Application of combination of baicalein and mulberroside C in the preparation of drugs for treating liver cancer

Abstract

The application belongs to the technical field of biological medicine, and provides application of combined use of tocopherol and mulberroside C in preparation of a drug for treating liver cancer, which is combined use of tocopherol and mulberroside C in preparation of a drug for treating liver cancer. Through in-vitro cell experiments, the growth inhibition effect of tocopherol and mulberroside C on liver cancer cells is evaluated; the results show that tocopherol and mulberroside C can inhibit the growth activity of liver cancer cells to achieve the purpose of treating liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the preparation of liver cancer drugs, specifically the application of the combined use of thymol and morinone C in the preparation of drugs for treating liver cancer. Background Technology

[0002] Liver cancer is one of the most common malignant tumors of the digestive tract worldwide. Hepatocellular carcinoma (HCC) accounts for over 80% of these cases. According to global cancer statistics, liver cancer ranks sixth in incidence and third in mortality among malignant tumors. Currently, common treatments for liver cancer include surgical resection, chemotherapy, radiotherapy, ablation therapy, and targeted therapy. Chemotherapy is a common treatment method, but its overall prognosis is not satisfactory. Chemotherapy drugs suffer from a narrow therapeutic window, significant toxic side effects, and a tendency to develop drug resistance, all of which affect their clinical efficacy. Therefore, conducting joint research on various active ingredients of traditional Chinese medicine is of great significance and promising application for developing safer and more effective drugs for the prevention and treatment of liver cancer.

[0003] Thymoquinone (TQ) is the main active ingredient in black cumin seeds. Studies have found that TQ has pharmacological effects such as immunomodulation, anti-inflammation and anti-oxidation. It can significantly inhibit the proliferation, migration and invasion of various tumor cells and has important potential in tumor prevention and treatment. However, there are still few studies on its effects and mechanisms on liver cancer cells.

[0004] Sanggenone C (SC) is a flavonoid compound extracted from mulberry bark. It has anti-inflammatory, lipid-lowering, and antioxidant effects, but there are few studies on its anti-tumor properties, especially in the field of liver cancer.

[0005] To address the aforementioned problems, this invention combines thymol and morinone C as a therapeutic agent for liver cancer, providing an application of the combined use of thymol and morinone C in the preparation of drugs for treating liver cancer, thus offering new insights into drug development for liver cancer treatment. Summary of the Invention

[0006] The purpose of this invention is to provide the application of thymol and morinone C in the preparation of drugs for treating liver cancer. The combined application of thymol and morinone C in the preparation of liver cancer drugs provides a new approach for anti-liver cancer drugs.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides the application of thymol and morinone C in the preparation of a drug for treating liver cancer, wherein the drug is used to inhibit the growth, migration and invasion of liver cancer cells.

[0009] Preferably, the concentration ratio of thymequinone to morinone C is 2:1 when used in combination.

[0010] Preferably, the thymol and mulberry root ketone C have a synergistic effect when used together to enhance the inhibitory effect on liver cancer cells.

[0011] Preferably, the drug comprises thymol, morinone C, and medically approved excipients.

[0012] Preferably, the drug comprises various acceptable dosage forms, such as injections, pills, capsules, granules, tablets, or oral liquids.

[0013] The beneficial effects of this invention are:

[0014] This invention evaluated the inhibitory effects of thymol and mulberry root ketone C on the growth of liver cancer cells through in vitro liver cancer cell experiments. The results showed that thymol and mulberry root ketone C can inhibit the growth activity of liver cancer cells and suppress their migration and invasion, providing a new drug for the treatment of liver cancer. Attached Figure Description

[0015] Figure 1 The activity and IC50 of Mahlavu cells after TQ treatment in this invention are... 50 Statistical results (A represents Mahlavu cell viability; B represents IC50) 50 value; *** P < 0.001 (compared with the 0 concentration group).

[0016] Figure 2 The activity and IC50 of MNK-45 cells after TQ treatment in this invention are... 50 Statistical results (A represents MNK-45 cell viability; B represents IC50 value) 50 value; * P < 0.05, *** P < 0.001 (compared with the 0 concentration group).

[0017] Figure 3 The activity and IC50 of HCT116 cells after TQ treatment in this invention are... 50 Statistical results (A represents HCT116 cell viability; B represents IC50) 50 value; *** P < 0.001 (compared with the 0 concentration group).

[0018] Figure 4 The activity and IC50 of MDA-MB-231 cells after TQ treatment in this invention are... 50 Statistical results (A represents MDA-MB-231 cell viability; B represents IC50) 50 value; ***P < 0.001 (compared with the 0 concentration group).

[0019] Figure 5 The activity and IC50 of PANC-1 cells after TQ treatment in this invention are... 50 Statistical results (A represents PANC-1 cell viability; B represents IC50) 50 value; *** P < 0.001 (compared with the 0 concentration group).

[0020] Figure 6 The activity and IC50 of HepG2 cells after TQ treatment in this invention are... 50 Statistical results (A represents HepG2 cell viability; B represents IC50) 50 value; ** P < 0.01, *** P < 0.001 (compared with the 0 concentration group).

[0021] Figure 7 The activity and IC50 of Huh-7 cells after TQ treatment in this invention are... 50 Statistical results (A represents Huh-7 cell viability; B represents IC50) 50 value; * P < 0.05, *** P < 0.001 (compared with the 0 concentration group).

[0022] Figure 8 The activity and IC50 of Mahlavu cells after treatment with mulberry root ketone C in this invention are... 50 Statistical results (A represents Mahlavu cell viability; B represents IC50) 50 value; *** P < 0.001 (compared with the 0 concentration group).

[0023] Figure 9 This is the result of the effect of thymol combined with morinone C on Mahlavu cell viability in this invention;

[0024] Figure 10 This is a schematic diagram of the combined effect of thymol and morinone C on Mahlavu cells in this invention.

[0025] Figure 11 This is a schematic diagram of the results of the scratch test in this invention to detect the migration of Mahlavu cells by thyme combined with morin C (A is the migration area; B is the migration microscopic observation).

[0026] Figure 12This is a schematic diagram of the Transwell assay results for detecting the effects of thymol combined with morin C on the migration and invasion of Mahlavu cells in this invention (A is the cell migration and invasion results; B is the statistical results of the number of migrating cells; C is the statistical results of the number of invading cells). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the Control group; *** P < 0.001 (compared to the Control group). Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] Example: In vitro pharmacodynamic study of thymequinone combined with morinone C against liver cancer.

[0031] Thymicin combined with mulberry root ketone C was used to culture liver cancer cells in vitro to verify the inhibitory effect of thymicin combined with mulberry root ketone C on the growth of Mahlavu liver cancer cells.

[0032] It should be noted that the thymol used in this invention was purchased from Maclean (model 490-91-5, purity 99%), and the mulberry root ketone C was purchased from Baoji Chenguang Biotechnology Co., Ltd. (model 80651-76-9, purity 98%). The amounts used are all converted to 100% purity.

[0033] 1. CCK-8 assay for detecting changes in cell proliferation

[0034] Tumor cells in the logarithmic growth phase were digested, centrifuged, and the supernatant was discarded. The cells were resuspended in complete culture medium, mixed, counted, and the concentration was adjusted. The cells were then seeded into 96-well plates at a density of 3 × 10³ cells per well and incubated overnight at 37°C with 5% CO2. After 24 hours of overnight incubation, PBS containing the corresponding drug was added to each well at a ratio of 200 μL, and the cells were incubated for another 48 hours. After incubation, the liquid in the wells was discarded. A mixture of CCK-8 and culture medium was prepared at a volume ratio of 1:10. 100 μL of the CCK-8 and culture medium mixture was added to each well and the cells were incubated at 37°C for 1 hour. The absorbance (OD) of each well was then measured at 450 nm using a microplate reader. Finally, the cell viability was calculated, and the cooperativity index (CI) was calculated using CompuSyn software.

[0035] 2. Detecting cell migration

[0036] Cell scratch assay

[0037] 2.6 × 10⁶ liver cancer cells in the logarithmic growth phase were collected. 5 Cells were seeded into 6-well plates and incubated overnight at 37 °C with 5% CO2 for 24 h, at which point the cell density reached 80%-90%. A straight line was drawn in each well using a 200 μL pipette tip, the old culture medium was aspirated, and the plates were washed twice with 1 mL of PBS to remove detached cells. Serum-free medium containing 4 μM TQ, 2 μM SC, or TQ+SC (the solution contains both 4 μM TQ and 2 μM SC; the TQ+SC mentioned below refers to this solution, with a concentration ratio of 2:1) was added to each well, 2 mL per well. The cell spacing was recorded under a microscope at 0 h. After photographing, the plates were incubated for 24 h and 48 h, and the cell spacing was recorded under a microscope after 24 h and 48 h. After the experiment, the images were processed using ImageJ software to obtain the cell migration distance and calculate the cell migration rate.

[0038] Transwell chamber assay for cell migration

[0039] Cells in the logarithmic growth phase were collected, resuspended in serum-free medium, and counted to a density of 50,000 cells. 200 μL of serum-free medium containing cells and drugs was added to the upper chamber, and cells were divided into a blank control group, TQ (4 μM), SC (2 μM), and TQ+SC. 800 μL of medium containing 10% FBS was added to the lower chamber, and the cells were incubated. After incubation, the medium from both chambers was aspirated. Residual cells in the upper chamber were wiped off with a cotton swab and washed with PBS. Cells were then fixed in the lower chamber with 4% paraformaldehyde, washed with PBS, and stained with 0.1% crystal violet. Excess stain was removed with PBS, and the chambers were inverted and air-dried. Cells were photographed using a fluorescence microscope, and cell migration was calculated using ImageJ.

[0040] 3. Transwell chamber detection of cell invasion

[0041] Pre-cool the Transwell chambers and pipette tips to 4°C, dissolve the matrix gel, dilute with serum-free medium, and add to the upper chamber. Incubate at 37°C until the matrix gel solidifies. Collect cells in the logarithmic growth phase, resuspend them with TQ (4 μM), SC (2 μM), and TQ+SC, and count to a final volume of 50,000 cells. Add the cell suspension and drug solution to the upper chamber to a volume of 200 μL, and add 800 μL of medium containing 10% FBS to the lower chamber. Incubate in an incubator. After incubation, aspirate the residual liquid from the upper chamber and wash with PBS. Fix the cells in the lower chamber with 4% paraformaldehyde for 30 min, stain with 0.1% crystal violet for 15 min, wash away excess stain, and air dry upside down. Take multi-field photographs using an inverted microscope, and calculate the cell invasion number using ImageJ.

[0042] 4. Experimental Results

[0043] 4.1 Effects of thymequinone on the viability of different tumor cells

[0044] Liver cancer cells (Mahlavu), thyroid cancer cells (MNK-45), colon cancer cells (HCT116), breast cancer cells (MDA-MB-231), and pancreatic cancer cells (PANC-1) were cultured in drug-containing medium (TQ: 0-200 μM). Cell viability was assessed using the CCK-8 assay at 48 h and 72 h. CCK-8 results and IC50 values ​​were recorded. 50 Statistical values ​​show ( Figures 1-5 With increasing TQ concentration and duration of action, the survival rates of Mahlavu, MNK-45, HCT116, MDA-MB-231, and PANC-1 cells all decreased in a gradient manner, suggesting that TQ can inhibit the in vitro proliferation activity of these tumor cells in a concentration- and time-dependent manner. Among them, Mahlavu cells were the most sensitive to TQ intervention, with an IC50 of 100% after 48 hours of TQ treatment. 50The value was 21.40 ± 1.55 μM, and the IC50 value was 72 h. 50 The value further decreased to 15.87±0.89 μM, indicating that TQ had a particularly significant inhibitory effect on the proliferation of Mahlavu liver cancer cells.

[0045] To further clarify the inhibitory effect of TQ on liver cancer cells, this embodiment also tested the IC50 of two other liver cancer cell types, HepG2 and Huh-7. 50 value( Figures 6-7 The results showed that at 48 h, the IC50 values ​​of HepG2 and Huh-7 cells were significantly higher. 50 The values ​​were 31.11±2.08 μM and 33.70±4.09 μM, respectively; after extending the treatment time to 72 h, the IC50 values ​​of all cells decreased significantly, decreasing to 27.85±5.58 μM and 29.36±3.16 μM, respectively. These results indicate that ( Figures 6-7 as well as Figure 1 TQ significantly inhibited the proliferation of three types of liver cancer cells, and this effect was time-dependent. Mahlavu cells were the most sensitive to TQ, exhibiting the lowest IC50 values ​​at both time points. 50 The in vitro proliferation inhibitory activity of thymoquinone (TQ) against this subtype of hepatocellular carcinoma cells was the strongest, and cell viability decreased in a concentration-dependent manner with increasing TQ concentration. Within the range of 0–200 μM, the maximum inhibitory rate of TQ against Mahlavu cells was 82.57 ± 0.84%, and the IC50 value after 48 h of TQ treatment was [not specified]. 50 21.40 μM ( Figure 1 Therefore, Mahlavu cells will be used as the research subject in subsequent experiments.

[0046] Morus root ketone C (SC) significantly inhibited the proliferation of Mahlavu liver cancer cells. The viability of Mahlavu cells decreased with increasing SC concentration, exhibiting a concentration-dependent effect. Within the range of 0–32 μM, the maximum inhibitory rate of SC on Mahlavu cells was 83.73 ± 0.63%. The IC50 value after 48 h of treatment with morus root ketone C was [not specified in the original text]. 50 16.52 μM ( Figure 8 The effect of thymequinone combined with morinda root extract on Mahlavu cells was detected by CCK-8 assay. Figure 9 The results showed that cell viability reached its lowest point when the concentrations of thymequinone (TQ) were 20.0 μmol / L and morula ketone C (SC) were 10.0 μmol / L, and further increases in the concentrations of both compounds did not decrease cell viability. Statistical data show that... Figure 10When the concentration of thymequinone (TQ) was 20.0 μmol / L and the concentration of mulberry root ketone C (SC) was 10.0 μmol / L, the combination index (CI) was 0.68475. Since a combination index less than 1 generally indicates a synergistic effect between the drugs, the combination of TQ and SC at this concentration may have a synergistic effect. Simultaneously, the Mahlavu effect was 0.156. Further experiments can be conducted at a TQ to SC concentration ratio of 2:1 (20:10) to explore the effects of this drug combination on subjects such as liver cancer.

[0047] 4.2 Cell scratches

[0048] To investigate the effects of TQ and SC on the migration of liver cancer cells, this study employed a scratch assay, detecting cell migration rates in each group at 24 h and 48 h. Experimental groups included: Control group, TQ group (4 μM), SC group (2 μM), and TQ+SC group (concentrations 4 μM and 2 μM, concentration ratio 2:1). Results showed ( Figure 11 In the study of TQ and SC treatments, at 24 h, the cell migration rate in the blank control group was 20.11%, while the migration rates in the TQ group and the SC monotherapy group decreased to 17.14% and 17.73%, respectively. The migration rate in the TQ and SC combined treatment group further decreased to 12.43%, significantly lower than the monotherapy groups and the control group. At 48 h, the migration rates in all groups increased. The migration rate in the blank control group was 40.40%, in the TQ group it was 31.39%, in the SC group it was 20.26%, while the migration rate in the combined treatment group was only 9.49%, significantly lower than the monotherapy groups. These results indicate that both TQ and SC can inhibit the migration ability of liver cancer cells to some extent, and the inhibitory effect is significantly enhanced when they are used in combination, exhibiting a clear synergistic effect.

[0049] 4.3 Transwell chamber assay for cell migration and invasion

[0050] To investigate the regulatory effects of TQ combined with SC on the migration and invasion of liver cancer cells, this study employed a Transwell assay, treating cells (5 × 10⁶ cells per hepatocellular carcinoma) with TQ (4 μM), SC (2 μM), or TQ + SC (4 μM + 2 μM, concentration ratio 2:1). 4 The effect of TQ on cell migration and invasion behavior was assessed by counting the number of cells that passed through the Transwell chambers over 24 h. The results showed that ( Figure 12Compared with the control group, both TQ and SC monotherapy significantly inhibited the migration and invasion of tumor cells (migration: P < 0.05, P < 0.01; invasion: P < 0.001), and the inhibitory effect of the combination of the two drugs was further enhanced, significantly better than either single drug (P < 0.001), suggesting that the combination of TQ and SC can synergistically inhibit the in vitro migration and invasion of cells. All the above results show that TQ+SC (concentration 4 μM + 2 μM, concentration ratio 2:1) at a volume of 200 μL significantly inhibited the migration and invasion of 5 × 10⁵ tumor cells. 4 It has an inhibitory effect on the growth of liver cancer cells.

[0051] In summary, this invention combines thymol and morula ketone C to prepare a drug for treating liver cancer. In vitro cell experiments were conducted to evaluate the inhibitory effects of thymol and morula ketone C on the growth of liver cancer cells. The results showed that the combined use of thymol and morula ketone C inhibited the growth of liver cancer cells. When the concentration of thymol and morula ketone C was 2:1, the treatment of liver cancer could be achieved by inhibiting the growth activity of liver cancer cells.

[0052] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of thymol and morinone C in combination in the preparation of drugs for treating liver cancer, characterized in that, The drug is used to inhibit the growth, migration, and invasion of liver cancer cells.

2. The application according to claim 1, characterized in that, The concentration ratio of thymequinone to mulberry root ketone C when used in combination is 2:

1.

3. The application according to claim 1, characterized in that, The thymequinone and mulberry root ketone C have a synergistic effect when used together to enhance the inhibitory effect on liver cancer cells.

4. The application according to claim 1, characterized in that, The drug includes thymol, morinone C, and medically approved excipients.

5. The application according to claim 1, characterized in that, The drug includes various acceptable dosage forms, such as injections, pills, capsules, granules, tablets, or oral liquids.

Images