CN118178361A

CN118178361A - Application of phyllitol in preparation of urinary system tumor medicine

Info

Publication number: CN118178361A
Application number: CN202410333028.0A
Authority: CN
Inventors: 唐正严; 彭经璇; 韩宇; 朱泉; 吴自强; 郑智桓
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2024-03-22
Filing date: 2024-03-22
Publication date: 2024-06-14

Abstract

The invention belongs to the technical field of medicine preparation, and in particular relates to application of phyllitol in preparation of a medicine for treating urinary system tumors. Animal experiments prove that the phyllitol has remarkable improvement effect on tumors caused by human bladder cancer cells, and can be used for treating urinary system tumors. The invention makes clear for the first time that the phyllitol has an inhibiting effect on the proliferation, migration and invasion of tumor cells of the urinary system.

Description

Application of phyllitol in preparation of urinary system tumor medicine

Technical Field

The invention belongs to the technical field of medicine preparation, and in particular relates to application of phyllitol in preparation of a medicine for treating urinary system tumors.

Background

Tumors are new organisms formed by abnormal proliferation of cells of local tissues under the action of various tumorigenic factors, and are often expressed as local tumors. Tumor cells have abnormal morphology and metabolic functions, grow vigorously, and often continue to grow.

The urinary system tumor refers to tumor occurring at any part of urinary system, including kidney, renal pelvis, ureter, bladder, urethra tumor. The viscera below the renal pelvis are ducted organs, the urinary tract epithelium is covered in the cavity, urine is in contact with the internal environment, and the carcinogen often causes tumors of the urinary tract epithelium through the urine, so that the tumors of the urinary tract epithelium of the renal pelvis, the ureter, the urinary bladder and the urethra have commonality and possibly cause multiple organ diseases. Bladder cancer is also most common due to the longest residence time of urine within the bladder. The renal clear cell carcinoma is an adenocarcinoma derived from tubular epithelial cells, and is frequently bleeding, necrosis, cystic changes and calcification, and occurs in kidney parenchyma, and after growing up, infiltrates, presses and destroys renal pelvis and renal calyx, and develops outside kidney capsule to form hemangioma embolism or metastasize to lymph nodes and other viscera. At present, the treatment of bladder cancer or renal clear cell carcinoma is mainly surgical treatment, radiotherapy, chemotherapy and biological treatment play an auxiliary role, and medicines for selecting medicine treatment are few and most of the medicines have higher cost.

Leaf alcohol (cis-3-Hexen-1-ol), also known as cis-3-hexenol, beta, gamma-hexenol, is found in tea, locust, radish, strawberry, grapefruit, etc. Patent CN116655455A discloses a method for synthesizing phyllitol, which takes crotonaldehyde and acetaldehyde as raw materials to synthesize phyllitol, and the method is easy to obtain raw materials, simple and mild in reaction conditions. No report on application of phyllitol in tumor cell inhibition is currently seen.

Disclosure of Invention

The search for the development of new anti-urinary system tumor drugs is one of the important tasks in the modern medical field. The invention aims at defining the inhibition effect of the phyllitol or the pharmaceutically acceptable salt thereof on the tumor cells of the urinary system, and is hopeful to provide a new candidate medicament for treating the tumor of the urinary system for clinic.

To achieve the above object, the present invention verifies the role of phyllitol in the treatment of urinary system tumors. The structural formula of the leaf alcohol is as follows:

First, the invention claims the application of phyllitol or its pharmaceutically acceptable salt in preparing urinary system tumor medicine.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ in the administration vehicle or dosage form manufacturing process, or by separately reacting the purified compound of the invention in its free form with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like. (see, e.g., berge et al (1977) "Pharmaceuti cal Salts", J.Pharm. Sci.66:1-19).

Based on the application of the invention, the phyllitol or the pharmaceutically acceptable salt thereof inhibits the activity of human renal clear cell adenocarcinoma cells or human bladder cancer cells.

In particular, the phyllitol or a pharmaceutically acceptable salt thereof is used for the treatment of human renal clear cell carcinoma or human bladder carcinoma.

Further, the phyllitol or a pharmaceutically acceptable salt thereof has an inhibitory effect on human renal clear cell adenocarcinoma cells or human bladder cancer cells.

Further, the concentration of the leaf alcohol ranges from 0 to 10 mu M, and the inhibition effect on the activity of the human renal clear cell adenocarcinoma cells or the human bladder cancer cells in the 24-hour action time is positively correlated with the concentration of the leaf alcohol.

Further, the IC50 of the leaf alcohol for inhibiting the activity of human renal clear cell adenocarcinoma cells is 2.337. Mu.M, and the IC50 of the leaf alcohol for inhibiting the activity of human bladder cancer cells is 2.117. Mu.M.

In particular, phyllitol inhibits proliferation, migration and invasion of human renal clear cell adenocarcinoma cells or human bladder cancer cells.

The research of the inventor of the patent shows that the phyllitol has remarkable improvement effect on a xenograft tumor model established under the skin of a nude mouse by using human bladder cancer cells, and the phyllitol is expected to provide a new candidate medicament for treating urinary system tumors clinically.

Based on research, the invention claims a drug for inhibiting urinary system tumor, and the active ingredients of the drug comprise the phyllitol or pharmaceutically acceptable salt thereof.

In particular, the invention claims a drug having therapeutic effect on human renal clear cell carcinoma or human bladder carcinoma. The active ingredient of the medicine comprises the leaf alcohol or the pharmaceutically acceptable salt thereof.

Further, the components of the medicament also comprise pharmaceutically acceptable carriers.

The invention adopts the phyllitol or the pharmaceutically acceptable salt thereof to treat the urinary system tumor. The medicine is prepared from an active component and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, such as organic or inorganic solid or liquid excipients suitable for parenteral and parenteral administration. The medicament is prepared into the form of conventional medicinal preparations, preferably tablets, pills, capsules, powder, granules and the like. The above pharmaceutical preparation may contain wetting agent, stabilizer, adjuvant and other common 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 preparation of the invention can be prepared according to the conventional preparation process of various preparations.

As a preferred embodiment of the pharmaceutical, the skilled artisan generally prepares the biologically active compound or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable carrier for use in pharmaceutical formulations. In order to use the phyllitol or the pharmaceutically acceptable salt thereof in preparing the urinary system tumor medicament, pharmaceutically acceptable preparation formulations comprise capsules, tablets, powder or granules and the like. It will be appreciated by those skilled in the art that the components of such medicaments, in addition to the leaf alcohol or a pharmaceutically acceptable salt thereof, include pharmaceutically acceptable carriers or adjuvants such as olive oil, corn starch, citric acid, stearic acid, ethylene glycol, lactose, terra alba, magnesium stearate, ascorbic acid, gelatin, agar and the like.

Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages:

(1) The invention verifies and confirms the inhibition effect of phyllitol on the tumor cells of the urinary system for the first time.

Leaf alcohol can inhibit the activity of human renal clear cell adenocarcinoma cells and human bladder cancer cells, and the inhibition effect is enhanced along with the increase of the action concentration of the leaf alcohol.

(2) Leaf alcohol can effectively inhibit the activity of 2 cells under the action condition of 2 mu M and 24 h. The scratch experiment and the Transwell experiment show that the phyllitol can obviously inhibit the migration and invasion capacity of the tumor cells of the urinary system.

(3) By establishing a xenograft tumor model and observing the growth condition of tumors, the result shows that the volume of the tumors treated by the phyllitol is obviously smaller than that of a control group, which proves that the phyllitol can obviously inhibit the growth of the transplanted tumors. In an animal in vivo experiment, the inhibition effect of the phyllitol on the human bladder cancer cells is proved to be obvious. The treatment effect of the phyllitol on the urinary system tumor is demonstrated, and the phyllitol is expected to provide a new candidate medicament for treating the urinary system tumor clinically.

Drawings

FIG. 1 shows statistics of cell viability after treatment of both 7860 and T24 urinary tumor cell lines for 24h at different concentrations of leaf alcohol. Wherein Cell viability represents Cell viability and IC50 represents the concentration of compound phytol at which half viability of the corresponding cancer cells is inhibited, < p <0.05, < p <0.01.

FIG. 2 shows the results of cells 7860 and T24 after leaf alcohol treatment in a colony formation experiment. Wherein NC is a control group, and phyllitol is an experimental group.

Fig. 3 shows the results of colony formation experiments 7860 and T24 cells, wherein Number of colonies represents the number of cell colonies, NC is control, phyllitol is experimental, n=3, p <0.05, p <0.01.

FIG. 4 shows the fluorescence detection results of 7860 of EdU experiments in the control group (leaf alcohol) control group (NC). Wherein DAPI and EdU represent the staining results of each fluorescent dye; merge is the staining result of the mixed dye of DAPI and EdU.

FIG. 5 shows the results of fluorescence detection in the experimental group (leaf alcohol) control group (NC) of T24 in EdU experiments. Wherein DAPI and EdU represent staining results of different fluorescent dyes; merge is the staining result of the mixed dye of DAPI and EdU.

FIG. 6 is a graph showing statistics of the growth rate of 7860 and T24 cells in EdU experiments. Wherein cell proliferation rate denotes the cell growth rate, NC is control, phyllitol is experimental, n=3, p <0.05, p <0.01.

FIG. 7 shows the staining results of the migration and invasion experiments of 7860 and T24 in the Transwell experiments. AInvasion is an attack experiment dyeing result, and B scale is a Migration experiment dyeing result; NC is control group and leaf alcohol is experimental group.

FIG. 8 is a graph showing the statistics of the number of cells in migration and invasion experiments in a Transwell experiment. Wherein, invaion represents an Invasion experimental result, proportion represents a Migration experimental result, count represents a number, NC is a control group, phyllitol is an experimental group, n=3, p <0.05, p <0.01.

FIG. 9 shows the results of cell scratch experiments. Wherein NC is a control group, and phyllitol is an experimental group.

Fig. 10 is a statistical result of wound healing rate of cell scratch test. Wherein Wound healing denotes the wound healing rate, NC is the control group, phyllitol is the experimental group, n=3, p <0.05, p <0.01.

FIG. 11 shows general specimens of nude mice subcutaneously transplanted in the leaf alcohol-treated group (5 mg/kg and 10 mg/kg), DMSO group and control group (NC).

Figure 12 is a weight statistic of weighed subcutaneous transplants. * p <0.05, p <0.01.

Detailed Description

The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples. The experimental methods and the detection methods in the following embodiments are all conventional methods unless otherwise specified; the medicaments and materials are commercially available unless specified; the index data are all conventional measurement methods unless specified.

To illustrate the effect of phyllitol on the treatment of urinary system tumors, the inventors of the present invention used human renal clear cell adenocarcinoma cells (7860) and human bladder cancer cells (T24) as experimental materials, and the specific experimental procedures and results were described below. 7860 and T24 referred to in the examples below are both purchased from Abiowell company. RPMI 1640 medium was purchased from Shanghai source culture Biotech Co.

Example 1

This example describes the statistics of the effect of varying concentrations of leaf alcohol on the viability of human renal clear cell adenocarcinoma cells (7860) and human bladder cancer cells (T24).

RPMI 1640 medium was used for the culture of both 7860 and T24 cell lines.

The culture system was 1640 medium supplemented with 1% penicillin-streptomycin (100X) (SP, new Salme Biotechnology Co., suzhou) and 10% fetal bovine serum (FBS, available from Biosciences). Cell wall-attached culture was performed on T24 and 7860 using a 6cm dish, the carbon dioxide concentration was controlled at 5%, the relative air humidity was controlled at 95%, and the culture was performed at 37 ℃. And replacing the cell culture solution every 1-2 d, and carrying out subculture of the cells when the cells grow to 80% -90% of the area of the covered culture dish.

Cell viability assays were performed on 7860 and T24 cell lines using the CCK-8 kit (New Saimei Biotechnology Co., st.). The CCK-8 kit contains a novel water-soluble tetrazolium salt WST-8, can exist in cells stably, can be reduced into yellow formazan dye by dehydrogenase in mitochondria, and has positive correlation with the more surviving cells or the stronger the activity of the cells, the darker the color of the dye.

Respectively digesting, centrifuging and re-suspending 7860 and T24 cell lines in the logarithmic growth phase; cell density of cell resuspension was measured by using a cell counting plate, and cells were seeded into 96-well plates (1×104/well) to give a cell density of about 10000 cells/well. She Chunnong degrees are 0, 0.25, 0.5, 1.25, 2.5, 5 and 10 mu M respectively, and are added into 96-well plates respectively, and incubated at 37 ℃ for 24 hours. Adding 20 mu L of water-soluble tetrazolium salt WST-8 solution into each hole, and incubating for 1-2 h at 37 ℃; absorbance per well was measured at 450nm using an microplate reader under different treatment conditions.

The experimental results are shown in FIG. 1. As can be seen from FIG. 1, under 24h action conditions, in the concentration range of 0-10. Mu.M, the cell activities of 7860 and T24 continuously decrease with the increase of the leaf alcohol concentration, which shows that the inhibition effect on the cell activities is continuously enhanced with the increase of the leaf alcohol concentration. Wherein 7860 has an IC50 of 2.337. Mu.M and T24 has an IC50 of 2.117. Mu.M, indicating that phytol can effectively inhibit the viability of 2 cells under the action of 2. Mu.M for 24 hours.

Example 2

This example describes experiments in which leaf alcohol affects proliferation of human renal clear cell adenocarcinoma cells (7860) and human bladder cancer cells (T24).

1. Cell clone formation assay

The clonogenic assay was used to examine the ability of individual cells to divide and proliferate to form cell colonies under different processing conditions. The experimental procedure was as follows:

(1) Respectively digesting, centrifuging, re-suspending and counting 7860 and T24 tumor cells;

(2) Respectively transferring 7860 and T24 1× ³ tumor cells into 6-well plate by pipetting gun, culturing with complete culture medium, and culturing in 37 deg.C incubator for 24 hr;

(3) After tumor cells are attached, changing the complete culture medium, adding leaf alcohol with the concentration of 0 mu M (NC) and 2 mu M (leaf alcohol) respectively, and changing the complete culture medium after 24 hours for continuous culture;

(4) After 10d of culture, the upper medium is discarded, the cell colony is gently washed once by phosphate buffer solution (PBS solution), fixed for 30min by using 4% paraformaldehyde solution, finally immersed into 0.1% crystal violet solution and dyed for 10min;

(5) Cell colony counts were performed with the aid of a microscope (single colony cell count >50 was considered as one monoclonal). Each set of experiments was set up with 3 replicates.

As can be seen from fig. 2, the number of experimental group (leaf alcohol) cell colonies was significantly smaller than that of control group (NC) for the same tumor cells under the same culture conditions, indicating that leaf alcohol can inhibit proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells. From the statistics of the number of cell clones, it can be seen from fig. 3 that the number of cell colonies in the experimental group 2 (leaf alcohol) of 7860 and T24 is significantly smaller than that in the control group (NC) corresponding thereto, further demonstrating that leaf alcohol has a significant inhibitory effect on proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

EdU test

Cell viability assays were performed using the EdU-594 cell proliferation assay kit (Biyun biotechnology Co., ltd.). The principle of the kit is based on the incorporation of thymidine (thymidine) analogue EdU (5-ethynyl-2' -deoxyuridine) during DNA synthesis, and the detection of cell proliferation is achieved by labeling EdU with Alexa Fluor594 by subsequent Click reaction (Click reaction). The experimental procedure was as follows:

(1) Digesting, centrifuging and re-suspending 7860 and T24 cell lines in the logarithmic growth phase;

(2) Measuring cell density of cell resuspension by using a cell counting plate, inoculating cells into a 96-well plate (1×10 ⁴/well) to make the cell density to be measured about 10000 cells/well;

(3) Preparing She Chunnong ℃ respectively 0 mu M (NC) and 2 mu M (leaf alcohol), adding the solution into a 96-well plate respectively, and carrying out mixed culture in a constant temperature box for 24 hours;

(4) Preparing an EdU working solution with the concentration of 20 mu M, continuously incubating the cells for 2 hours, and adding the EdU working solution and the culture solution into a 96-well plate in an equal volume; after EdU labeling of cells was completed, the culture solution was removed, and 10. Mu.L of a fixing solution was added thereto, followed by fixation at room temperature for 15min; removing the fixing solution, and washing the cells with 100 mu L of washing solution for 3 times each time for 3-5min; removing the washing liquid, and incubating with 100 mu L of penetrating liquid for 10-15min at room temperature; removing the permeation solution, washing the cells with 100 μl of washing solution for 1-2 times each for 3-5min;

(5) Adding 100 mu L of Click reaction liquid into each hole, lightly shaking the culture plate to ensure that the reaction mixture can uniformly cover the sample, incubating for 30min at room temperature in a dark place, then sucking the Click reaction liquid, and washing 3 times with washing liquid for 3-5min each time;

(6) Cell nuclei were stained using Hoechst33342, and 100 μl of 1x Hoechst33342 solution was added to each well and incubated at room temperature in the dark for 10min;

(7) After 1X Hoechst 33342 solution was pipetted off, the solution was washed 3 times for 3-5min each time, followed by fluorescence detection. The maximum excitation wavelength of Azide is 590nm and the maximum emission wavelength is 615nm. Hoechst 33342 is blue fluorescence, the maximum excitation wavelength is 346nm, and the maximum emission wavelength is 460nm. Each set of experiments was set up with 3 replicates.

As can be seen from fig. 4 and 5, the numbers of cells fluorescent stained in both the experimental groups 7860 and T24 treated with leaf alcohol were significantly lower than in the control group (NC). The results show that the phyllitol has obvious inhibition effect on proliferation of human renal transparent adenocarcinoma cells and human bladder cancer cells. From the statistics of the cell clone numbers, it can be seen from fig. 6 that the cell colony numbers of the 2 experimental groups (leaf alcohol) of 7860 and T24 are significantly smaller than those of the corresponding control group (NC), further demonstrating that leaf alcohol has a significant inhibitory effect on proliferation of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

Example 3

This example describes experiments in which leaf alcohols affect 7860 and T24 migration and invasion capacity.

Transwell experiment

Transwell migration experiments: a Transwell cell (available from Corning corporation, usa) was placed in the 24-well plate, the bottom surface of the cell divided the space in the 24-well plate into upper and lower layers, and the bottom surface of the cell was perforated with small holes, and only cells having a strong migration ability could be deformed and moved to pass through. Are commonly used to examine changes in tumor cell migration capacity. The experimental procedure was as follows:

(1) 7860 and T24 cells were digested, centrifuged, resuspended in RPMI 1640 medium (no FBS), and after cell counting was completed, 5×10 ⁴ cells (solution volume controlled to no more than 200 μl) were transferred into the upper cavity of the Transwell chamber;

(2) 500 μl of complete RPMI 1640 medium (10% FBS) was added into the Transwell chamber lower cavity to serve as a chemoattractant; leaf alcohol was added to the lower Transwell chamber and the lower Transwell chamber to a final concentration of 0. Mu.M (NC) and 2. Mu.M (leaf alcohol), respectively, and allowed to stand in an incubator at 37℃for 24 hours;

(3) Removing culture solution in the upper cavity and the lower cavity of the small chamber, and repeatedly and gently scraping tumor cells remained in the upper cavity of the small chamber by using a cotton swab; for the cells that completed migration in the subchamber, they were fixed with 4% paraformaldehyde for 30min, then immersed in 0.1% crystal violet solution for 10min, and a micrograph of the cells that completed migration was taken with a microscope.

The results are shown in FIG. 7B.

Transwell invasion experiments: a layer of Matrigel glue (purchased from BD company in the united states) was spread on the bottom of the Transwell chamber to simulate the stromal environment of normal tissue, and tumor cells with high invasive ability were able to express more matrix metalloproteinases to digest the local Matrigel glue, thereby further completing migration. For testing changes in tumor cell invasiveness.

Spreading Matrigel gel (100 μL after being diluted by FBS-free RPMI 1640 culture medium 4:1) on the upper layer of a Transwell chamber, and solidifying on ice for standby; RPMI 1640 medium (no FBS) containing 8×10 ⁴ cells was added to the upper chamber of the Transwell chamber and the rest of the procedure was the same as for the Transwell migration experiment. Each set of experiments was set up with 3 replicates.

The results are shown in FIG. 7A.

As can be seen from fig. 7, the experimental group (leaf alcohol) with 7860 and T24 cell lines added with leaf alcohol had significantly lower cell migration and invasion capacity than the control group (NC) for the same tumor cells under the same culture conditions, indicating that leaf alcohol was able to significantly inhibit migration and invasion capacity of human renal clear cell adenocarcinoma cells and human bladder cancer cell urinary system tumors. As can be seen from fig. 8, the number of cell migration and invasion of the experimental group 2 (phyllitol) was significantly lower than that of the corresponding control group, and further phyllitol had a significant inhibitory effect on the migration and invasion abilities of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

2. Cell scratch assay

Uniformly scribing transverse lines on the back of the 6-hole plate by using a marker pen, traversing through holes every 0.5cm, and enabling each hole to penetrate through at least 5 lines; about 5X 10 ⁵ cells were added to the wells and incubated for 12h; scribing with a transverse line perpendicular to the back of the gun head; washing cells with PBS for 3 times, removing the scraped cells, and adding a serum-free culture medium; leaf alcohol was added to the above culture system to a final concentration of 0. Mu.M (NC) and 2. Mu.M (leaf alcohol), and incubated in a 37℃5% CO ₂ incubator at constant temperature, sampled at 0, 12, 24h, and photographed. Each set of experiments was set up with 3 replicates.

The result of the cell scratch experiment is shown in fig. 9, and the statistical result is shown in fig. 10. As can be seen from fig. 9, the experimental group (phyllitol) showed significantly lower cell migration ability than the control group (NC) for the same tumor cells under the same culture conditions, indicating that phyllitol can inhibit the migration ability of human bladder cancer cells and human renal clear cell adenocarcinoma cells. As can be seen from fig. 10, the phytol at the experimental concentration can significantly inhibit scratch repair ability and Transwell cell penetration ability of the tumor cells of the urinary system, indicating that the migration ability of the cells is significantly inhibited. It is proved that the phyllitol has remarkable inhibiting effect on the migration capacity of human bladder cancer cells and human renal clear cell adenocarcinoma cells.

Example 4

This example describes a method for establishing a nude mouse subcutaneous graft model.

Examples 1-3 demonstrate that phyllitol can inhibit the viability and proliferation of tumor cells in the urinary system. To further verify whether phyllitol can inhibit tumor growth in vivo animal experiments, xenograft tumor models were created subcutaneously in nude mice using T24.

This example uses 4-week-old female nude mice (Jiangsu Jiugang Biotech Co., ltd.) to build a subcutaneous xenograft tumor model. The method comprises the following specific steps:

(1) T24 tumor cells in the logarithmic growth phase were taken, digested, centrifuged, and resuspended using 4℃PBS for cell counting. Approximately 2X 10 ⁶ T24 tumor cells were finally suspended using 100. Mu.L of PBS solution and aspirated into a 1ml insulin injection needle. The suspension was subcutaneously injected into the flank of nude mice.

(2) Tumor volumes were recorded and calculated every 3d using vernier calipers, with the calculation formula: v=0.5×l×w ² (V, volume; L, length; W, width).

(3) After 10d, microscopic tumors (v=100 mm ³ or so) were formed on the flank of nude mice, and nude mice were randomly divided into 4 groups.

(4) All nude mice were randomly divided into 4 groups of 5 nude mice in each group. The treatment method of the nude mice in the leaf alcohol group is as follows: the leaf alcohol solution was diluted with PBS and injected into the peritoneal cavity of nude mice (i.p.), with doses and frequency of 5 mg/kg/every three days (n=5) and 10 mg/kg/every three days (n=5). The control group nude mice were treated by injecting into the peritoneal cavity of the nude mice with equal volumes of PBS buffer (NC) and dimethyl sulfoxide (DMSO), respectively, once every three days (n=5).

(5) The change of the subcutaneous tumor volume is recorded by using a vernier caliper during the administration period, and after 4-5 weeks of administration, nude mice are sacrificed after cervical removal, and the subcutaneous transplantation tumor specimens of the nude mice are collected.

Nude mice were randomly divided into 4 groups after the oncological model was built, and experimental animals were given phyllitol (5 mg/kg and 10 mg/kg), DMSO group treatment or control group (NC) treatment. From the collection of nude mice subcutaneous engrafted tumor specimens in FIG. 11, the leaf alcohol treated group showed significantly less tumor than the DMSO group and the control group (NC), demonstrating that leaf alcohol can inhibit tumor growth in animal experiments. From the weight statistics of the transplanted tumors in FIG. 12, the weight of the transplanted tumors treated with the leaf alcohol (5 mg/kg and 10 mg/kg) is obviously lower than that of the transplanted tumors in the DMSO group and the control group (NC), which indicates that the leaf alcohol treatment can obviously reduce the weight of the transplanted tumors under the skin of the nude mice.

The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims

1. Application of phyllitol or its pharmaceutically acceptable salt in preparing urinary system tumor medicine is provided.

2. The use according to claim 1, wherein said phyllitol or a pharmaceutically acceptable salt thereof inhibits viability of human renal clear cell adenocarcinoma cells or human bladder cancer cells.

3. The use according to claim 2, wherein said phyllitol inhibits proliferation, migration or invasion capacity of human renal clear cell adenocarcinoma cells or human bladder cancer cells.

4. The use according to claim 1, wherein said phyllitol or a pharmaceutically acceptable salt thereof is for use in the treatment of human renal clear cell carcinoma or human bladder carcinoma.

5. The use according to claim 4, wherein said phyllitol or a pharmaceutically acceptable salt thereof has an inhibitory effect on human renal clear cell adenocarcinoma cells or human bladder cancer cells.

6. The use according to claim 5, wherein the concentration of said leaf alcohol ranges from 0 to 10 μm and the inhibition of the viability of human renal clear cell adenocarcinoma cells or human bladder cancer cells over a 24 hour period of action is positively correlated with the concentration of said leaf alcohol.

7. The use according to claim 5, wherein said leaf alcohol has an IC50 of 2.337 μm and said leaf alcohol has an IC50 of 2.117 μm for inhibiting human renal clear cell adenocarcinoma cell viability.

8. A medicament for inhibiting urinary system tumors, characterized in that the active ingredient of the medicament comprises the phyllitol or a pharmaceutically acceptable salt thereof according to claim 1.

9. A medicament having a therapeutic effect on human renal clear cell carcinoma or human bladder carcinoma, characterized in that the active ingredient of the medicament comprises the leaf alcohol or a pharmaceutically acceptable salt thereof according to claim 1.

10. The medicament of claim 8, or claim 9, further comprising a pharmaceutically acceptable carrier.