Method for preparing liver-protecting medicine by utilizing fructus broussonetiae extract and application of liver-protecting medicine

Abstract

The invention discloses a method for preparing a liver-protecting medicine by utilizing a paper mulberry fruit extract, which comprises the following steps: S1, extracting effective components from paper mulberry fruits by adopting a proper extraction process to obtain the paper mulberry fruit extract rich in various pharmacological active components; s2, mixing the fructus broussonetiae extract with pharmaceutically acceptable auxiliary materials according to a certain proportion, and preparing the liver protection medicine through a preparation process. The invention further discloses application of the liver-protecting medicine prepared by the method for preparing the liver-protecting medicine from the fructus broussonetiae extract. The traditional Chinese medicine composition can effectively inhibit abnormal proliferation and migration of liver cells and promote apoptosis of the liver cells, so that the effects of protecting the liver and treating HCC are achieved.

Description

Technical Field

[0001] The invention relates to a method for preparing a liver protection drug by utilizing a mulberry seed extract and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the common malignant tumors with high morbidity and mortality, which seriously threatens human health. At present, the treatment methods of HCC mainly include surgical resection, liver transplantation, chemotherapy and targeted therapy, but these methods often have problems such as limited efficacy and large side effects. Therefore, the development of new, efficient and low-toxic liver protection drugs has become an important direction of current research.

[0003] As a traditional Chinese medicine, Cuscuta australis has many functions such as nourishing the kidney, strengthening tendons and bones, clearing the liver and improving eyesight, strengthening the spleen and promoting diuresis, etc. It is widely used in ancient books to treat diseases related to the liver, kidney and spleen. Modern pharmacological studies have found that Cuscuta australis extract is rich in a variety of active ingredients, such as fatty oils, amino acids, polysaccharides, quercetin, rhein, etc. These ingredients show significant pharmacological activity in anti-oxidation, lowering blood lipids, and inhibiting tumor growth.

[0004] Therefore, a method for preparing a liver-protecting drug using a Cucurbitacin seed extract and its application are provided. Summary of the invention

[0005] The purpose of the present invention is to overcome the existing defects and provide a method and application of preparing a liver protection drug using a Cucurbitacin seed extract, which can effectively inhibit the abnormal proliferation and migration of liver cells and promote liver cell apoptosis, thereby playing a role in protecting the liver and treating HCC.

[0006] The technical solution to achieve the above purpose is: A method for preparing a liver-protecting drug using a mulberry seed extract of the present invention comprises: Step S1, extracting effective ingredients from the seeds of Cucurbitacis chinensis by using an appropriate extraction process to obtain a Cucurbitacis chinensis chinensis extract rich in multiple pharmacologically active ingredients; Step S2, mixing the Cucurbitacinum chinense extract with pharmaceutically acceptable excipients in a certain proportion, and preparing the liver protection drug through a preparation process.

[0007] Preferably, in step S1, the extraction process includes but is not limited to solvent extraction and ultrasonic-assisted extraction.

[0008] Preferably, in step S2, the dosage forms of the liver-protecting drug include but are not limited to tablets, capsules, granules and oral solutions.

[0009] The second aspect of the present invention is a method for preparing a liver-protective drug using a paper mulberry seed extract, and the liver-protective drug prepared is used for preventing and treating liver diseases.

[0010] The beneficial effects of the present invention are as follows: the present invention adopts the Cucurbitacin Seed Extract rich in multiple pharmacologically active ingredients, the Cucurbitacin Seed Extract can promote the apoptosis of liver cells by inhibiting the abnormal proliferation and migration of liver cells, and effectively protect the liver from damage; when used in combination with existing liver protection drugs or anticancer drugs, the therapeutic effect can be significantly enhanced, and the drug dosage and side effects can be reduced; the liver protection drug provided by the present invention is not only suitable for the treatment of HCC, but also can be used for the prevention and treatment of other liver diseases, and has a wide range of application potentials. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a flow chart of a method for preparing a liver-protecting drug using a Cuscuta australis seed extract according to the present invention; Figure 2 It is a curve diagram of the inhibition rate of the papaya seed extract on HepG2 cell proliferation in the present invention; Figure 3 It is a curve diagram of the inhibition rate of HepG2 cell proliferation by the combined administration of sorafenib and the extract of Cucurbitacis chinensis seeds and sorafenib in the present invention; Figure 4 It is a schematic diagram of the half inhibitory concentration of the paper mulberry fruit extract on HepG2 cells in the present invention; Figure 5 It is a schematic diagram of the half inhibitory concentration of sorafenib and the combined administration of the extract of Cucurbitacis chinensis seeds and sorafenib on HepG2 cells in the present invention; Figure 6 This is a flow cytometric measurement diagram of HepG2 cells in the control group (Control) of the present invention; Figure 7 This is a flow cytometric measurement diagram of HepG2 cells in the Broomcorn Millet Extract Group (BLE) of the present invention; Figure 8 This is a flow cytometric measurement diagram of HepG2 cells in the Sorafenib group (Sorafenib) of the present invention; Fig. 9 This is a flow cytometric measurement diagram of HepG2 cells in the combined treatment group (Combined) of the present invention; Fig.10 It is a curve diagram showing the effects of the control group (Control), Bleucocarpus chinensis extract (BLE), Sorafenib (Sorafenib) and combined treatment (Combined) on the apoptosis rate of HepG2 cells in the present invention; Fig.11 It is a schematic diagram of liver index of the model group (Normal, Model), Bleu corylifolia extract group (BLE), sorafenib group (Sorafenib) and combined drug administration group (Combined) in the present invention; Fig.12It is a schematic diagram of AFP in the model group, the paper mulberry fruit extract group, the sorafenib group and the combined drug administration group in the present invention; Fig.13 Schematic diagram of CEA in the model group, paper mulberry extract group, sorafenib group and combined drug administration group in the present invention; Fig.14 It is a schematic diagram of AST measured after the end of medication in the model group, the paper mulberry fruit extract group, the sorafenib group and the combined medication group in the present invention; Fig.15 It is a schematic diagram of ALT measured after the end of medication in the model group, the paper mulberry fruit extract group, the sorafenib group and the combined medication group in the present invention; Fig.16 It is a schematic diagram of AKP measured after the end of medication in the model group, the paper mulberry fruit extract group, the sorafenib group and the combined drug administration group in the present invention; Fig.17 It is a schematic diagram of γ-GT measured after the end of medication in the model group, the paper mulberry fruit extract group, the sorafenib group and the combined medication group in the present invention. DETAILED DESCRIPTION

[0012] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0013] The present invention will be further described below in conjunction with the accompanying drawings.

[0014] like Figure 1 As shown, a method for preparing a liver-protecting drug using a mulberry seed extract comprises: Step S1, extracting effective ingredients from the seeds of Cucurbitacis chinensis by using a suitable extraction process to obtain a Cucurbitacis chinensis chinensis extract rich in multiple pharmacologically active ingredients.

[0015] In the embodiments, the extraction process includes but is not limited to solvent extraction and ultrasonic assisted extraction.

[0016] Step S2, mixing the Cucurbitacinum chinense extract with pharmaceutically acceptable excipients in a certain proportion, and preparing the liver protection drug through a preparation process.

[0017] In the embodiments, the dosage forms of the liver-protecting drug include but are not limited to tablets, capsules, granules and oral solutions.

[0018] A liver protection drug prepared by a method for preparing a liver protection drug using a mulberry seed extract is used for preventing and treating liver diseases.

[0019] The present invention explores a combined medication scheme of the Cuscuta australis seed extract and existing liver-protecting drugs or anti-cancer drugs (such as sorafenib).

[0020] The specific implementation is as follows: Experimental methods 1. MTT assay After HCC cells were cultured to the logarithmic growth phase, they were digested and counted, and the cells were inoculated in a 96-well plate at a concentration of 104 / mL. After overnight culture, the cells were divided into groups and treated with different drugs: blank administration group, paper mulberry extract group, sorafenib group, and paper mulberry extract combined with sorafenib group. After 48 hours of action, 20 μL of MTT solution (5 mg / ml, Sigma) was added to each well for color development. After further incubation for 4 hours, DMSO (150 μL) was added to each well to dissolve the crystals. The absorbance value was detected at 490 nm using an enzyme-linked immunosorbent assay, and a cell growth curve was drawn. The cytotoxicity of paper mulberry extract on HepG2 cells was detected by MTT assay, and the half inhibitory concentration (IC50) was calculated.

[0021] 2. Flow Cytometry After HCC cells were cultured to the logarithmic growth phase, they were digested and counted, and the cells were inoculated in a 6-well plate at a concentration of 2*105 / well. After overnight culture, they were divided into groups and treated with different drugs: blank administration group, paper mulberry extract group, sorafenib group, and paper mulberry extract combined with sorafenib group. All cells were collected after 48 hours of treatment and washed with PBS. Dye was added and treated with light at room temperature for ten minutes. The cell suspension was detected by flow cytometry for apoptosis analysis.

[0022] 3. Animal model construction and drug administration C57BL / 6 mice were gavaged with diethylnitrosamine solution (prepared with saline) once a day, and the normal group was gavaged with the same proportion of saline, and the other feeding conditions were the same. Weigh and observe their survival every week. After 4 weeks, 2 mice were randomly selected from each group for dissection and the liver morphology was observed with the naked eye. The liver showed an increase in volume, nodules of varying sizes on the surface, and the cut surface was grayish white, indicating that the model was successfully established. After the model was successfully established, the mice were randomly divided into groups for drug administration: blank administration group, paper mulberry extract group, sorafenib group, and paper mulberry extract combined with sorafenib group. The blank administration group was given an equal volume of saline. Once a day for 30 consecutive days. The condition of the mice was observed every day, and the weight and food and water intake of the mice were recorded. After the end of the administration, the mice were killed, and the liver index, tumor markers and liver function indicators of each group of mice were detected.

[0023] Experimental Results 1. Effect of Cucurbitacin Extract on HepG2 Cell Proliferation The results showed that BLE inhibited the growth of HepG2 cells in a dose-dependent manner, and the combined treatment of BLE and sorafenib could significantly inhibit the growth of HepG2 cells. Figure 2 , 3 The half inhibitory concentration (IC50) of the extract of Corydalis chinensis fruit was 10.21 mg / mL. Figure 4 As shown in Figure 2, the IC50 of Sorafenib alone (Sorafenib) and Sorafenib combined with Paper Mulberry Extract (Combined) were 12.61μM and 5.56μM, respectively. Figure 5 As shown, it was shown that the inhibitory effect of sorafenib combined with papaya extract on HepG2 cells was stronger than that of single administration.

[0024] 2. Effect of Cucurbitacin Extract on Apoptosis of HepG2 Cells In order to further verify that the combined administration of sorafenib and B. chinensis extract is more effective than sorafenib alone, the research team used flow cytometry to detect the effects of B. chinensis extract and sorafenib alone or in combination on HepG2 cell apoptosis. The results showed that the apoptosis rates of HepG2 cells in the control group (Control), B. chinensis extract (BLE), sorafenib (Sorafenib) and combined treatment (Combined) were 6.08%, 27.38%, 34.69% and 52.61%, respectively. Figure 6-10 These results together confirmed that the extract of Cucurbitacin combined with sorafenib treatment can significantly affect the biological process of HCC cells.

[0025] 3. Effects of Cucurbitacin Extract on Liver Index, Tumor Markers and Liver Function Indexes AST, ALT, AKP and γ-GT in Mice with Hepatocellular Carcinoma The study found that the liver index of the BLE group, Sorafenib group and combined group was significantly lower than that of the model group (Normal, Model). Fig.11 As shown. CEA (alpha-fetoprotein) and AFP (carcinoembryonic antigen) are commonly used tumor markers in the diagnosis and prognosis of liver cancer. The results showed that the serum levels of AFP and CEA in the model group (Normal, Model) were significantly higher than those in the control group. Compared with the model group (Normal, Model), the levels of AFP and CEA in the paper mulberry extract group (BLE), sorafenib group (Sorafenib) and combined administration group (Combined) were significantly reduced, as shown in Fig.12 , 13After treatment, the levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), AKP (alkaline phosphatase) and γ-GT (γ-glutamyl transferase) in different groups were measured. It was found that the levels of AST, ALT, AKP and γ-GT in the model group were significantly increased. At the same time, the levels of AST, ALT, AKP and γ-GT in the BLE group, Sorafenib group and combined group were significantly decreased, as shown in Figure 2. Figure 14-17 shown.

[0026] The experimental results showed that the combination of Cucurbitacin seed extract and sorafenib can significantly enhance the inhibitory effect on HCC cells, reduce tumor marker levels, and improve liver function indicators.

[0027] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a liver-protecting drug using a mulberry seed extract, It is characterized in that include: Step S1, extracting effective ingredients from the seeds of Cucurbitacis chinensis by using an appropriate extraction process to obtain a Cucurbitacis chinensis chinensis extract rich in multiple pharmacologically active ingredients; Step S2, mixing the Cucurbitacinum chinense extract with pharmaceutically acceptable excipients in a certain proportion, and preparing the liver protection drug through a preparation process.

2. A method for preparing a liver-protecting drug using the extract of Cuscuta australis seed according to claim 1, It is characterized in that In step S1, the extraction process includes but is not limited to solvent extraction and ultrasonic assisted extraction.

3. A method for preparing a liver-protecting drug using the extract of Cuscuta australis seed according to claim 1, It is characterized in that In step S2, the dosage forms of the liver-protecting drug include but are not limited to tablets, capsules, granules and oral solutions.

4. A liver-protecting drug prepared by the method for preparing a liver-protecting drug using a Cucurbitacin fruit extract according to claims 1-3, which is used for preventing and treating liver diseases.

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