cis-3-hexenol or β,γ-hexenol, has been found in plants such as tea, locust, radish, strawberry, and grapefruit. Patent CN116655455A discloses a method for synthesizing cis-3-hexenol using crotonaldehyde and acetaldehyde as raw materials. The raw materials are readily available, the method is simple, and the reaction conditions are mild. Currently, there are no reports on the application of cis-3-hexen-1-ol in inhibiting tumor cells. Summary of the Invention

[0005] Exploring and developing new drugs to treat urinary system tumors is one of the important tasks in modern medicine. The purpose of this invention is to clarify the inhibitory effect of leaf alcohol or its pharmaceutically acceptable salts on urinary system tumor cells, and to provide a potential new drug candidate for the clinical treatment of urinary system tumors.

[0006] To achieve the above objectives, this invention verified the role of leaf alcohol in the treatment of urinary tract tumors. The structural formula of leaf alcohol is:

[0007] First, this invention seeks protection for the use of leaf alcohol or a pharmaceutically acceptable salt thereof in the preparation of urinary system tumor drugs.

[0008] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic and organic acid addition salt of the compounds of the present invention. These salts can be prepared in situ during the application mordant or dosage form manufacturing process, or by independently reacting a purified compound of the present invention in its free form with a suitable organic or inorganic acid, and then separating the resulting salt during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, hydrogen sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthate, methanesulfonate, gluconate, lactobionate, and laurylsulfonate, etc. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

[0009] Based on the application described in this invention, the leaf alcohol or its pharmaceutically acceptable salt inhibits the activity of human clear cell adenocarcinoma cells or human bladder cancer cells.

[0010] Specifically, the leaf alcohol or a pharmaceutically acceptable salt thereof is used for the treatment of human clear cell carcinoma of the kidney or human bladder cancer.

[0011] Furthermore, the leaf alcohol or a pharmaceutically acceptable salt thereof has an inhibitory effect on human clear cell adenocarcinoma cells of the kidney or human bladder cancer cells.

[0012] Furthermore, the concentration range of the leaf alcohol is 0–10 μM, and the inhibitory effect on the activity of human renal clear cell adenocarcinoma cells or human bladder cancer cells within a 24-hour treatment period is positively correlated with the concentration of the leaf alcohol.

[0013] Furthermore, the IC50 of the leaf alcohol for inhibiting the activity of human renal clear cell adenocarcinoma cells is 2.337 μM, and the IC50 of the leaf alcohol for inhibiting the activity of human bladder cancer cells is 2.117 μM.

[0014] Specifically, leaf alcohol inhibits the proliferation, migration, and invasion of human renal clear cell adenocarcinoma cells or human bladder cancer cells.

[0015] The inventors of this patent discovered that leaf alcohol has a significant improving effect on xenograft tumor models established subcutaneously in nude mice using human bladder cancer cells, indicating that leaf alcohol is expected to provide a new candidate drug for the clinical treatment of urinary system tumors.

[0016] Based on research findings, this invention seeks protection for a medicament for inhibiting urinary system tumors, the active ingredient of which comprises the aforementioned leaf alcohol or a pharmaceutically acceptable salt thereof.

[0017] Specifically, this invention claims protection for a medicament that has therapeutic effects on human clear cell renal cell carcinoma or human bladder cancer. The active ingredient of the medicament includes the aforementioned leaf alcohol or a pharmaceutically acceptable salt thereof.

[0018] Furthermore, the drug components also include pharmaceutically acceptable carriers.

[0019] This invention utilizes leaf alcohol or its pharmaceutically acceptable salts to treat urinary system tumors. The drug is composed of an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is, for example, an organic or inorganic solid or liquid excipient suitable for oral and parenteral administration. The drug of this invention is prepared in the form of conventional pharmaceutical preparations, preferably, tablets, pills, capsules, powders, granules, etc. The above-mentioned pharmaceutical preparations may contain wetting agents, stabilizers, excipients, and other commonly used additives, such as olive oil, corn starch, citric acid, stearic acid, ethylene glycol, lactose, gypsum powder, magnesium stearate, ascorbic acid, gelatin, agar, etc. The pharmaceutical preparations of this invention can be prepared according to various conventional preparation processes.

[0020] As a preferred pharmaceutical method, those skilled in the art typically process a biologically active compound or its pharmaceutically acceptable salt into a pharmaceutical formulation using a pharmaceutically acceptable carrier. For the preparation of a urinary system tumor drug using the leaf alcohol or its pharmaceutically acceptable salt described in this invention, pharmaceutically feasible dosage forms include capsules, tablets, powders, or granules. Those skilled in the art should understand that, in addition to containing leaf alcohol or its pharmaceutically acceptable salt, the components of such a drug also include pharmaceutically acceptable carriers or excipients, such as olive oil, corn starch, citric acid, stearic acid, ethylene glycol, lactose, gypsum powder, magnesium stearate, ascorbic acid, gelatin, agar, etc.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0022] (1) This invention is the first to verify and clarify the inhibitory effect of leaf alcohol on urinary system tumor cells.

[0023] Leaf alcohol can inhibit the activity of human renal clear cell adenocarcinoma cells and human bladder cancer cells, and the inhibitory effect increases with the increase of leaf alcohol concentration.

[0024] (2) Leaf alcohol effectively inhibited the viability of both cell types under the conditions of 2 μM and 24 h. The results of the scratch assay and Transwell assay showed that leaf alcohol could significantly inhibit the migration and invasion ability of urinary system tumor cells.

[0025] (3) By establishing a xenograft tumor model and observing tumor growth, it was found that the tumor volume treated with leaf extract was significantly smaller than that in the control group, indicating that leaf extract can significantly inhibit the growth of xenograft tumors. In animal in vivo experiments, leaf extract was found to have a significant inhibitory effect on human bladder cancer cells. This demonstrates the therapeutic effect of leaf extract on urinary system tumors and is expected to provide a new candidate drug for the clinical treatment of urinary system tumors. Attached Figure Description

[0026] Figure 1 The results of cell viability analysis for two urinary tract tumor cell lines, 7860 and T24, after treatment with different concentrations of leaf extract for 24 hours were statistically analyzed. Cell viability represents cell viability, and IC50 represents the concentration of leaf extract at which the corresponding cancer cell viability is reduced to half. *p<0.05, **p<0.01.

[0027] Figure 2 The results of 7860 and T24 cells after treatment with leaf alcohol are shown in the clonogenic assay. NC represents the control group, and leaf alcohol represents the experimental group.

[0028] Figure 3 The results of colony counts for 7860 and T24 cells in the colony formation assay are shown. In the colony count, Number of colonies represents the number of cell colonies, NC is the control group, and Yedan is the experimental group. n = 3, *p < 0.05, **p < 0.01.

[0029] Figure 4 The results show the fluorescence detection results of the experimental group (leaf alcohol) and the control group (NC) of 7860 in the EdU experiment. Among them, DAPI and EdU represent the staining results of each fluorescent dye; Merge represents the staining results of the mixed dye DAPI and EdU.

[0030] Figure 5 The results show the fluorescence detection results of the experimental group (leaf alcohol) and the control group (NC) of T24 in the EdU experiment. Among them, DAPI and EdU represent the staining results of different fluorescent dyes; Merge represents the staining results of a mixture of DAPI and EdU dyes.

[0031] Figure 6 The statistical results of cell proliferation rate for 7860 and T24 cells in the EdU experiment are shown. Cell proliferation rate represents the cell growth rate; NC is the control group; and leaf alcohol is the experimental group. n = 3; *p < 0.05; **p < 0.01.

[0032] Figure 7The staining results for the 7860 and T24 migration and invasion assays in the Transwell assay are shown. A(Invasion) represents the staining results for the invasion assay, and B(Migration) represents the staining results for the migration assay; NC represents the control group, and leaf alcohol represents the experimental group.

[0033] Figure 8 This section presents the statistical results of cell numbers in the Transwell assays for migration and invasion. In this section, Invasion represents the invasion assay result, Migration represents the migration assay result, Count represents the number of cells, NC represents the control group, and leaf alcohol represents the experimental group. n = 3, *p < 0.05, **p < 0.01.

[0034] Figure 9 The results are from the cell scratch assay. NC represents the control group, and leaf alcohol represents the experimental group.

[0035] Figure 10 The results show the statistical results of wound healing rate in the cell scratch assay. Wound healing represents the wound healing rate, NC is the control group, and leaf alcohol is the experimental group. n = 3, *p < 0.05, **p < 0.01.

[0036] Figure 11 Gross specimens of subcutaneous xenografts from nude mice in the leaf alcohol treatment group (5 mg / kg and 10 mg / kg), the DMSO group, and the control group (NC).

[0037] Figure 12 The results show the statistical results of weighing subcutaneous xenografts. *p<0.05, **p<0.01. Detailed Implementation

[0038] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially; unless otherwise specified, the index data are conventional measurement methods.

[0039] To illustrate the efficacy of leaf alcohol in the treatment of urinary system tumors, the inventors used human clear cell adenocarcinoma cells of the kidney (7860) and human bladder cancer cells (T24) as experimental materials. The specific experimental procedures and results are described below. Both 7860 and T24 cells mentioned in the following examples were purchased from Abiowell. RPMI 1640 culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

[0040] Example 1

[0041] This embodiment describes the statistical effects of different concentrations of leaf alcohol on the viability of human renal clear cell adenocarcinoma cells (7860) and human bladder cancer cells (T24).

[0042] RPMI 1640 medium was used for the culture of two cell lines, 7860 and T24.

[0043] The culture system consisted of 1% penicillin-streptomycin (100X) (SP, Suzhou Xinsaimei Biotechnology Co., Ltd.) and 10% fetal bovine serum (FBS, purchased from Biosciences) added to 1640 medium. T24 and 7860 cells were cultured in 6cm culture dishes with a carbon dioxide concentration of 5%, relative humidity of 95%, and a culture temperature of 37℃. The cell culture medium was changed every 1–2 days. Once the cells had grown to cover 80%–90% of the culture dish area, they were passaged.

[0044] Cell viability was assessed in 7860 and T24 cell lines using the CCK-8 assay kit (Suzhou Xinsaimei Biotechnology Co., Ltd.). The CCK-8 kit contains a novel water-soluble tetrazolium salt, WST-8, which is stably present in cells. This compound can be reduced to yellow formazan dye by mitochondrial dehydrogenases. The more surviving cells or the stronger the cell viability, the deeper the dye color, showing a positive correlation between the two.

[0045] Cell lines 7860 and T24, in logarithmic growth phase, were digested, centrifuged, and resuspended. Cell density in the resuspended cells was determined using a cell counting chamber. Cells were seeded into 96-well plates (1×10⁴ cells / well) to achieve a density of approximately 10,000 cells / well. Leaf alcohol concentrations of 0, 0.25, 0.5, 1.25, 2.5, 5, and 10 μM were prepared and added to 96-well plates, incubated at 37°C for 24 h. 20 μL of water-soluble tetrazolium salt WST-8 solution was added to each well, and the plates were incubated at 37°C for 1–2 h. The absorbance of each well under different treatment conditions was measured at 450 nm using a microplate reader.

[0046] The experimental results are shown in Figure 1 .Depend on Figure 1 It was found that under 24-hour treatment conditions, within the concentration range of 0-10 μM, the cell viability of both 7860 and T24 decreased continuously with increasing leaf alcohol concentration, indicating that the inhibitory effect on cell viability increased with increasing leaf alcohol concentration. Specifically, the IC50 of 7860 was 2.337 μM, and the IC50 of T24 was 2.117 μM, indicating that leaf alcohol effectively inhibited the viability of both cell types under 2 μM and 24-hour treatment conditions.

[0047] Example 2

[0048] This example describes an experiment in which leaf alcohol affects the proliferation of human renal clear cell adenocarcinoma cells (7860) and human bladder cancer cells (T24).

[0049] 1. Cell colony formation experiment

[0050] The colony formation assay is used to detect the ability of a single cell to divide and proliferate to form cell colonies under different treatment conditions. The experimental steps are as follows:

[0051] (1) Digest, centrifuge, resuspend, and count the 7860 and T24 tumor cells, respectively;

[0052] (2) Use a pipette to transfer 1×10⁻⁶ of 7860 and T24 respectively. 3 One tumor cell was added to a 6-well plate and cultured in complete culture medium at 37°C for 24 hours.

[0053] (3) After the tumor cells adhered to the culture medium, the complete culture medium was replaced and leaf alcohol at concentrations of 0 μM (NC) and 2 μM (leaf alcohol) was added respectively. After 24 hours, the complete culture medium was replaced and the cells were cultured again.

[0054] (4) After culturing for 10 days, discard the upper culture medium, gently wash the cell colonies once with phosphate buffer (PBS solution), fix with 4% paraformaldehyde solution for 30 min, and finally immerse in 0.1% crystal violet solution for staining for 10 min.

[0055] (5) Cell colony counting was performed under a microscope (a single colony with a cell count >50 was considered a single clone). Each experiment was repeated in 3 replicates.

[0056] Depend on Figure 2 It can be seen that, for the same type of tumor cells, under the same culture conditions, the number of cell colonies in the experimental group (leafol) was significantly less than that in the control group (NC), indicating that leafol can inhibit the proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells. Figure 3 As can be seen from the statistical results of cell clone count, the number of cell colonies in the two experimental groups (leaf alcohol) of 7860 and T24 was significantly less than the number of cell colonies in their respective control groups (NC), further indicating that leaf alcohol has a significant inhibitory effect on the proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

[0057] 2. EdU Experiment

[0058] Cell viability was detected using the EdU-594 cell proliferation assay kit (Beyotime Biotechnology Co., Ltd.). This kit works by incorporating the thymidine deoxyribonucleoside analog EdU (5-ethynyl-2'-deoxyuridine) during DNA synthesis, followed by a click reaction that labels EdU with Alexa Fluor594, thus enabling the detection of cell proliferation. The experimental steps are as follows:

[0059] (1) Digest, centrifuge, and resuspend the 7860 and T24 cell lines in the logarithmic growth phase;

[0060] (2) Calculate the cell density of the cell resuspension using a cell counting chamber, and seed the cells into 96-well plates (1×10⁻⁶ cells / well). 4 / well), so that the density of cells to be tested is approximately 10,000 cells / well;

[0061] (3) Prepare leaf alcohol concentrations of 0 μM (NC) and 2 μM (leaf alcohol), add them to 96-well plates respectively, and incubate them in a constant temperature incubator for 24 h;

[0062] (4) Prepare 20 μM EdU working solution and continue incubating cells for 2 h. Add an equal volume of EdU working solution and culture medium to a 96-well plate. After EdU labeling of cells, remove the culture medium and add 10 μL of fixative. Fix at room temperature for 15 min. Remove the fixative and wash each well with 100 μL of washing buffer 3 times for 3-5 min each time. Remove the washing buffer and incubate each well with 100 μL of permeabilizing buffer for 10-15 min at room temperature. Remove the permeabilizing buffer and wash each well with 100 μL of washing buffer 1-2 times for 3-5 min each time.

[0063] (5) Add 100 μL of Click reaction solution to each well, gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample, incubate at room temperature in the dark for 30 min, then remove the Click reaction solution and wash with washing solution 3 times, 3-5 min each time.

[0064] (6) Use Hoechst 33342 for nuclear staining. Add 100 μL of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 min.

[0065] (7) After removing the 1X Hoechst 33342 solution, wash three times with washing buffer for 3-5 minutes each time, followed by fluorescence detection. The maximum excitation wavelength of Azide 594 is 590 nm, and the maximum emission wavelength is 615 nm. Hoechst 33342 is a blue fluorescent compound with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. Each experiment was performed in triplicate.

[0066] Depend on Figure 4 and Figure 5 It was found that the number of cells stained with fluorescent dye in the 7860 and T24 experimental groups treated with leaf alcohol was significantly lower than that in the control group (NC). This indicates that leaf alcohol has a significant inhibitory effect on the proliferation of human renal clear adenocarcinoma cells and human bladder cancer cells. Figure 6 As can be seen from the statistical results of cell clone count, the number of cell colonies in the two experimental groups (leaf alcohol) of 7860 and T24 was significantly less than the number of cell colonies in their respective control groups (NC), further indicating that leaf alcohol has a significant inhibitory effect on the proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

[0067] Example 3

[0068] This example describes an experiment on the effects of leaf alcohol on the migration and invasion abilities of 7860 and T24.

[0069] 1. Transwell experiment

[0070] Transwell migration assay: Transwell chambers (purchased from Corning, USA) are placed in 24-well plates. The bottom of each chamber divides the space within the wells into upper and lower layers. Small pores are present on the bottom of each chamber, allowing only highly migratory cells to deform and move through. This assay is commonly used to examine changes in the migration ability of tumor cells. The experimental procedure is as follows:

[0071] (1) Digest, centrifuge, and resuspend 7860 and T24 cells in RPMI 1640 medium (FBS-free). After cell counting, transfer 5 × 10⁶ cells to a container. 4 One cell (solution volume controlled not exceeding 200 μL) was transferred into the upper chamber of the Transwell;

[0072] (2) 500 μL of complete RPMI 1640 medium (10% FBS) was added into the lower chamber of the Transwell as a chemical inducer; leaf alcohol was added to the lower chamber of the Transwell and the lower chamber of the Transwell respectively, so that the final concentrations were 0 μM (NC) and 2 μM (leaf alcohol), and then placed in a 37℃ incubator for 24 h.

[0073] (3) Remove the culture medium from the upper and lower chambers, and gently scrape the tumor cells remaining in the upper chamber with a cotton swab repeatedly; for the cells that have completed migration in the lower chamber, fix them with 4% paraformaldehyde for 30 min, then immerse them in 0.1% crystal violet solution for 10 min, and take pictures of the cells that have completed migration with a microscope.

[0074] The results are as follows Figure 7 As shown in B.

[0075] Transwell invasion assay: A layer of Matrigel (purchased from BD Biosciences, USA) is laid flat on the bottom of a Transwell chamber to simulate the matrix environment of normal tissue. Highly invasive tumor cells express more matrix metalloproteinases to digest the local Matrigel, thereby further completing their migration. This assay is used to examine changes in the invasive ability of tumor cells.

[0076] Spread 100 μL of Matrigel gel (diluted 4:1 with FBS-free RPMI 1640 medium) evenly on the upper layer of a Transwell chamber and allow it to solidify on ice for later use; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 4 RPMI 1640 medium (FBS-free) was added to the upper chamber of the Transwell chamber for each cell, and the remaining steps were the same as for the Transwell migration assay. Each experiment was performed in triplicate.

[0077] The results are as follows Figure 7 As shown in Figure A.

[0078] Depend on Figure 7 It can be seen that, for the same type of tumor cells, under the same culture conditions, the migration and invasion abilities of the 7860 and T24 cell lines supplemented with leaf extract (leaf extract) were significantly lower than those of the control group (NC), indicating that leaf extract can significantly inhibit the migration and invasion abilities of human renal clear cell adenocarcinoma cells and human bladder cancer cells, urinary system tumors. Figure 8 It can be seen that the number of cells migrating and invading in the two experimental groups (leaf alcohol) was significantly lower than that in their respective control groups, further demonstrating that leaf alcohol has a significant inhibitory effect on the migration and invasion ability of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

[0079] 2. Cell scratch assay

[0080] Using a marker pen, draw evenly spaced horizontal lines on the back of the 6-hole board, one line every 0.5 cm, passing through each hole, with at least 5 lines passing through each hole; add approximately 5×10 mm of marker material into each hole. 5 Cells were cultured for 12 hours; a horizontal line was made perpendicular to the back of the cell using a pipette tip; cells were washed three times with PBS to remove the marked cells, and serum-free medium was added; leaf alcohol was added to the above culture system to a final concentration of 0 μM (NC) and 2 μM (leaf alcohol), respectively, and incubated at 37℃ with 5% CO2. Samples were taken and photographed at 0, 12, and 24 hours. Each experiment was performed in triplicate.

[0081] See cell scratch assay results for photographs. Figure 9 The statistical results are shown in Figure 10 .Depend on Figure 9It can be seen that, for the same type of tumor cells under the same culture conditions, the migration ability of the experimental group (leafol) was significantly lower than that of the control group (NC), indicating that leafol can inhibit the migration ability of human bladder cancer cells and human renal clear cell adenocarcinoma cells. Figure 10 The experimental concentration of leaf extract significantly inhibited the scratch repair ability and Transwell chamber penetration ability of urinary tract tumor cells, indicating that cell migration was significantly suppressed. This confirms that leaf extract has a significant inhibitory effect on the migration ability of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

[0082] Example 4

[0083] This embodiment describes a method for establishing a subcutaneous xenograft model in nude mice.

[0084] Examples 1-3 have demonstrated that leaf alcohol can inhibit the viability and proliferation of urinary system tumor cells. To further verify whether leaf alcohol can inhibit tumor growth in animal experiments, a xenograft tumor model was established subcutaneously in nude mice using T24.

[0085] This embodiment uses 4-week-old female nude mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to establish a subcutaneous xenograft tumor model. The specific steps are as follows:

[0086] (1) T24 tumor cells in the logarithmic growth phase were collected, digested, centrifuged, and resuspended in PBS solution at 4°C for cell counting. Approximately 2 × 10⁶ cells were finally counted using 100 μL of PBS solution. 6 One T24 tumor cell was suspended and aspirated into a 1ml insulin injection syringe. The suspension was then subcutaneously injected into the flank of a nude mouse.

[0087] (2) The tumor volume was recorded and calculated every 3 days using calipers. The calculation formula is: V = 0.5 × L × W 2 (V, volume; L, length; W, width).

[0088] (3) After 10 days, tiny tumors (V=100mm) were visible to the naked eye on the flanks of nude mice. 3 (Left and right), and then the nude mice were randomly divided into 4 groups.

[0089] (4) In this experiment, all nude mice were randomly divided into 4 groups, with 5 mice in each group. The nude mice in the leaf alcohol group were treated as follows: leaf alcohol solution was diluted with PBS and injected into the peritoneal cavity of the nude mice (ip), with a dosage and frequency of 5 mg / kg / every three days (n=5) and 10 mg / kg / every three days (n=5). The nude mice in the control group were treated by injecting equal volumes of PBS buffer (NC) and dimethyl sulfoxide (DMSO) into the peritoneal cavity of the nude mice, once every three days (n=5).

[0090] (5) During the administration period, the changes in the volume of the subcutaneous tumor were recorded using a vernier caliper. After 4 to 5 weeks of administration, the nude mice were euthanized by cervical dislocation, and the subcutaneous xenograft specimens of the nude mice were collected.

[0091] After establishing the tumorigenesis model, nude mice were randomly divided into four groups, receiving either leaf alcohol (5 mg / kg and 10 mg / kg), DMSO, or a control group (NC). Figure 11 Subcutaneous xenograft tumor specimens collected from nude mice showed that the tumors in the leaf alcohol-treated group were significantly smaller than those in the DMSO group and the control group (NC), indicating that leaf alcohol can inhibit tumor growth in animal experiments. Figure 12 The results of tumor weight analysis showed that the tumors treated with leaf extract (5 mg / kg and 10 mg / kg) weighed significantly less than those in the DMSO group and the control group (NC), indicating that leaf extract treatment can significantly reduce the weight of subcutaneous tumors in nude mice.

[0092] As described above, the present invention can be well implemented. The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The use of leaf alcohol in the preparation of a medicament for inhibiting renal clear cell adenocarcinoma or bladder cancer.

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