Use of tibetan tea or extract thereof for the preparation of a chemotherapeutic drug sensitizer

Abstract

The application provides a use of Tibetan tea or an extract thereof in preparation of a chemotherapy drug sensitizer, and belongs to the field of medicines. The application first finds that the water extract of Tibetan tea can significantly down-regulate the expression of a multi-drug resistance transporter P-gp in human hepatoma HepG2 cells, inhibit the proliferation of the human hepatoma HepG2 cells, reduce the drug resistance of the HepG2 cells, and enhance the sensitivity of the hepatoma cells to a chemotherapy drug paclitaxel. The application also first finds that the water extract of Yaan Tibetan tea and the paclitaxel play a synergistic effect in inhibiting the expression of the multi-drug resistance transporter P-gp in the human hepatoma HepG2 cells. The water extract of the Tibetan tea combined with the paclitaxel is used for treating hepatoma, and has a good application prospect.

Description

Use of Tibetan tea or its extracts in the preparation of chemotherapy drug sensitizers Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the use of Tibetan tea or its extract in the preparation of chemotherapy drug sensitizers. Background Technology

[0002] Liver cancer is a very common digestive system tumor, often referred to as the "king of cancers." my country has a high prevalence of liver cancer, with most patients diagnosed at an advanced stage, at which point surgery is no longer an option. With advancements in medical technology, treatment methods for liver cancer are constantly evolving. Currently, the main treatment methods for liver cancer include: surgery, liver transplantation, ablation therapy, interventional therapy, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Among these, chemotherapy was once a crucial treatment for advanced liver cancer, as the drugs have a relatively direct inhibitory effect on cancer cells.

[0003] Paclitaxel is one of the most potent natural broad-spectrum anticancer drugs discovered to date. It is widely used in the treatment of malignant tumors such as liver cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, gastric cancer, head and neck tumors, and soft tissue sarcomas. Studies have shown that it inhibits the proliferation of various tumor cells and induces apoptosis. However, on the one hand, paclitaxel has poor specificity for tumor cells and exhibits significant toxicity, often resulting in varying degrees of adverse reactions, typically including allergic reactions, bone marrow suppression, neurotoxicity, cardiovascular toxicity, joint and muscle pain, gastrointestinal reactions, liver toxicity, and hair loss. On the other hand, long-term use of paclitaxel can easily lead to drug resistance in tumor cells, reducing its efficacy and causing significant inconvenience to clinical treatment.

[0004] Multidrug resistance (MDR) is one of the main causes of chemotherapy failure in cancer. P-glycoprotein (P-gp or Pgp) is a typical MDR transporter with broad specificity. Many commonly used drugs, including vincristine alkaloids, taxanes, tyrosine kinase inhibitors, and some PARP inhibitors, are P-glycoprotein substrates. Cells transport these drugs out of the cell via P-glycoprotein-mediated active transport, reducing drug accumulation within the cell, leading to decreased drug efficacy and drug resistance, ultimately resulting in drug resistance. P-glycoprotein expression has been found in various solid tumors, and its expression increases after chemotherapy, causing drug resistance and being associated with poor patient prognosis, thus serving as one of the prognostic indicators for clinical oncology patients. Therefore, developing drugs that can effectively inhibit P-gp expression is of great significance for increasing the sensitivity of chemotherapeutic drugs.

[0005] Ya'an Tibetan tea is a local specialty dark tea, named after its primary production area, Ya'an, Sichuan Province, and its long history of popularity in the Qinghai-Tibet Plateau region. In a broader sense, Ya'an Tibetan tea includes traditional Tibetan tea (also known as Southern Route Border Tea, Border-Sold Tea, Kangzhuan Tea, and Jinjian Tea) and newer types of Tibetan tea. Ya'an Tibetan tea is rich in tea polyphenols, tea polysaccharides, and organic acids. Current research on the bioactivity of Ya'an Tibetan tea mainly focuses on its lipid-lowering, weight-loss, antioxidant, and anti-radiation effects. However, tea polysaccharides have been reported to have anti-tumor activity, and since Ya'an Tibetan tea contains tea polysaccharides, it is speculated that it has certain anti-tumor potential. However, there are currently no reports of Ya'an Tibetan tea being used as a chemotherapy drug sensitizer for cancer treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a new use for Tibetan tea in the preparation of chemotherapy drug sensitizers.

[0007] This invention provides the use of Tibetan tea or its extracts in the preparation of chemotherapeutic drug sensitizers.

[0008] Furthermore, the Tibetan tea mentioned is Ya'an Tibetan tea.

[0009] Furthermore, the extract is an aqueous extract or an organic solvent extract.

[0010] Furthermore, the chemotherapy drug is a vincristine alkaloid, a taxane, a tyrosine kinase inhibitor, or a PARP inhibitor.

[0011] Furthermore, the taxane drug is paclitaxel.

[0012] Furthermore, the chemotherapy drug sensitizer is a reagent that enhances the sensitivity of cancer cells to chemotherapy drugs, wherein the cancer is liver cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, gastric cancer, head and neck tumors, or soft tissue sarcoma.

[0013] The chemotherapeutic drug sensitizer is a reagent that inhibits the expression of multidrug resistance transporter proteins.

[0014] Furthermore, the multidrug resistance transporter is a P-gp protein.

[0015] Furthermore, the chemotherapy drug sensitizer is a reagent that inhibits the expression of Bcl-2 protein.

[0016] Furthermore, the chemotherapeutic drug sensitizer is a reagent that enhances the expression of Bax protein.

[0017] The present invention also provides a combination medicament for the prevention and / or treatment of cancer, comprising Tibetan tea or its extracts in the same or different strength units for simultaneous or separate administration, and a chemotherapy drug, as well as a pharmaceutically acceptable carrier.

[0018] Furthermore, the Tibetan tea is Ya'an Tibetan tea, and the extract is a water extract or an organic solvent extract;

[0019] The chemotherapy drugs are vincristine alkaloids, taxanes, tyrosine kinase inhibitors, or PARP inhibitors.

[0020] Furthermore, the mass ratio of the Tibetan tea or its extract to the chemotherapy drug is (200-800):0.8, preferably 600:0.8.

[0021] Furthermore, the preparation method of the Tibetan tea water extract includes the following steps: weighing Tibetan tea, grinding it into powder, adding boiling water for extraction, collecting the extract, and drying it to obtain the Tibetan tea water extract.

[0022] Furthermore, the drying method is freeze-drying.

[0023] Furthermore, the mass-to-volume ratio of the powder to water is 1:10 g / mL.

[0024] This invention is the first to discover that Tibetan tea water extract can significantly downregulate the expression of multidrug resistance transporter P-gp in human hepatocellular carcinoma HepG2 cells, inhibit the proliferation of human hepatocellular carcinoma HepG2 cells, reduce the drug resistance of HepG2 cells, and enhance the sensitivity of hepatocellular carcinoma cells to the chemotherapeutic drug paclitaxel.

[0025] This invention also reveals for the first time that the combined use of Ya'an Tibetan tea extract and paclitaxel has a synergistic effect in inhibiting the expression of the multidrug resistance transporter P-gp in human hepatocellular carcinoma HepG2 cells.

[0026] This invention combines Tibetan tea water extract with paclitaxel for the treatment of liver cancer, showing promising application prospects.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1. Western blot images (A) and quantitative results (B) of the effects of Ya'an Tibetan tea water extract alone, paclitaxel alone, and Ya'an Tibetan tea water extract combined with paclitaxel on protein expression levels in HepG2 cells. Note: Compared with paclitaxel alone, ▲ P < 0.05.

[0030] Figure 2. Inhibition rates of HepG2 cells by using 400 μg / mL Ya'an Tibetan tea water extract (i.e., the freeze-dried powder group in the figure), 0.8 μg / mL paclitaxel alone, and 400 μg / mL Tibetan tea water extract combined with 0.8 μg / mL paclitaxel.

[0031] Figure 3. Inhibition rate of HepG2 cells by using 600 μg / mL Ya'an Tibetan tea water extract alone, 0.8 μg / mL paclitaxel alone, and 600 μg / mL Ya'an Tibetan tea water extract combined with 0.8 μg / mL paclitaxel.

[0032] Figure 4. Effects of Ya'an Tibetan tea extract alone, paclitaxel alone, and Ya'an Tibetan tea extract combined with paclitaxel on HepG2 cell proliferation. A. Effects of each experimental group on the colony formation ability of HepG2 cells; B. Cell colony formation rate of each experimental group. Note: Compared with the control group, *p<0.05, **p<0.01; compared with the combined group... # p < 0.05, ## p < 0.01.

[0033] Figure 5. Effects of Ya'an Tibetan tea extract alone, paclitaxel alone, and Ya'an Tibetan tea extract combined with paclitaxel on apoptosis induction in HepG2 cells. A. Apoptosis in each experimental group under flow cytometry; B. Apoptosis rate in each experimental group. Note: *p<0.05, **p<0.01 compared with the control group; compared with the combined group. # p < 0.05, ## p < 0.01. Detailed Implementation

[0034] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0035] The Ya'an Tibetan tea water extract used in the following examples was prepared according to the following method:

[0036] Weigh 5.5g of Ya'an Tibetan tea, grind it into powder using a crusher to obtain 5g of tea powder, add 50mL of boiled deionized water, let stand for 30s, then centrifuge at 5000r / min for 5min, and collect the supernatant (or tea water). Repeat this process three times. The last two times, the tea water was obtained in the same way, but the standing time was 60s and 90s respectively, resulting in a total of 140mL of tea water. Pre-freeze the obtained tea water at -40℃ for 24h, then quickly transfer it to a vacuum freeze dryer at -45℃ and a vacuum degree <15pa for 60h. The resulting solid powder is the freeze-dried powder, named Ya'an Tibetan tea water extract.

[0037] The solvents used to dilute paclitaxel and Ya'an Tibetan tea extract in the following examples are the cell culture media used in the corresponding examples.

[0038] The data analysis and processing methods used in the following examples were as follows: each experiment was repeated three times, and the results were processed using Excel software. Data are expressed as mean ± standard deviation (± standard), and differences between groups were analyzed using t-tests. P < 0.05 was considered statistically significant.

[0039] Example 1: Western blot detection of Bcl-2, Bax, and P-gp protein expression levels in HepG2 cells

[0040] 1. Experimental Methods

[0041] With 1.8×10 4 Cells were seeded at a rate of 10 cells / well in 6-well plates, and RPMI 1640 complete medium was added to 2 mL. Cell adhesion and growth were observed to reach over 60%. Experimental groups were as follows: blank control group, paclitaxel 0.8 μg / mL, Ya'an Tibetan tea extract (concentration gradients of 0 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL), and a combination of Ya'an Tibetan tea extract and paclitaxel: 600 μg / mL Ya'an Tibetan tea extract + 0.8 μg / mL paclitaxel. Cells were observed under a microscope every 4 hours. When approximately 30% of cells at a drug concentration of 800 μg / mL died, protein samples were prepared. The culture medium and the cell suspension after trypsin digestion were collected, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The pellet was resuspended in 1 mL PBS, transferred to a 1.5 mL EP tube, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. Add the appropriate volume of BSA lysis buffer according to the amount of precipitate, and lyse on ice for 30 min. During lysis, remove the EP tube every 10 min to mix thoroughly, and immediately place it in an ice box after mixing. After lysis, centrifuge at 12000 rpm for 30 min at 4 °C, and transfer the supernatant to a new EP tube, which is the cellular protein. Determine the protein concentration according to the BCA protein concentration assay kit instructions, and store at -20 °C for later use. Prepare SDS-PAGE gels. Electrophoresis conditions: stacking gel 80 V, 40 min; separating gel 120 V, 50 min. Transfer membrane, block, and incubate with primary antibody at 4 °C overnight. Incubate with secondary antibody for 2 h. Develop with a chemiluminescent reagent A:B in a 1:1 ratio, expose using a gel imaging scanner, and quantify protein expression using ImageJ software.

[0042] 2. Experimental Results

[0043] Western blot analysis (Figure 1A) showed that, compared with the paclitaxel monotherapy group, the expression of Bcl-2 protein was weakened, the expression of Bax protein was enhanced, and the expression of P-gp was weakened in the Ya'an Tibetan tea water extract and paclitaxel combination group.

[0044] Further quantitative data in Figure 1B show that, compared with the paclitaxel monotherapy group, the combination of Ya'an Tibetan tea extract and paclitaxel significantly downregulated P-gp expression in human liver cancer cells HepG2 (P < 0.05), indicating that Ya'an Tibetan tea extract can significantly inhibit P-gp expression and has a chemosensitizing effect on paclitaxel.

[0045] Table 1. P-gp expression levels and P-gp inhibition rates in each group

[0046]

[0047]

[0048] Note: In Table 1, “P-gp expression level” is the ordinate of each group in Figure 1B (the ordinate is the ratio of the expression level of the target protein to the internal reference protein); “P-gp expression inhibition rate” = (P-gp expression level of blank control group - P-gp expression level of experimental group) / P-gp expression level of blank control group × 100%.

[0049] As shown in Table 1, the inhibition rate of P-gp expression was 17% in the 600 μg / mL Ya'an Tibetan tea extract alone group, 58% in the 0.8 μg / mL paclitaxel alone group, and 81% in the 600 μg / mL Ya'an Tibetan tea extract + 0.8 μg / mL paclitaxel combination group, which was higher than the sum of the inhibition rates of the 600 μg / mL Ya'an Tibetan tea extract alone group and the 0.8 μg / mL paclitaxel alone group. This indicates that the combination of 600 μg / mL Ya'an Tibetan tea extract and 0.8 μg / mL paclitaxel played a synergistic role in inhibiting P-gp expression.

[0050] The above experimental results show that: (1) Ya'an Tibetan tea water extract can significantly inhibit the expression of P-gp and has a chemosensitizing effect on paclitaxel; (2) 600 μg / mL Ya'an Tibetan tea water extract combined with 0.8 μg / mL paclitaxel has a synergistic effect in inhibiting P-gp expression. Example 2: In vitro inhibitory effect of Ya'an Tibetan tea water extract combined with paclitaxel on the proliferation of human liver cancer cells HepG2.

[0051] 1. Experimental Methods

[0052] HepG2 cells in good growth condition were collected, counted by flow cytometry, and seeded into 96-well plates with a volume of 100 μL per well (3000 cells). Experimental groups were as follows: blank control group, Ya'an Tibetan tea extract 400 μg / mL, Ya'an Tibetan tea extract 600 μg / mL, paclitaxel 0.8 μg / mL, and Ya'an Tibetan tea extract and paclitaxel combination group: 400 μg / mL Ya'an Tibetan tea extract + 0.8 μg / mL paclitaxel, 600 μg / mL Ya'an Tibetan tea extract + 0.8 μg / mL paclitaxel. Cell suspensions were thoroughly mixed according to the desired drug concentration gradient and seeded into the plates to ensure even distribution. Four replicates were set up for each group. Finally, a ring of PBS or culture medium was added around each well of the 96-well plate for sealing. Cells were incubated at 37℃ in a 5% CO2 incubator for 24 h. Cells that were uniformly distributed and in good condition under a microscope were then grouped. Ya'an Tibetan tea extract was dissolved at the highest concentration, mixed thoroughly, filtered through a filter membrane, and then serially diluted according to the required concentration. 100 μL of the extract was added to each well and incubated at 37°C with 5% CO2 for 24 h, 36 h, and 48 h. A preliminary experiment was then conducted: reagents were added according to the kit instructions, and the cells were incubated at 37°C with 5% CO2. OD values ​​were measured at 1 h, 2 h, 3 h, and 4 h, and the optimal reaction time was determined by selecting an OD value between 1 and 2. 10 μL of CCK-8 solution was prepared into a suspension according to the specified order and proportions and added to the wells of the plate in sequence. The plate was gently tapped to ensure thorough mixing. Light exposure must be avoided. The 96-well plate was placed in a 37°C incubator, and the OD values ​​of different components were determined using an enzyme-labeled method.

[0053] 2. Experimental Results

[0054] As shown in Figures 2 and 3 (compared with the single-use group, *P<0.05, **P<0.01), the water extract of Ya'an Tibetan tea had a significant inhibitory effect on HepG2 cells, and its inhibition rate increased after being combined with paclitaxel (P<0.01).

[0055] Example 3: Inhibitory effect of Ya'an Tibetan tea water extract combined with paclitaxel on the clonal formation of human hepatocellular carcinoma HepG2 cells.

[0056] 1. Experimental Methods

[0057] Cells were digested with trypsin and resuspended, then counted using flow cytometry. A plate colony assay was performed in 6-well plates, with 1200 cells added to each well. Experimental groups included: a blank control group, a group receiving 400 μg / mL Tibetan tea extract and 0.1 μg / mL paclitaxel, and a group receiving a combination of Ya'an Tibetan tea extract and paclitaxel (400 μg / mL Ya'an Tibetan tea extract + 0.1 μg / mL paclitaxel), with three replicates per group. Cell suspensions were added to each well to ensure single cell distribution. Cells were incubated at 37°C, with the culture medium changed every three days. Cell growth was observed during culture until visible colonies appeared and cells were dispersed. Staining was then performed. The culture plate was cleaned with PBS, and cells were fixed with methanol for at least 30 minutes. Crystal violet staining solution was then applied to the cells, with the staining time determined by the staining concentration. The staining solution was gently washed away with RO water, and the cells were observed and photographed for preservation.

[0058] 2. Experimental Results

[0059] After drug treatment, the colony-forming ability of HepG2 cells was detected using crystal violet staining. The experimental results (Figure 4) showed that the inhibitory effects of Ya'an Tibetan tea extract alone and the combination of Ya'an Tibetan tea extract and paclitaxel on HepG2 cell colony formation were significantly better than the control group (P<0.05). Furthermore, compared with paclitaxel or Ya'an Tibetan tea extract alone, the combination of Ya'an Tibetan tea extract and paclitaxel significantly enhanced the inhibitory effect on HepG2 cell colony formation.

[0060] Example 4: Apoptosis-inducing effect of Ya'an Tibetan tea water extract combined with paclitaxel on human hepatocellular carcinoma HepG2 cells.

[0061] 1. Experimental Methods

[0062] HepG2 cells in the logarithmic growth phase with a density of 80% or higher were washed twice with 3 mL PBS, digested with 1 mL of 0.25% trypsin, and centrifuged at 800 rpm for 5 minutes after digestion was terminated. The supernatant was removed, and the precipitate was resuspended in 1 mL DMEM. After 10 dilutions, the cells were counted by flow cytometry. The experimental groups were: blank control group, Ya'an Tibetan tea extract 400 μg / mL, paclitaxel 0.8 μg / mL, and combination therapy group: Ya'an Tibetan tea extract 400 μg / mL + paclitaxel 0.8 μg / mL. 2 mL DMEM was added to each well of a 12-well plate; then, the cells were injected at a rate of 4.5 × 10⁻⁶ cells per well. 4Seed a quantity of cells into 12-well plates (3 replicates per group); observe under a microscope to check if the cell density in each well is uniform and if the distribution is even. If the distribution is uneven, shake in a figure-eight motion to mix. Once uniform distribution is observed, gently place the plates in a 37°C, 5% CO2 incubator for approximately 12 hours until the cells adhere. Discard the original culture medium, add 1 mL of PBS to each well and gently wash once, then aspirate as much PBS as possible from each well. Add the appropriate drug at a volume of 2 mL per well, observe the cell status under a microscope, and return the plates to a 37°C, 5% CO2 incubator for 24 hours, handling with care.

[0063] After 24 hours of drug treatment, the cell density, adherent cell status, and number of rounded, brightened, and floating apoptotic cells in each group were observed under a microscope. The culture medium of each group was collected into 15 mL centrifuge tubes. 1 mL of PBS was added to each well and the cells were gently shaken once. Digestion was terminated with 1 mL of DMEM. After digestion was terminated, starting from the first digested well, the cells in each well were gently digested with a cell scraper. The cells in each group were collected into the corresponding centrifuge tubes and centrifuged at 700 g for 10 min.

[0064] During centrifugation, according to the well markings, take the corresponding number of 1.5 mL centrifuge tubes, label them accordingly, and add 180 μL of PBS to each tube for later use. After centrifugation, discard the supernatant, resuspend the pellet in 1 mL of PBS in each tube, and take 20 μL of the cell suspension from each tube into the corresponding labeled 1.5 mL centrifuge tube containing 180 μL of PBS. After mixing, count the cells using a flow cytometer (only count viable cells). Based on the counting results, take 8 × 10⁸ cells from each group. 4 Transfer each cell to a newly labeled 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 5 min. Discard the supernatant and resuspend the pellet in each tube with 1 mL PBS. Prepare a mixed staining solution of Annexin-V, PI, and Binding Bμffer at a ratio of 1:1:100. Prepare 100 μL of the mixed staining solution for each of the three replicates in each group and store it in the dark. Resuspend the pellet in the mixed staining solution, stain for 20 min, and then analyze.

[0065] 2. Experimental Results

[0066] Twenty-four hours after drug administration, cells in different states were counted using flow cytometry after double staining with Annexin and PI (Figure 5). (In the figure, Q1 represents necrotic cells, Q2 represents late-stage apoptosis, Q3 represents normal cells, and Q4 represents early-stage apoptosis). The results showed that no obvious apoptosis was observed in the HepG2 cells of the negative control group after staining. Compared with the use of paclitaxel or Ya'an Tibetan tea extract alone, the combined use of Ya'an Tibetan tea extract and paclitaxel significantly increased the proportion of early-stage apoptotic cells.

[0067] In summary, this invention provides the use of Tibetan tea or its extracts in the preparation of chemotherapeutic drug sensitizers, belonging to the pharmaceutical field. This invention is the first to discover that Tibetan tea aqueous extract can significantly downregulate the expression of the multidrug resistance transporter P-gp in human hepatocellular carcinoma HepG2 cells, inhibit the proliferation of HepG2 cells, reduce the drug resistance of HepG2 cells, and enhance the sensitivity of hepatocellular carcinoma cells to the chemotherapeutic drug paclitaxel. This invention also is the first to discover that the combination of Ya'an Tibetan tea aqueous extract and paclitaxel exerts a synergistic effect in inhibiting the expression of the multidrug resistance transporter P-gp in human hepatocellular carcinoma HepG2 cells. The combination of Tibetan tea aqueous extract and paclitaxel in this invention for the treatment of hepatocellular carcinoma has promising application prospects.

Claims

1. The use of Tibetan tea water extract in the preparation of paclitaxel sensitizers, characterized in that, The paclitaxel sensitizer is a drug that inhibits the expression of multidrug resistance transporters, and the multidrug resistance transporter is P-gp protein; the paclitaxel sensitizer is a drug that enhances the sensitivity of cancer cells to paclitaxel, and the cancer is liver cancer; the mass ratio of Tibetan tea extract to paclitaxel is 600:0.

8.

2. The use according to claim 1, characterized in that: The Tibetan tea mentioned is Ya'an Tibetan tea.

3. A combination drug for the prevention and / or treatment of liver cancer, characterized in that: It contains Tibetan tea water extract and paclitaxel in the same or different unit sizes for simultaneous or separate administration, as well as a pharmaceutically acceptable carrier; the mass ratio of the Tibetan tea water extract to paclitaxel is 600:0.8.

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