Chinese-western medicine pharmaceutical composition containing extract of six-rehmannia and its application in preparing medicine for treating liver cancer
By combining Liuwei Dihuang extract with lenvatinib, the treatment challenges of patients with intermediate and advanced liver cancer have been addressed, achieving significant synergistic anti-liver cancer effects and improving liver function, providing a new treatment option combining traditional Chinese and Western medicine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIAXING UNIV
- Filing Date
- 2023-10-31
- Publication Date
- 2026-07-07
AI Technical Summary
In the current technology, patients with mid-to-late stage liver cancer lose the opportunity for radical treatment due to the depletion of qi and blood by pathogenic factors and the deficiency of yin in the liver and kidneys. In addition, conventional treatment methods are limited, and new combination drug regimens are needed to improve the treatment effect.
The extract of Liuwei Dihuang Pills is used in combination with lenvatinib. The aqueous or alcoholic extract of Liuwei Dihuang Pills is combined with lenvatinib in a certain proportion to form a drug composition. It can be used in combination or administered separately. It is prepared by ultrasonic extraction and filtration to make solutions or suspensions of different concentrations for the treatment of liver cancer.
It significantly enhances the therapeutic effect of lenvatinib on liver cancer, reduces the dosage, and has a significant synergistic effect. In particular, the combined use of Liuwei Dihuang Pill alcohol extract and lenvatinib has the most significant effect, which can significantly inhibit the growth of liver cancer cells and improve liver function indicators.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of combined use of traditional Chinese medicine, specifically to a combination of traditional Chinese and Western medicine containing Liuwei Dihuang extract and its application in the preparation of drugs for treating liver cancer. Background Technology
[0002] Liver cancer, also known as "primary liver cancer," mainly includes several different pathological types such as hepatocellular carcinoma (HCC) and cholangiocarcinoma. In my country, most liver cancer patients have a background of hepatitis B virus infection / cirrhosis, and most are diagnosed at an intermediate or advanced stage, characterized by a large tumor burden in the liver, a high probability of portal vein tumor thrombosis, and poor liver function. Most patients have already lost the opportunity for radical treatment when they seek medical attention.
[0003] Traditional Chinese medicine, as a traditional medicine in my country, has always played an important role in the treatment and prevention of diseases. Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill) originated from "Xiao'er Yaozheng Zhijue" (Straightforward Explanation of Pediatric Drug Syndromes) by Qian Yi, a physician of the Song Dynasty. It is composed of six Chinese medicinal herbs, namely Rehmannia glutinosa, Cornus officinalis, Dioscorea opposita, Alisma plantago-aquatica, Paeonia suffruticosa, and Poria cocos, in a certain proportion. It has the effects of nourishing kidney yin and replenishing kidney essence, and is a classic Chinese medicine formula for treating kidney yin deficiency. Summary of the Invention
[0004] From the perspective of Traditional Chinese Medicine (TCM), liver cancer falls under the categories of "liver accumulation," "abdominal masses," and "accumulation," resulting from insufficient vital energy (Qi) and dysfunction of the internal organs, leading to Qi stagnation, blood stasis, phlegm accumulation, and toxin accumulation. In the middle and late stages of liver cancer, the main pathogenesis is characterized by the depletion of Qi and blood by pathogenic factors, and deficiency of liver and kidney Yin. Liuwei Dihuang Wan (Six-Ingredient Rehmannia Pill) is a widely used TCM formula for tonifying the liver and kidneys. The combined use of TCM and Western medicine in the treatment of liver cancer is widely applied clinically. Its extracts refer to the products obtained by dissolving it in different types of solvents.
[0005] This application attempts to combine Liuwei Dihuang extract (water extract or alcohol extract) with lenvatinib for administration. Results show that both the water extract and alcohol extract of Liuwei Dihuang significantly enhance the efficacy of lenvatinib in treating liver cancer. Based on this, this application provides a traditional Chinese and Western medicine composition containing Liuwei Dihuang extract and its application in the preparation of drugs for treating liver cancer.
[0006] A pharmaceutical composition comprising lenvatinib and a Liuwei Dihuang extract, wherein the Liuwei Dihuang extract is an aqueous extract or an alcoholic extract of Liuwei Dihuang pills.
[0007] In the pharmaceutical composition described above, lenvatinib and Liuwei Dihuang extract can be mixed together to form a single dosing unit, or they can be used separately as dosing units.
[0008] Optionally, the mass ratio of lenvatinib to Liuwei Dihuang extract is 1:30 to 350.
[0009] The mass of the Liuwei Dihuang extract can be understood as the mass of the dissolved water extract: Liuwei Dihuang pill powder is dispersed in the corresponding solvent for ultrasonic extraction, and then the obtained extract is filtered to obtain filtrate and residue. The mass difference between the obtained residue and the original drug is the mass of the dissolved water extract.
[0010] Optionally, the mass ratio of lenvatinib to Liuwei Dihuang extract is 1:300-350.
[0011] More preferably, the Liuwei Dihuang extract is an alcoholic extract of Liuwei Dihuang pills; the mass ratio of lenvatinib to the alcoholic extract of Liuwei Dihuang pills is 1:300-350.
[0012] Optionally, in the alcohol extract of Liuwei Dihuang Pills, the content of mononoside is not less than 20 ug / mL and the content of loganin is not less than 25 ug / mL; in the water extract of Liuwei Dihuang Pills, the content of mononoside is not less than 5 ug / mL and the content of loganin is not less than 10 ug / mL.
[0013] Optionally, the preparation of the water extract of Liuwei Dihuang Pill includes:
[0014] Liuwei Dihuang Pill powder was dispersed in ultrapure water and extracted by ultrasonication. The resulting extract was then filtered to obtain filtrate and residue. The filtrate was concentrated, dried, and mixed with sterile water. After mixing, it was filtered through a microporous membrane to obtain the final product.
[0015] Optionally, the preparation of the ethanol extract of Liuwei Dihuang Pill includes:
[0016] The filter residue was dispersed in ethanol and extracted by ultrasonication. The resulting extract was then filtered. The filtrate was concentrated and dried, then mixed with DMSO and sterile water, and finally filtered through a microporous membrane to obtain the final product.
[0017] Optionally, the conditions for ultrasonic extraction are: ultrasonication at 60℃ for 90 minutes.
[0018] Optionally, the filtration method is depressurization filtration.
[0019] Optionally, the microporous filter membrane is a 0.22 μm microporous filter membrane.
[0020] More specifically, here is a concrete example of preparing the water extract of Liuwei Dihuang Pills: Accurately weigh 15g of Liuwei Dihuang Pill powder, add 400mL of ultrapure water, sonicate at 60℃ for 90min, filter under reduced pressure, dry and weigh the filter residue, and the difference in mass between the filter residue and the original drug is the mass of the dissolved water extract. After the filtrate is concentrated and dried, add sterile water to dissolve it to 5mL, mix well, and filter through a 0.22μm microporous membrane. Prepare a stock solution of a certain concentration according to the mass of the dissolved water extract, and dilute it with DMEM medium when used in cell experiments.
[0021] A specific example of preparing the ethanol extract of Liuwei Dihuang Pills: Add 400 mL of ethanol to the above-mentioned water extraction residue (i.e., the filter residue in the water extraction process), sonicate at 60℃ for 90 min, filter under reduced pressure, dry and weigh the filter residue, and the mass difference between the two filter residues is the mass of the dissolved ethanol extract. Concentrate the filtrate and add 3 mL of DMSO and 2 mL of sterile water to dissolve it. After mixing, filter it through a 0.22 μm microporous membrane. Prepare a stock solution of a certain concentration according to the mass of the dissolved water extract. Dilute it with DMEM medium when using it in cell experiments.
[0022] This application also provides the use of the pharmaceutical composition in the preparation of an anti-liver cancer drug.
[0023] This application also provides an anti-liver cancer drug, comprising a therapeutically effective amount of the pharmaceutical composition and a pharmaceutically acceptable carrier. The therapeutically effective amount of the pharmaceutical composition may be incorporated into a pharmaceutically acceptable carrier, which may be a diluent, binder, disintegrant, lubricant, flavoring agent, fragrance, etc.
[0024] Optionally, the dosage form of the drug may be an oral preparation. The oral preparation can be formulated as an oral liquid, tablet, granule, capsule, powder, drop, or micro-pellet, but is not limited to oral preparations. All drugs in these dosage forms can be prepared using current pharmaceutical formulation methods.
[0025] This application also provides a medicine box for treating liver cancer, comprising:
[0026] Preparations containing lenvatinib;
[0027] A preparation containing Liuwei Dihuang extract, wherein the Liuwei Dihuang extract is an aqueous extract or an alcoholic extract of Liuwei Dihuang pills;
[0028] The lenvatinib and Liuwei Dihuang extract are administered simultaneously, separately, or sequentially.
[0029] Optionally, the formulation containing lenvatinib can be a direct lenvatinib drug or an oral formulation of lenvatinib with pharmaceutically acceptable excipients.
[0030] Optionally, the preparation containing Liuwei Dihuang extract can be a product obtained by concentrating and drying Liuwei Dihuang extract, or it can be a solution containing Liuwei Dihuang extract.
[0031] This application also provides a method for preparing the aforementioned medicine box, comprising:
[0032] Preparation of formulations containing lenvatinib;
[0033] Prepare formulations containing Liuwei Dihuang extract.
[0034] This application has at least one of the following beneficial effects:
[0035] (1) This application found that the water extract or alcohol extract of Liuwei Dihuang has a significant effect on promoting the treatment of liver cancer with lenvatinib.
[0036] (2) This application found that the combination of lenvatinib and Liuwei Dihuang Pill extract has a significant synergistic effect on the inhibition of liver cancer, among which LLMeOH (lenvatinib combined with high concentration of Liuwei Dihuang Pill alcohol extract) has the most obvious synergistic effect.
[0037] (3) This invention can be translated into clinical practice, providing a possibility for reducing the dosage of lenvatinib in clinical use, which is helpful for the treatment of liver cancer. Attached Figure Description
[0038] Figure 1 Figure 1 shows the results of the combined effects of Liuwei Dihuang Pill extract and lenvatinib on body weight and tumor growth in H22 tumor-bearing mice (a: body weight growth curve of each group of tumor-bearing mice; b: tumor volume change curve of each group of tumor-bearing mice; c: in vitro tumor size of tumor-bearing mice; d: tumor mass of each group of tumor-bearing mice).
[0039] Figure 2 Figure 1 shows the SOD activity results of each group of KM tumor-bearing mice;
[0040] Figure 3 Figure showing the changes in serum AST and AFP levels in tumor-bearing mice after drug administration (a: AST enzyme activity; b: AST / ALT ratio; c: serum alpha-fetoprotein (AFP) content).
[0041] Figure 4 Figure 1 shows the effect of Liuwei Dihuang Pill on the IC50 of lenvatinib (a: IC50 of HepG2 cells treated with lenvatinib alone; b: IC50 of HepG2 cells treated with the water extract of Liuwei Dihuang Pill alone; c: IC50 of HepG2 cells treated with the alcohol extract of Liuwei Dihuang Pill alone).
[0042] Figure 5 Figure 1 shows the results of the combined use of Liuwei Dihuang Pill extract and its inhibitory effect on the proliferation of liver cancer cells (a: IC50 of HepG2 cells treated with lenvatinib (water extract of Liuwei Dihuang Pill); b: IC50 of HepG2 cells treated with lenvatinib (ethanol extract of Liuwei Dihuang Pill); c: Inhibitory effect of Liuwei Dihuang Pill extract on the proliferation of HepG2 cells).
[0043] Figure 6 This is a graph showing the results of quantitative RT-qPCR detection of the P53 gene in human liver cancer cells in Example 2. Detailed Implementation
[0044] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0046] Preparation of water extract of Liuwei Dihuang Pills: Accurately weigh 15g of Liuwei Dihuang Pill powder, add 400mL of ultrapure water, sonicate at 60℃ for 90min, filter under reduced pressure, dry and weigh the filter residue. The difference in mass between the filter residue and the original drug is the mass of the dissolved water extract. After concentrating and drying the filtrate, add sterile water to prepare a solution of a certain concentration for animal experiments. After mixing, filter through a 0.22μm microporous membrane. Prepare a stock solution of a certain concentration according to the dissolved mass. Dilute with DMEM medium for cell experiments.
[0047] Preparation of the alcohol extract of Liuwei Dihuang Pill: Add 400 mL of ethanol to the above-mentioned water extraction residue (i.e., the filter residue from the water extraction process), sonicate at 60℃ for 90 min, filter under reduced pressure, dry and weigh the filter residue. The mass difference between the two filter residues is the mass of the dissolved alcohol extract. After concentrating the filtrate, add 5-10 mL of sterile water to prepare a suspension of a certain concentration for animal experiments. Alternatively, add 3 mL of DMSO and 2 mL of sterile water to dissolve (for cell experiments), mix well, filter through a 0.22 μm microporous membrane, and prepare a stock solution of a certain concentration. Dilute with DMEM medium before use in cell experiments.
[0048] The powder used for Liuwei Dihuang Pills can be any commercially available product. The Liuwei Dihuang Pills powder used in the following examples is the commercially available product Beijing Tongrentang Liuwei Dihuang Pills (water-honey pills) [Approval Number] National Medicine Approval Number Z11021283.
[0049] Lenvatinib can be any commercially available product, including active pharmaceutical ingredient (API) and finished drug product. In the following examples, the lenvatinib used is an analytically pure 99% API produced by Aladdin.
[0050] Experimental Example 1
[0051] The preparation of the drug delivery solution in this embodiment is as follows:
[0052] (1) Preparation of high-concentration water extract of Liuwei Dihuang Pills: Accurately weigh 15g of Liuwei Dihuang Pills powder, add 400mL of ultrapure water, sonicate at 60℃ for 90min, filter under reduced pressure, dry and weigh the filter residue, the difference between the mass of the filter residue and the mass of Liuwei Dihuang Pills powder is the mass of the dissolved water extract, concentrate the filtrate and prepare 0.1g / mL of high-concentration water extract of Liuwei Dihuang Pills with sterile water based on this mass, that is, the mass of dissolved water extract as described above in each milliliter of Liuwei Dihuang Pills water extract is 0.1g;
[0053] (2) Preparation of the alcohol extract of Liuwei Dihuang Pill: Add 400 mL of ethanol to the above water extraction residue, sonicate at 60℃ for 90 min, filter under reduced pressure, dry and weigh the filter residue. The difference in mass between the two filter residues is the dissolved mass of the alcohol extract. After the filtrate is concentrated, prepare 0.1 g / mL (high concentration, i.e., high concentration alcohol extract of Liuwei Dihuang Pill) and 0.01 g / mL (low concentration, i.e., low concentration alcohol extract of Liuwei Dihuang Pill) solutions with sterile water according to this mass. As understood above, the dissolved alcohol extract mass in each milliliter of high concentration alcohol extract of Liuwei Dihuang Pill is 0.1 g, and the dissolved alcohol extract mass in each milliliter of low concentration alcohol extract of Liuwei Dihuang Pill is 0.01 g.
[0054] (3) Preparation of lenvatinib solution: Accurately weigh 72 mg of lenvatinib, add 24 mL of 0.15 wt% CMC-Na solution to prepare a 3 mg / mL suspension, and vortex sonicate for 30 min to dissolve it.
[0055] Laboratory animals and grouping of drugs:
[0056] Healthy male KM mice, weighing 18–22 g, were used in the experiment. Cancer cells were injected into the backs of the mice to establish a tumor model (tumor models were established in all mice except the control group). After one week of culture, the mice were randomly divided into groups as follows:
[0057] Normal control group: 0.4 mL of distilled water was administered by gavage daily;
[0058] Tumor control group (control group): 0.4 mL of distilled water was administered by gavage daily;
[0059] Lenvatinib group (Letb group): 0.2 mL of 30 mg / kg lenvatinib was administered by gavage first, followed by 0.2 mL of normal saline by gavage 1 hour later;
[0060] Lenvatinib combined with high-concentration ethanol extract of Liuwei Dihuang Pills (LLMeOH group): Lenvatinib 0.2 mL 30 mg / kg was first administered by gavage, followed by high-concentration ethanol extract of Liuwei Dihuang Pills 0.2 mL by gavage 1 hour later;
[0061] Lenvatinib combined with low-concentration ethanol extract of Liuwei Dihuang Pills (LLE group): Lenvatinib 0.2 mL 30 mg / kg was first administered by gavage, followed by 0.2 mL of low-concentration ethanol extract of Liuwei Dihuang Pills by gavage after 1 hour;
[0062] Lenvatinib combined with high-concentration aqueous extract of Liuwei Dihuang Pills (LLH2O group): Lenvatinib 0.2 mL 30 mg / kg was first administered by gavage, followed by high-concentration aqueous extract of Liuwei Dihuang Pills 0.2 mL by gavage after 1 hour;
[0063] The group receiving high-concentration ethanol extract of Liuwei Dihuang Pills alone (HE group): 0.2 mL of high-concentration ethanol extract of Liuwei Dihuang Pills was administered by gavage first, followed by 0.2 mL of normal saline by gavage after 1 hour.
[0064] The group receiving high-concentration water extract of Liuwei Dihuang Pills alone (HH group): 0.2 mL of high-concentration water extract of Liuwei Dihuang Pills was administered by gavage first, followed by 0.2 mL of physiological saline by gavage 1 hour later.
[0065] The length, width, and weight of the tumor were measured and recorded every other day.
[0066] After 14 consecutive days of administration, blood was collected from the fundus of each mouse 2 hours after administration on the last day. After standing at room temperature for 2 hours, the serum was centrifuged at 13000 rpm for 5 minutes and the supernatant serum was placed in a 1.5 mL centrifuge tube.
[0067] Tumor volume and tumor inhibition rate calculation:
[0068] Every other day after drug administration, the size of the subcutaneous tumor model was measured using calipers, and the mice were weighed. Fourteen days later, the mice were euthanized by cervical dislocation, and the livers were surgically removed and immediately frozen at -80°C. Simultaneously, intact tumor tissue was removed for photographing, dried on absorbent paper, weighed, and the tumor weight was recorded. The tumor tissue was then stored at -80°C for subsequent experiments.
[0069] The tumor volume was calculated based on the recorded data of the major diameter L (mm) and minor diameter W (mm) of the tumor in each group of mice:
[0070]
[0071] Tumor inhibition rate (IR%) = [average tumor volume in the model group - average tumor volume in the drug-treated group] / average tumor volume in the model group × 100%.
[0072] HE staining of tumor tissue from tumor-bearing mice:
[0073] Tumor tissue immersed in formalin was dehydrated using a gradient ethanol process, cleared with xylene, embedded in paraffin, sectioned to a thickness of 3-5 μm, and stained with hematoxylin and eosin. Under a microscope, cell nuclei were stained blue-purple, while cytoplasm and non-cellular components were stained red to varying degrees.
[0074] SOD kit for detecting serum SOD activity:
[0075] In the experiment, mouse blood samples were collected and allowed to stand at room temperature for 2 hours, then centrifuged at 10,000 rpm for 10 minutes. All the supernatant serum was collected and frozen in batches at -80°C. The -80°C frozen serum was used to conduct a preliminary experiment according to the kit instructions to determine the optimal 1:5 dilution ratio for the formal experiment.
[0076] ALT and AST kits were used to detect serum levels:
[0077] Serum stored at -80℃ was used for experimental determination according to the ALT and AST kit methods.
[0078] ELISA method for detecting serum AFP, IFN-γ, and TNF-α levels:
[0079] The levels of AFP, IFN-γ, and TNF-α cytokines in serum were detected using ELISA. The specific procedure was performed according to the kit instructions.
[0080] Experimental results:
[0081] (1) Changes in mouse body weight and tumors:
[0082] Before combined administration, there was no significant difference in body weight among the groups (P > 0.05); after administration, the body weight gain in the model group was also not significantly different from that in the treatment group (P > 0.05), indicating that neither lenvatinib nor Liuwei Dihuang pills affected the body weight of tumor-bearing mice. (See results below.) Figure 1 a.
[0083] The in vivo anti-hepatocellular carcinoma activity of lenvatinib combined with Liuwei Dihuang pills was detected in an H22 hepatocellular carcinoma mouse model. Fourteen days after administration, the in vitro tumor size in each group of tumor-bearing mice was as follows: Figure 1 c in the middle, from Figure 1 As shown in Figure c, the tumor volume was smallest in the group treated with the combined Liuwei Dihuang Pill alcohol extract (including high-concentration and low-concentration groups), demonstrating a more significant inhibitory effect on tumor growth compared to the lenvatinib alone group. The tumor volume change curves for each group were measured every other day for 14 consecutive days. Figure 1 (b) shows that the tumor volume growth was slower in the treatment groups than in the control group. The group with ethanol extract of Liuwei Dihuang Pill combined with lenvatinib showed even smaller tumor growth than the other groups, indicating that lenvatinib combined with ethanol extract of Liuwei Dihuang Pill can significantly inhibit the growth of subcutaneous xenografts of liver cancer in KM tumor-bearing mice.
[0084] from Figure 1As can be seen from the data in Figure d, compared with the control group, the tumor tissue weight in the treatment group was significantly reduced by more than half, indicating that both lenvatinib and Liuwei Dihuang pills are effective for subcutaneous xenografts. Among them, the tumor tissue weight in the group using the combined ethanol extract of Liuwei Dihuang pills was the lowest, and the tumor weight in the group using the high-concentration ethanol extract of Liuwei Dihuang pills was significantly lower than that in the group using the low-concentration ethanol extract of Liuwei Dihuang pills.
[0085] In the tumor inhibition rate results (Table 1), compared with the lenvatinib alone group and the Liuwei Dihuang Pill extract alone group, the tumor inhibition rate of LLMeOH (lenvatinib combined with high-concentration Liuwei Dihuang Pill ethanol extract group) was significantly better than the sum of the tumor inhibition rates of the lenvatinib alone group and the high-concentration Liuwei Dihuang Pill extract alone group; the tumor inhibition rate of LLH2O (lenvatinib combined with high-concentration Liuwei Dihuang Pill water extract group) was also better than the sum of the tumor inhibition rates of the lenvatinib alone group and the high-concentration Liuwei Dihuang Pill water extract alone group. This demonstrates that the combination of lenvatinib and Liuwei Dihuang Pill extract has a significant synergistic effect on the inhibition of liver cancer, with the synergistic effect of LLMeOH (lenvatinib combined with high-concentration Liuwei Dihuang Pill ethanol extract group) being the most significant, further indicating that the ethanol extract of Liuwei Dihuang Pill has a better promoting effect of lenvatinib in the treatment of liver cancer.
[0086] Table 1. Comparison of tumor inhibition rates in different groups of tumor-bearing mice treated with Liuwei Dihuang Pills combined with lenvatinib.
[0087]
[0088]
[0089] (2) Effect of combined drug administration on serum SOD in tumor-bearing mice:
[0090] The SOD activity results for each group are as follows: Figure 2 Compared with the healthy control group, the SOD activity of the drug intervention groups was increased to varying degrees. Among them, the SOD activity of the lenvatinib alone group (p<0.05), the high-dose combination with alcohol extract group (p<0.01), and the low-dose combination with alcohol extract group (p<0.05) was significantly increased, with obvious differences. In the combination drug groups, there was no difference in the effect of Liuwei Dihuang Pill water extract and water extract and alcohol extract alone on SOD compared with the control group.
[0091] (3) Serum AST and ALT levels in tumor-bearing mice after drug administration:
[0092] The results of mouse serum AST detection showed that ( Figure 3 Compared with the control group, except for the lenvatinib monotherapy group and the high-concentration Liuwei Dihuang pill alcohol extract group, the AST content in the other treatment groups was significantly reduced (P<0.05), with the combined water extract and alcohol extract groups showing a more significant decrease.
[0093] AST / ALT is an indicator of liver function impairment; a higher ratio indicates greater liver damage. Among the AST / ALT ratios, the control group had the highest ratio. The ratio in the combined alcohol extract group was significantly lower than that in the control group (P < 0.05). There was no difference between the high-dose and low-dose groups. However, the combined alcohol-water extract, water extract alone, and alcohol extract alone groups showed no difference compared to the healthy group.
[0094] Alpha-fetoprotein (AFP) is a glycoprotein mainly synthesized by fetal hepatocytes. Its concentration in adult serum is extremely low. AFP is closely related to the development and progression of liver cancer and various other tumors, exhibiting high concentrations in multiple tumors and serving as a positive indicator for various cancers. Clinically, it is primarily used as a serum biomarker for primary liver cancer, for diagnosis and monitoring of treatment efficacy. Figure 3 c showed that serum AFP was higher in the control group, and all treatment groups could reduce AFP, with the most significant decreases observed in the combination of ethanol extract and the ethanol extract alone compared to the control group (P < 0.01).
[0095] The results of tumor size and serum biomarkers in different treatment groups of H22 tumor-bearing mice showed that, compared with lenvatinib alone, the combination of Liuwei Dihuang pills and lenvatinib significantly reduced tumor volume and mass, and achieved the highest tumor inhibition rate. Serum AST, AST / ALT, and AFP levels were also lowest in the ethanol extract, indicating that the ethanol extract of Liuwei Dihuang pills can significantly improve the therapeutic efficacy of lenvatinib on liver cancer in H22 tumor-bearing mice.
[0096] Experiment Example 2
[0097] Cellular experiments:
[0098] (1) Effect of Liuwei Dihuang Pill on the IC50 of Lenvatinib
[0099] HepG2 cells in the logarithmic growth phase were selected for the cck8 assay. Lenvatinib (Letb) was designed with concentration gradients of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μmol / mL; the designed concentrations of the aqueous extract of Liuwei Dihuang Pills (LH2O) were 30, 3, 1.5, 0.75, 0.3, and 0.06 mg / mL; the designed concentrations of the ethanolic extract of Liuwei Dihuang Pills (LMeOH) were 30, 3, 1.5, 0.75, 0.3, and 0.06 mg / mL; in the combination therapy group (LLH2O and LLMeOH), the concentration of the Liuwei Dihuang Pills extract was fixed at 0.1 mg / mL, and the lenvatinib concentration was set in the same manner. 24 hours after drug administration, the culture medium was discarded, and 200 μL of complete culture medium containing 10 μL of CCK8 solution was added. The mixture was incubated at 37°C for 2 hours, and the OD450 absorbance was measured using a microplate reader. Three replicates were set up for each group. The relative inhibition rate of the drug in each group was calculated.
[0100] (2) Effect of Liuwei Dihuang Pill combined with Lenvatinib on the inhibition of liver cancer cell proliferation
[0101] HepG2 cells were randomly divided into a blank group containing only culture medium and no cells, a control group without drugs (control), a lenvatinib group (Letb), a group treated with a combination of Liuwei Dihuang water extract and lenvatinib (LLH2O), and a group treated with a combination of Liuwei Dihuang pill alcohol extract and lenvatinib (LLMeOH). The concentration of Liuwei Dihuang pill was 0.1 mg / mL, and the concentration of lenvatinib was 0.001, 0.01, 0.1, 1, 10, and 100 μmol / mL. The absorbance at 450 nm was measured using an ELISA reader. The experiment was repeated three times, and the relative inhibition rate of cells in each group was calculated.
[0102] The cell proliferation inhibition rate was calculated as (D experimental group - D blank group) / (D control group - D blank group) × 100%.
[0103] Experimental results
[0104] (1) The effect of Liuwei Dihuang Pill extract on lenvatinib in hepatocellular carcinoma cells was determined by CCK8 assay, and it was found that the optimal effect was achieved when Liuwei Dihuang Pill extract was used in combination with lenvatinib at a concentration of 0.1 mg / mL in HepG2 cells. Figure 4 and Figure 5 As shown, the IC50 of lenvatinib alone in HepG2 cells was 115.60 μmol / mL, the IC50 of the water extract of Liuwei Dihuang Pills alone was 82.97 mg / mL, and the IC50 of the alcohol extract alone was 4.20 mg / mL. When combined with the water extract (0.1 mg / mL), the IC50 of lenvatinib significantly decreased to 61.83 μmol / mL. Figure 5(a) In combination with the alcohol extract (0.1 mg / mL), the IC50 of lenvatinib also decreased significantly to 48.9 μmol / mL. Figure 5 (b)
[0105] The above results show that when the concentration of the aqueous or ethanolic extract of Liuwei Dihuang Pills is 0.1 mg / mL, the extract alone does not affect the proliferation of HepG2 cells (IC50 of the aqueous or ethanolic extract is 1 / 100-1 / 20 of the concentration). Compared with lenvatinib alone, the combination group showed a 50% inhibition rate within the measured lenvatinib concentration range (0.001-100 μg / mL). Figure 5 (c) The alcohol extract showed a greater inhibition rate than the water extract. This indicates that Liuwei Dihuang extract can synergistically enhance the inhibitory effect of lenvatinib on liver cancer cells in vitro.
[0106] like Figure 6 As shown, the P53 gene in human hepatocellular carcinoma cells was quantitatively detected by RT-qPCR. In HepG2 cells, the combination of the alcohol extract and lenvatinib significantly upregulated the P53 gene compared to lenvatinib alone (P < 0.05). P53 is a tumor suppressor closely related to tumors, which can regulate cell cycle arrest, induce apoptosis, regulate metabolism, and thus inhibit tumors. When intracellular P53 activity decreases, expression is reduced, or pathways are impaired, the anti-tumor activity of P53 will weaken, leading to the occurrence and development of cancer. Cellular experiments showed that the combination of Liuwei Dihuang Wan extract and lenvatinib can promote P53 expression and increase the anti-cancer effect of lenvatinib, suggesting that the synergistic effect of Liuwei Dihuang Wan alcohol extract on the anti-hepatocellular carcinoma effect of lenvatinib may be related to its regulation of P53.
[0107] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. The use of a combination of traditional Chinese and Western medicine in the preparation of an anti-liver cancer drug, characterized in that, The traditional Chinese and Western medicine composition consists of lenvatinib and Liuwei Dihuang extract. The Liuwei Dihuang extract is an alcohol extract of Liuwei Dihuang pills. In the alcohol extract of Liuwei Dihuang pills, the content of mononoside is not less than 20 ug / mL, and the content of loganin is not less than 25 ug / mL. The mass ratio of lenvatinib to Liuwei Dihuang extract is 1:30~350.
2. The use according to claim 1, characterized in that, The mass ratio of lenvatinib to Liuwei Dihuang extract is 1:300~350.
3. The use according to claim 1, characterized in that, Preparation of the alcohol extract of Liuwei Dihuang Pill: Liuwei Dihuang Pill powder was dispersed in ultrapure water and extracted by ultrasonication. The resulting extract was then filtered to obtain filtrate and residue. The residue was dispersed in ethanol and extracted by ultrasonication. The resulting extract was then filtered. The filtrate was concentrated and dried, then mixed with dimethyl sulfoxide and sterile water, and finally filtered through a microporous membrane to obtain the final product.