Paris polyphylla extract and application of paris polyphylla extract in preparation of antitumor drugs

Abstract

The invention discloses a paris polyphylla extract which is prepared by the following steps: extracting crushed paris polyphylla for 1-3 times by adopting a cyclodextrin solution at 30-70 DEG C under ultrasonic waves, collecting and combining an extracting solution, separating and purifying the extracting solution through macroporous resin, eluting the extracting solution through an ethanol solution with the volume concentration of 70%, and concentrating and drying the eluent, thereby obtaining the paris polyphylla extract. The paris polyphylla extract is applied to treatment of bladder cancer, experimental results show that the paris polyphylla extract has an obvious inhibiting effect on proliferation of human bladder cancer cells T24 and UM-UC-3, a theoretical basis is provided for deep research on the anti-tumor action mechanism of paris polyphylla, and the paris polyphylla extract is of great significance in revealing research on anti-tumor effective active ingredients of paris polyphylla and has a wide application prospect. The invention provides a new way and means for treating tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor medicine, and specifically relates to a method for preparing a Paris polyphylla extract and application of the extract in preparing an anti-bladder cancer drug. Background Art

[0002] Tumor is one of the diseases that seriously threaten human health, and its incidence and mortality rates are increasing year by year. Among them, bladder cancer is a cancer that originates from bladder cells, usually originating from the urothelial cells in the inner layer of the bladder. It is one of the tenth most common cancers in the world. The high recurrence rate of bladder cancer after treatment is a major problem faced by the clinical treatment of bladder cancer. At present, the treatment methods for tumors mainly include surgery, radiotherapy, chemotherapy and targeted therapy. However, chemotherapy drugs have great toxic side effects and are prone to drug resistance. Targeted drugs are expensive and have a limited scope of application. Natural medicines have gradually become a hot spot in the research and development of anti-tumor drugs due to their wide sources, diverse activities, and fewer toxic side effects.

[0003] Paris polyphylla Paris polyphylla var. yunnanensis (Franch.) Hand.-Mzt.) is a plant of the genus Paris of the Veratraceae family. It has the effects of clearing away heat and detoxifying, relieving swelling and relieving pain, cooling the liver and calming the nerves. It can be used to treat carbuncles, sore throat, snake bites, bruises, cold wind convulsions and other diseases.

[0004] The Pharmacopoeia of the People's Republic of China (2025 Edition) uses the method of ethanol heating and reflux to extract saponins from Paris polyphylla, and uses the total content of Paris polyphylla saponins I, II and VII as the standard for testing the quality of Paris polyphylla. In the "Orthogonal Experimental Optimization Design of Four Saponin Extraction Methods in Paris polyphylla", "Shizhen Traditional Chinese Medicine and Pharmacy 2020 Issue 7", the industrial extraction of saponins from Paris polyphylla mainly uses organic solvents such as ethanol and methanol. Organic solvents are flammable, explosive, and volatile, and have high requirements for equipment and safety, posing a threat to human health and environmental safety. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for preparing a Paris polyphylla extract, in order to solve the problems of large toxic and side effects of existing anti-tumor drugs and easy drug resistance. The present invention uses a cyclodextrin solution to extract the crushed Paris polyphylla 1-3 times at 30-70°C under ultrasound, collects and combines the extracts, separates and purifies the extracts through a macroporous resin, elutes with a 70% volume concentration ethanol solution, and concentrates and dries the eluent to obtain a Paris polyphylla extract.

[0006] The cyclodextrin is β -Cyclodextrin or Hydroxypropyl- β -Cyclodextrin, the mass concentration of the cyclodextrin solution is 0.5-3%.

[0007] The macroporous resin is selected from HPD600, HPD100, D101, NKA-9, HP20, AB-8, H103, and SP825L.

[0008] Another object of the present invention is to apply the above-mentioned Paris vietnamensis extract in the preparation of a therapeutic drug for bladder cancer.

[0009] The composition (or active ingredient) of the therapeutic drug for bladder cancer of the present invention is the Paris vietnamensis extract, and one or more pharmaceutically acceptable excipients can also be added, or it can be compounded with other active ingredients to exert an inhibitory effect; in addition to being made into tablets, the preparation can also be made into various pharmaceutically acceptable forms such as pills, powders, capsules, granules, oral liquids, and injections.

[0010] Compared with the prior art, the present invention has the following advantages: In the present invention, cyclodextrin solution and ultrasonic assistance are used to extract Paris vietnamensis. Cyclodextrin can form inclusion complexes with active ingredients, significantly increasing the water solubility of the active ingredients and greatly improving the bioavailability of the active ingredients. The obtained Paris vietnamensis extract has strong efficacy and low toxicity. When the Paris vietnamensis extract is applied in the treatment of bladder cancer, the experimental results show that it has a relatively obvious inhibitory effect on the proliferation of human bladder cancer cells T24 and UM-UC-3. The present invention provides a theoretical basis for the in-depth study of the anti-tumor mechanism of Paris vietnamensis and is of great significance for revealing the effective anti-tumor active ingredients of Paris vietnamensis. The present invention provides a new way and means for the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the extraction results of total saponins from Paris vietnamensis with different extraction solvents; Figure 2 shows the purification results of the Paris vietnamensis extract with different macroporous resins; among them, Figure A shows the adsorption rate results of total saponins with different macroporous resins, Figure B shows the desorption rate results of total saponins with different macroporous resins, and Figure C shows the recovery rate results of total saponins with different macroporous resins; Figure 3 shows the IC 50 value results of the MTT cell proliferation experiment of PPT on human bladder cancer cells T24 and UM-UC-3; among them, Figure A is the IC 50 value of PPT on T24 cells, and Figure B is the IC 50 value of PPT on UM-UC-3; Figure 4 shows the results of the cell clone formation experiment of PPT on human bladder cancer cells T24 and UM-UC-3; Figure 5 shows the flow cytometry detection results of the effect of PPT on the mitochondrial membrane potential of human bladder cancer cells T24 and UM-UC-3; Figure 6 It is the statistical result of the effect of PPT on the mitochondrial membrane potential of human bladder cancer cells T24 and UM-UC-3; Figure 7 It is the flow cytometry detection result of the apoptosis of human bladder cancer cell line T24 induced by PPT; Figure 8 It is the flow cytometry detection result of the apoptosis of human bladder cancer cell line UM-UC-3 induced by PPT; Figure 9 It is the result of Western blot analysis of T24 and UM-UC-3 cells treated with different concentrations of PPT. The upper figure is the electrophoresis pattern and the lower figure is the statistical graph. Detailed implementation manners

[0012] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation manners, but these embodiments shall not be used to interpret the limitations of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available unless otherwise specified; after reading the description of the present invention, those skilled in the art's various equivalent modifications, alterations, and decorations all fall within the scope defined by the claims of the present invention.

[0013] In the embodiment, the dry rhizome of Paris vietnamensis (Takht.) H. Li ( Paris polyphylla ) was purchased from the Yunnan Kunming Chinese herbal medicine market and identified as authentic.

[0014] Reagents: MTT reagent (Solarbio, Beijing, China), RPMI 1640 medium (Guangzhou Saiku Biotechnology Co., Ltd.), MEM medium (Guangzhou Saiku Biotechnology Co., Ltd.), fetal bovine serum (Guangzhou Saiku Biotechnology Co., Ltd.), trypsin (Gibco, USA), JC-10 detection kit (Jiangsu KeyGen Biotech Co., Ltd.), cell apoptosis detection kit (Jiangsu KeyGen Biotech Co., Ltd.), β β-cyclodextrin (Solarbio, Beijing, China) and hydroxypropyl-β- β -cyclodextrin (Aladdin, Shanghai, China).

[0015] Cell lines: bladder cancer cell lines T24 and UM-UC-3, purchased from Guangzhou Saiku Biotechnology Co., Ltd.

[0016] Example 1: Preparation of the extract of Paris vietnamensis (Takht.) H. Li 1. The dry rhizome of Paris vietnamensis (Takht.) H. Li was pulverized and passed through a 200-mesh sieve to obtain coarse powder; 2. According to the ratio of material to liquid of g:mL being 1:10, β-cyclodextrin solution with a mass concentration of 0.5 - 3% and hydroxypropyl-β- with a mass concentration of 0.5 - 3% were respectively added to the coarse powder of Paris vietnamensis (Takht.) H. Li β -cyclodextrin solutionβ Mix with β-cyclodextrin solution, and then perform ultrasonic extraction (power 180W) at 50 °C for 30 minutes; filter, add extraction reagent to the solid and repeat the extraction once. Collect and combine the filtrates, and use high performance liquid chromatography to detect the total saponin content in the extract. Chromatographic conditions: The mobile phase is acetonitrile (A) and water (B), and the gradient elution program is: 0 - 22 min, 35% - 53% acetonitrile. The flow rate is 1.0 mL / min, the injection volume is 10 μL, the column temperature is 35 °C, and the detection wavelength is 203 nm. Calculate the extraction rate of total saponins from Paris vietnamensis (Takht.) H. Li with different solvents; at the same time, use the extraction with added water and 75% ethanol as controls; The results are shown in Figure 1 , as can be seen from the figure, when water is used as the extraction solvent for Paris vietnamensis (Takht.) H. Li, the extraction rate is the lowest; when the mass concentration of β-cyclodextrin solution is 0.5 - 3%, the total saponin content in the obtained extract of Paris vietnamensis (Takht.) H. Li is higher than that with water as the extractant, and when using 1.5% β -cyclodextrin solution and 2.5% hydroxypropyl- β -cyclodextrin solution as the extraction solvent for Paris vietnamensis (Takht.) H. Li, the extraction rate is better, comparable to that with 75% ethanol. Using cyclodextrin solution for extraction is safer. Cyclodextrin can form inclusion compounds with active ingredients, significantly increasing the water solubility of active ingredients and greatly improving the bioavailability of active ingredients; 3. Pass the extract obtained by extracting with 2% hydroxypropyl- β -cyclodextrin in step 2 through macroporous resins HPD600, HPD100, D101, NKA-9, HP20, AB-8, H103, SP825L columns respectively, elute with 70% ethanol solution by volume, and use high performance liquid chromatography to detect the total saponin content in the eluate. Chromatographic conditions: The mobile phase is acetonitrile (A) and water (B), and the gradient elution program is: 0 - 22 min, 35% - 53% acetonitrile. The flow rate is 1.0 mL / min, the injection volume is 10 μL, the column temperature is 35 °C, and the detection wavelength is 203 nm. Calculate the adsorption rate, desorption rate and recovery rate of total saponins in Paris vietnamensis (Takht.) H. Li by different resins; The results are shown in Figure 2 , as can be seen from the figure, there are differences in the adsorption rate, desorption rate and recovery rate of the extract of Paris vietnamensis (Takht.) H. Li by different macroporous resins; among the 8 macroporous resins, the adsorption rates of macroporous resins HPD600, HPD100, D101, NKA-9, HP20, AB-8, SP825L for the extract of Paris vietnamensis (Takht.) H. Li are significantly higher than that of H103; when eluting with 70% ethanol solution, the desorption rates of NKA-9 and HPD600 macroporous resins are better than other resins, and the total saponin recovery rates of NKA-9 and HPD600 macroporous resins are better than other resins.

[0017] Example 2: Preparation of extract of Paris vietnamensis (Takht.) H. Li 1. Crush the dried rhizome of Paris vietnamensis, sieve it through a 200-mesh sieve to obtain coarse powder; 2. According to the ratio of material-liquid ratio g:mL of 1:10, add a 2% (mass concentration) hydroxypropyl-β-cyclodextrin solution to the coarse powder of Paris vietnamensis. After mixing, perform ultrasonic extraction (power 180W) at 50 °C for 30 minutes, separate the solid and liquid. Add the solid to the hydroxypropyl-β-cyclodextrin solution for re-extraction twice. Collect and combine the extraction solutions. Pass the extraction solution through a macroporous resin HPD600 column, elute with a 70% (volume concentration) ethanol solution, concentrate the eluate at 40 °C, and freeze-dry to obtain the extract of Paris vietnamensis (PPT). β -β-cyclodextrin solution, mix well, and then perform ultrasonic extraction (power 180W) at 50 °C for 30 minutes, separate the solid and liquid, and add the solid to the hydroxypropyl- β -β-cyclodextrin solution for re-extraction twice. Collect and combine the extraction solutions. Pass the extraction solution through a macroporous resin HPD600 column, elute with a 70% (volume concentration) ethanol solution, concentrate the eluate at 40 °C, and freeze-dry to obtain the extract of Paris vietnamensis (PPT).

[0018] Example 3: Inhibition experiment of the extract of Paris vietnamensis in Example 2 on bladder cancer cells 1. Inhibitory effect of the extract of Paris vietnamensis on the proliferation of tumor cells Uniformly seed the bladder cancer cell lines T24 and UM-UC-3 cells into a 96-well plate (4000 cells / well, 100 μL per well), place it in a cell culture incubator, and culture at 37 °C and 5% CO2 for 24 hours. After the cells are completely adherent, treat them with different concentrations (1.5625 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL) of the extract of Paris vietnamensis (PPT) and cisplatin CDDP (0.625 μΜ / L, 1.25 μΜ / L, 2.5 μΜ / L, 5 μΜ / L, 10 μΜ / L, 20 μΜ / L) for 48 hours. Then add 30 μL of MTT solution (5 mg / mL) to the 96-well plate, place it in the incubator, react at 37 °C and 5% CO2 for 5 hours. Then discard the liquid in the well plate, add 100 μL of DMSO solution (purity 99.99%), shake on a shaker for 10 minutes (20 revolutions / min), detect the absorbance value at 570 nm, calculate the cell survival rate, draw a dose-effect curve, and calculate the IC50 value; The results are shown in Figure 3 , it can be seen from the figure that under the action of the extract of Paris vietnamensis, the proliferation of bladder cancer cells is inhibited, and it shows concentration-dependent inhibition. The IC 50 values of the extract of Paris vietnamensis in Example 2 for T24 and UM-UC-3 cells are 5.405 μg / mL and 4.283 μg / mL respectively. The results indicate that the extract of Paris vietnamensis can significantly inhibit the proliferation of bladder cancer cells, and the inhibitory effect is comparable to that of cisplatin.

[0019] 2. Colony formation assay Uniformly inoculate the logarithmically growing T24 and UM-UC-3 bladder cancers into a 6-well plate. After the cells adhere, culture for another 3 - 4 days, and then add drugs for treatment in different groups. The specific grouping is as follows: (1)Control group: Cells were treated with only complete 1640 medium (T24) or complete MEM medium (UM-UC-3). (2)PPT (2.5 μg / mL) group: Cells were treated with 2.5 μg / mL PPT. (3)PPT (5 μg / mL) group: Cells were treated with 5 μg / mL PPT. (4)PPT (10 μg / mL) group: Cells were treated with 10 μg / mL PPT. The drugs of each group were prepared with fresh complete medium. The final volume of the reaction system in each well was 2 mL. After adding the corresponding drugs, the cells were cultured for 12 days. The cell medium was renewed every three days. The supernatant was discarded and the cells were washed once with PBS, then fixed with 4% paraformaldehyde for 20 minutes, washed 3 times with PBS, stained with crystal violet for 30 minutes, the staining solution was discarded, washed 3 times with PBS, and photographed for record. Colony formation is an experiment to evaluate cell survival, which is used to evaluate the ability of a single cell to proliferate and form colonies after drug treatment. The results are shown in Figure 4 , and it can be seen from the figure that the extract of Paris vietnamensis inhibits the colony formation of bladder cancer cells in a dose-dependent manner.

[0020] 3. Measurement of mitochondrial membrane potential (ΔΨm) T24 and UM-UC-3 bladder cancer cells in the logarithmic growth phase were evenly seeded in 6-well plates, placed in a cell culture incubator, and cultured at 37 °C and 5% CO2 for 24 hours. After the cells were completely adherent, they were treated with PPT at different concentrations (7.5 μg / mL, 15 μg / mL, 30 μg / mL) for 24 h, then centrifuged to remove the supernatant. The cells were resuspended with 500 μL of JC-10 staining working solution in the JC-10 detection kit and incubated at room temperature for 10 - 30 min in the dark. Detection was performed using a flow cytometer (FL1 and FL2 channels). The results are shown in Figure 5 , 6 , and it can be seen from the figure that the ratio of green fluorescence (ΔΨm index) in the group treated with Paris vietnamensis increased significantly and showed a concentration-dependent manner, indicating that the extract of Paris vietnamensis damaged the mitochondrial membrane potential of bladder cancer cells and caused changes.

[0021] 4. Cell apoptosis analysis (1) Uniformly inoculate logarithmic-phase T24 and UM-UC-3 bladder cancer cells into 6-well plates, place them in a cell culture incubator, and culture them at 37°C and 5% CO2 for 24 hours. After the cells are completely adherent, treat them with different concentrations (7.5 μg / mL, 15 μg / mL, 30 μg / mL) of PPT and Z-VAD-FMK (20 μM) for 24 hours, then gently digest them with 0.25% trypsin (without EDTA) to avoid false positives caused by over-digestion; add serum-containing medium to neutralize trypsin, centrifuge at 1000 rpm for 5 minutes, wash twice with PBS, resuspend the cells with PBS, take 100 μL of cell suspension (about 1×10 5 cells) and add it to a flow cytometry tube, add 5 μL of Annexin V-FITC, gently mix, incubate in the dark at room temperature for 15 minutes, add 5 μL of PI (final concentration 1 - 2 μg / mL), incubate in the dark for 5 minutes, and immediately add 400 μL of Binding Buffer, avoiding long-term placement (detect by machine within 1 hour).

[0022] (2) Protein immunoblotting assay Uniformly inoculate logarithmic-phase T24 and UM-UC-3 bladder cancer cells into 6-well plates, place them in a cell culture incubator, and culture them at 37°C and 5% CO2 for 24 hours. After the cells are completely adherent, treat them with different concentrations (7.5 μg / mL, 15 μg / mL, 30 μg / mL) of PPT for 24 hours, then extract total cellular proteins and perform protein immunoblotting assay to detect the expression levels of c-PARP and c-cas-7 proteins.

[0023] The results are shown in Figures 7 - 9 . It can be seen from the figure that Paris vietnamensis extract can significantly promote the apoptosis of bladder cancer cells. Through flow cytometry detection, it is found that the Paris vietnamensis extract treatment group can inhibit the proliferation of bladder cancer cells in a concentration-dependent manner and induce cell apoptosis. After adding the apoptosis inhibitor Z-VAD-FMK, the ability of Paris vietnamensis extract to promote apoptosis can be reversed. The Western blot results show that the levels of key apoptotic proteins c-PARP and c-cas-7 increase, suggesting that Paris vietnamensis can promote the apoptosis of bladder cancer cells.

[0024] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A Paris vietnamensis extract, characterized in that: Using a cyclodextrin solution, pulverized Paris vietnamensis is extracted 1 - 3 times at 30 - 70 °C under ultrasonic conditions, and the extraction solutions are collected and combined. The extraction solution is separated and purified by macroporous resin, eluted with an ethanol solution with a volume concentration of 70%, and the eluate is concentrated and dried to obtain the Paris vietnamensis extract.

2. The Paris vietnamensis extract according to claim 1, characterized in that: The cyclodextrin is β β-cyclodextrin or hydroxypropyl- β β-cyclodextrin.

3. The Paris vietnamensis extract according to claim 2, wherein: The mass concentration of the cyclodextrin solution is 0.5 - 3%.

4. The Paris vietnamensis extract according to claim 1, characterized in that: The macroporous resin models are HPD600, HPD100, D101, NKA-9, HP20, AB-8, H103, SP825L.

5. Use of the Paris vietnamensis extract according to claim 1 in the preparation of a therapeutic drug for bladder cancer.

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