Regulation and control medicine for inhibiting liver cancer angiogenesis and application of regulation and control medicine

Abstract

The invention relates to a regulation medicine for inhibiting liver cancer angiogenesis and application thereof, and belongs to the technical field of tumor biology. The medicine takes moringa seed polysaccharide as an active ingredient, the molecular weight range of the moringa seed polysaccharide is 5-10 kDa, and the moringa seed polysaccharide medicine is prepared through the steps of ultrasonic-assisted water extraction, Sevage reagent deproteinization, dialysis, ultrafiltration membrane grading purification and the like. Experimental results show that the moringa seed polysaccharide can significantly inhibit angiogenesis induced by liver cancer cells and has no obvious toxicity to normal cells. In addition, the moringa seed polysaccharide and the sorafenib are combined for use, so that a remarkable synergistic effect can be achieved, and the inhibition effect on the growth of the liver cancer is further enhanced. The invention has the advantages of simple preparation process, high safety and the like, provides a new drug choice for liver cancer treatment, and has important clinical application value and social benefit.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor biology, and particularly relates to a regulatory drug for inhibiting angiogenesis in liver cancer and its application. Background Art

[0002] Hepatocellular Carcinoma (HCC) is a common malignant tumor clinically, and its incidence has shown an increasing trend in recent years. According to relevant statistical data, the number of HCC patients in China accounts for about 55% of the total global cases, making China one of the countries with the heaviest burden of HCC globally. This severe situation not only poses a huge threat to public health but also raises higher requirements for clinical treatment.

[0003] Currently, the treatment strategies for HCC mainly include traditional methods such as surgical resection, radiotherapy (RT), and chemotherapy (CT). However, these treatment methods all have certain limitations in clinical applications. For example, although surgical resection is the main treatment method for early HCC, due to the strong invasiveness of the tumor or impaired liver function of the patient, it is often difficult to completely remove the tumor tissue, and the postoperative recurrence rate is relatively high. Although RT and CT can directly kill tumor cells, their non-specific toxicity often leads to significant adverse reactions, such as myelosuppression, gastrointestinal symptoms, and decreased immune function, seriously affecting the quality of life of patients.

[0004] In this context, researchers have gradually focused on this key link of tumor angiogenesis. Research shows that the growth, invasion, and metastasis of tumors highly depend on the formation of new blood vessels, that is, tumor angiogenesis. By inhibiting tumor angiogenesis, the nutrient supply and metabolic waste excretion channels of tumor cells can be cut off, thereby effectively curbing the growth and spread of tumors. This mechanism provides a new idea for the treatment of HCC and promotes the research and application of anti-angiogenic drugs. In recent years, the treatment strategy targeting vascular endothelial growth factor (VEGF) and its receptor (VEGFR) has shown certain potential in clinical practice, but it still needs to be further optimized to improve the efficacy and reduce the risk of drug resistance.

[0005] In summary, aiming at the deficiencies of traditional methods in the treatment of HCC, exploring new treatment strategies based on inhibiting tumor angiogenesis not only helps to overcome the limitations of existing therapies but also lays a theoretical foundation for the development of more efficient and safe treatment regimens. Summary of the Invention

[0006] The present invention aims to provide a regulatory drug capable of effectively inhibiting the angiogenesis of liver cancer, thereby achieving effective inhibition of the growth of liver cancer. In addition, by using this drug in combination with existing liver cancer treatment drugs, the inhibitory effect on the growth of liver cancer can be further enhanced, achieving a better therapeutic effect.

[0007] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides a regulatory drug for inhibiting the angiogenesis of liver cancer, the drug contains an effective dose of moringa seed polysaccharide as an active ingredient, and the molecular weight range of the moringa seed polysaccharide is 5 - 10 kDa; The moringa seed polysaccharide is prepared by the following preparation method: (1) After defatting the dried moringa seed powder, ultrasonic-assisted water extraction is used to extract crude moringa seed polysaccharide; (2) Remove proteins with Sevage reagent and dialyze to remove small molecule impurities; (3) Use ultrafiltration membrane for fractional purification, collect the fraction with a molecular weight of 5 - 10 kDa, and freeze-dry to obtain moringa seed polysaccharide.

[0008] Preferably, in the above preparation method: The material-liquid ratio of the ultrasonic-assisted water extraction method is 1:20; the parameters of the ultrasonic-assisted water extraction method are frequency 40 kHz, power 200 W, temperature 40 °C, and time 30 minutes; The molecular weight cut-off for dialysis is 3.5 kDa, and the dialysis time is 48 h; The ultrafiltration membrane fractional purification is to filter successively with ultrafiltration membranes with a molecular weight cut-off of 50 kDa, 10 kDa, and 5 kDa, and collect the 5 - 10 kDa retentate.

[0009] Preferably, in the regulatory drug, the effective dose of the moringa seed polysaccharide is 50 - 200 μg / mL.

[0010] Preferably, the detailed preparation method of the moringa seed polysaccharide is as follows: (1) Wash fresh moringa seeds with deionized water to remove surface impurities, then put them in an oven and dry until the moisture content ≤ 10%, and then grind them into 60-mesh moringa seed powder with a ball mill; (2) Add petroleum ether to the moringa seed powder at a material-liquid ratio of 1:5, carry out defatting treatment at 60 °C for 2 hours, filter and collect the powder; add petroleum ether again at the same material-liquid ratio, filter after defatting at 60 °C for 2 hours, and place the powder in a fume hood to volatilize the residual solvent to obtain defatted moringa seed powder; (3) Mix the defatted Moringa oleifera seed powder with deionized water at a solid-liquid ratio of 1:20, and perform ultrasonic-assisted extraction for 30 minutes in an intermittent mode of ultrasonic wave for 5 seconds and pause for 2 seconds under the conditions of a frequency of 40 kHz, a power of 200 W, and a temperature of 40 °C; (4) Centrifuge at 5000 rpm for 15 minutes, and collect the supernatant, namely the Moringa oleifera seed extract; (5) Add an equal volume of Sevage reagent (the ratio of chloroform to n-butanol is 4:1) to the Moringa oleifera seed extract, shake vigorously for 30 minutes, then centrifuge at 4000 rpm for 10 minutes, collect the upper aqueous phase, and repeat this operation 5 times to obtain a protein-free Moringa oleifera seed extract; (6) Load the protein-free Moringa oleifera seed extract into a dialysis bag with a molecular weight cut-off of 3.5 kDa, and dialyze against running water for 48 hours to obtain a crude extract of Moringa oleifera seed polysaccharide; (7) Concentrate the crude extract of Moringa oleifera seed polysaccharide to 1 / 10 of its original volume using a rotary evaporator to obtain a concentrated solution; (8) First, filter the concentrated solution through a 50 kDa ultrafiltration membrane, then filter the filtrate a obtained through a 10 kDa ultrafiltration membrane, and then filter the filtrate b obtained through a 5 kDa ultrafiltration membrane, and collect the retentate c separately to obtain Moringa oleifera seed polysaccharide; (9) Place the Moringa oleifera seed polysaccharide in a freeze dryer and freeze-dry it to obtain Moringa oleifera seed polysaccharide.

[0011] Secondly, the present invention provides the use of Moringa oleifera seed polysaccharide in the preparation of a drug for treating liver cancer, and the Moringa oleifera seed polysaccharide is prepared by the above preparation method.

[0012] Preferably, in the drug for treating liver cancer, the effective dose of the Moringa oleifera seed polysaccharide is 50 - 200 μg / mL; The drug for treating liver cancer is used to inhibit the proliferation of liver cancer cells and the angiogenesis induced by liver cancer cells.

[0013] Preferably, the inhibition of the proliferation of liver cancer cells is the inhibition of the proliferation of HepG2 cells. HepG2 cells are a commonly used human liver cancer cell line and are derived from the liver tissue of liver cancer patients.

[0014] Preferably, the angiogenesis induced by liver cancer cells is the angiogenesis of vascular endothelial cells induced by liver cancer cells. Vascular endothelial cells (HUVEC): that is, human umbilical vein endothelial cells, which are a representative of vascular endothelial cells.

[0015] In addition, the present invention provides a drug composition for synergistically inhibiting the growth of liver cancer, and the drug composition is composed of Moringa oleifera seed polysaccharide and sorafenib; In the pharmaceutical composition, the moringa seed polysaccharide is prepared by the above preparation method; the concentration of the moringa seed polysaccharide is 50 - 200 μg / mL; The concentration of sorafenib is ≥5 μM.

[0016] Preferably, the pharmaceutical composition inhibits the growth of liver cancer by inhibiting angiogenesis induced by liver cancer cells.

[0017] Synergistic inhibition means that when two or more drugs are used in combination, their efficacy is better than the sum of the efficacy of each drug used alone. In the present invention, the combined use of moringa seed polysaccharide and sorafenib can play a synergistic role and more effectively inhibit the growth and angiogenesis of liver cancer.

[0018] Finally, the present invention provides an application of a pharmaceutical composition in the preparation of a drug for synergistically inhibiting the growth of liver cancer, characterized in that the pharmaceutical composition is composed of moringa seed polysaccharide and sorafenib; The moringa seed polysaccharide is prepared by the above preparation method; In the pharmaceutical composition, the concentration of the moringa seed polysaccharide is 50 - 200 μg / mL; The concentration of sorafenib is ≥5 μM.

[0019] Preferably, the pharmaceutical composition achieves the effect of inhibiting the growth of liver cancer by synergistically inhibiting angiogenesis induced by liver cancer cells.

[0020] The beneficial effects of the present invention are as follows: 1. The present invention for the first time discovers that moringa seed polysaccharide C (molecular weight 5 - 10 kDa) has a significant inhibitory effect on liver cancer angiogenesis, providing a novel natural - sourced candidate drug for the treatment of liver cancer. At the same time, moringa seed polysaccharide C has no significant toxicity to normal cells, indicating its selective effect on tumor cells and avoiding non - specific damage to normal tissues.

[0021] 2. The present invention further explores the combined application of moringa seed polysaccharide and the existing liver cancer treatment drug sorafenib, and finds that they have a significant synergistic effect. According to the Bliss independence model analysis, the actual inhibitory effect of the combined drug use is significantly higher than the expected inhibitory effect. For example, in in vitro experiments, the actual inhibition rate of combined group c is 77.35%, much higher than the expected 65.97%; in in vivo experiments, the actual inhibition rate of the combined group is 68.27%, significantly higher than the expected 51.15%. This synergistic effect not only improves the curative effect but also overcomes the drug resistance problem of single - drug use. Description of the Drawings

[0022] Figure 1 : Effect of moringa seed polysaccharide C on the viability of human umbilical vein endothelial cells (HUVEC); Figure 2 : In vitro inhibitory effect of Moringa oleifera seed polysaccharide C on angiogenesis induced by hepatocellular carcinoma cells; Figure 3 : In vitro anti-angiogenic effect of combined use of Moringa oleifera seed polysaccharide C and sorafenib; Figure 4 : In vivo anti-angiogenic effect of combined use of Moringa oleifera seed polysaccharide C and sorafenib; Figure 4 Among them, A is the actual detection result of angiogenesis in the chick embryo chorioallantoic membrane, and B is the statistical histogram of angiogenesis in the chick embryo chorioallantoic membrane. Specific implementation mode

[0023] To clearly illustrate the technical characteristics of this solution, the following elaborates on this solution through specific implementation modes.

[0024] In existing research, Moringa oleifera seed polysaccharide has mainly been applied in the fields of anti-inflammatory and antioxidant, and its potential biological functions also mainly focus on these aspects. However, regarding the role of Moringa oleifera seed polysaccharide in the treatment of liver cancer, there has been no systematic research or clear report so far. Based on this, this study aims to explore the role of Moringa oleifera seed polysaccharide in the treatment of liver cancer, with a view to providing a scientific basis and new research direction for its application in the field of tumor treatment.

[0025] Example 1: Obtaining of Moringa oleifera seed polysaccharide 1. Raw material selection and treatment Select fresh Moringa oleifera seeds without mildew and pollution, and use deionized water to rinse to remove surface impurities; Place the Moringa oleifera seeds in an oven and dry until the moisture content ≤ 10%; Use a ball mill to grind to 60 mesh to obtain Moringa oleifera seed powder.

[0026] 2. Defatting treatment Add petroleum ether according to a solid-liquid ratio of 1:5, and perform defatting treatment at 60 °C for 2 hours; Filter and collect the Moringa oleifera seed powder, and add petroleum ether again according to a solid-liquid ratio of 1:5, and perform defatting treatment at 60 °C for 2 hours; Filter and collect the powder, and place it in a fume hood to volatilize the residual solvent to obtain defatted Moringa oleifera seed powder.

[0027] 3. Ultrasonic-assisted extraction Mix the defatted Moringa oleifera seed powder with deionized water according to a solid-liquid ratio of 1:20, and perform ultrasonic-assisted extraction according to a frequency of 40 kHz, a power of 200 W, a temperature of 40 °C, a time of 30 minutes, and an intermittent mode: ultrasonic for 5 seconds and pause for 2 seconds; Centrifuge (5000 rpm, 15 minutes) to collect the supernatant to obtain Moringa oleifera seed extract.

[0028] 4. Protein removal Add an equal volume of Sevage reagent (chloroform: n-butanol = 4:1) to the Moringa oleifera seed extract, and shake vigorously for 30 minutes; Centrifuge (4000 rpm, 10 minutes), and collect the upper aqueous phase; In the aqueous phase, add an equal volume of Sevage reagent again and repeat 5 times to obtain a protein-free Moringa oleifera seed extract.

[0029] 5. Dialysis to remove salts and small molecules Load the protein-free Moringa oleifera seed extract into a dialysis bag (cut-off molecular weight 3.5 kDa), and dialyze against running water for 48 hours to obtain a crude extract of Moringa oleifera seed polysaccharide.

[0030] 6. Ultrafiltration membrane fractionation and purification Concentrate the crude extract of Moringa oleifera seed polysaccharide to 1 / 10 of its original volume using a rotary evaporator to obtain a concentrated crude extract of Moringa oleifera seed polysaccharide; Filter the concentrated crude extract of Moringa oleifera seed polysaccharide using a 50 kDa ultrafiltration membrane, and collect the retentate a to obtain a solution of Moringa oleifera seed polysaccharide A; Filter the filtrate a using a 10 kDa ultrafiltration membrane, and collect the retentate b to obtain a solution of Moringa oleifera seed polysaccharide B; Filter the filtrate b using a 5 kDa ultrafiltration membrane, and collect the retentate c and the filtrate c to obtain solutions of Moringa oleifera seed polysaccharide C and Moringa oleifera seed polysaccharide D; Place the obtained solutions of Moringa oleifera seed polysaccharide A, Moringa oleifera seed polysaccharide B, Moringa oleifera seed polysaccharide C, and Moringa oleifera seed polysaccharide D in a freeze dryer, and freeze-dry at -50 °C and 10 Pa to obtain Moringa oleifera seed polysaccharide A, Moringa oleifera seed polysaccharide B, Moringa oleifera seed polysaccharide C, and Moringa oleifera seed polysaccharide D.

[0031] Example 2 Detect the inhibitory effect of the 4 different molecular weight Moringa oleifera seed polysaccharides prepared in Example 1 on the growth of liver cancer cells.

[0032] 1. Dissolve the 4 polysaccharides separately in serum-free DMEM medium to 2 mg / mL; 2. Dilute the stock solutions of the 4 polysaccharides with serum-free medium to 50, 100, 200, 400 μg / mL, prepare fresh before use, and add 10% FBS as needed; 3. Digest the HepG2 cells in the logarithmic growth phase with trypsin, and terminate the digestion with serum-containing medium; 4. Count after trypan blue staining, and adjust the cell density to 5×10 4 cells / mL; 5. Add 100 μL of cell suspension (containing 5×103 cells), and the edge wells were filled with sterile PBS to reduce edge effects; 6. Incubate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere to the surface.

[0033] 7. Treat the cells according to the following grouping settings: Experimental group: Treat with Moringa oleifera seed polysaccharides A, B, C, and D at final concentrations of 50, 100, and 200 μg / mL, respectively; Control group: Treat with DMEM medium containing 10% FBS; Blank control group: Only contains medium and no cells; Set 3 replicate wells for each treatment; 8. Discard the old medium, and after treatment according to the grouping, place it in the cell incubator and continue to culture for 48 h.

[0034] 9. After the culture is completed, add 10 μL of CCK-8 reagent to each well, gently mix, and then place it in the cell incubator and continue to incubate for 2.5 h; 10. Place it in an enzyme-linked immunosorbent assay (ELISA) reader and measure the absorbance of each well at a wavelength of 450 nm. The results obtained are shown in Table 1.

[0035] Table 1 Detection results of the inhibitory effect of Moringa oleifera seed polysaccharide A

[0036] Table 2 Detection results of the inhibitory effect of Moringa oleifera seed polysaccharide B

[0037] Table 3 Detection results of the inhibitory effect of Moringa oleifera seed polysaccharide C

[0038] Table 4 Detection results of the inhibitory effect of Moringa oleifera seed polysaccharide D

[0039] As can be seen from the results in Tables 1-4, Moringa oleifera polypeptides A and Moringa oleifera seed polysaccharide D did not have a significant inhibitory effect on the growth of hepatocellular carcinoma cells HepG2. In contrast, Moringa oleifera seed polysaccharides B and C could effectively inhibit the growth of HepG2 cells, and among them, the effect of Moringa oleifera seed polysaccharide C was more significant.

[0040] Example 3: Moringa oleifera seed polysaccharide C has no cytotoxicity to normal cells 1. Digest human umbilical vein endothelial cells in the logarithmic growth phase with trypsin and terminate the digestion with serum-containing medium; 2. Count after trypan blue staining and adjust the cell density to 1×105 cells / mL; 3. Add 100 μL of cell suspension (containing 1×10 4 cells) to each well of a 96-well plate, and fill the edge wells with sterile PBS to reduce edge effects; 6. Incubate in a 37°C, 5% CO2 incubator until the cell confluence reaches over 90%; 7. Treat the cells according to the following grouping settings: Experimental group: Treat with Moringa oleifera seed polysaccharide C at final concentrations of 50, 100, and 200 μg / mL respectively; Control group: Treat with DMEM medium containing 10% FBS; Blank control group: Only contains medium and no cells; Set 3 replicate wells for each treatment; 8. Discard the old medium, place it in the cell incubator after treatment according to the grouping, and continue to culture for 24 h.

[0041] 9. After the culture is completed, add 10 μL of CCK-8 reagent to each well, gently mix, and then place it in the cell incubator and continue to incubate for 2.5 h; 10. Place it in an enzyme-linked immunosorbent assay (ELISA) reader, detect the absorbance of each well at a wavelength of 450 nm, calculate the cell viability, and the results obtained are as Figure 1 shown.

[0042] From Figure 1 the results, it can be seen that within the concentration range detected in the present invention, no obvious toxicity is exhibited to normal cells.

[0043] Example 4: Moringa oleifera seed polysaccharide C reduces angiogenesis induced by hepatoma cells 1. Digest HepG2 cells in the logarithmic growth phase with trypsin and terminate the digestion with serum-containing medium; 2. Count after trypan blue staining and adjust the cell density to 5×10 4 cells / mL; 3. Add 2 mL of cell suspension to each well of a 6-well plate and incubate in a cell incubator until the cell density is greater than 80%; 4. Pretreat the cells with 50, 100, and 200 μg / mL of Moringa oleifera seed polysaccharide C without serum for 12 h, and pretreat the control group with serum-free medium; Replace with fresh serum-free medium, continue to culture for 24 h, and centrifuge to collect the supernatant; 5. Resuspend human umbilical vein endothelial cells (2×10 5cells / mL), 100 μL of the cell suspension was inoculated onto Matrigel matrix gel and incubated in a cell culture incubator for 4 hours. Ten fields of view were randomly selected to calculate the number of tube formations, and the results obtained were as Figure 2 shown in Table 5.

[0044] Table 5 Inhibitory effect of Moringa oleifera seed polysaccharide C on angiogenesis induced by liver cancer cells

[0045] From Figure 2 the results shown in Table 5, it can be seen that the ability of liver cancer cells treated with Moringa oleifera seed polysaccharide C to induce angiogenesis was significantly reduced, indicating that Moringa oleifera seed polysaccharide C prepared in the present invention has the effect of inhibiting angiogenesis induced by liver cancer cells.

[0046] Example 5: Inhibition of angiogenesis induced by liver cancer cells by Moringa oleifera seed polysaccharide C combined with sorafenib Sorafenib is a first-line targeted drug widely used in the treatment of liver cancer. As a multi-kinase inhibitor, sorafenib can delay disease progression by inhibiting tumor cell proliferation and angiogenesis. However, there are many limitations in the clinical application of this drug: firstly, its price is expensive, bringing a heavy economic burden to patients and their families; secondly, the incidence of adverse reactions is relatively high. Therefore, the present invention attempts to combine Moringa oleifera seed polysaccharide C with sorafenib to inhibit angiogenesis caused by liver cancer cells.

[0047] 1. Digest HepG2 cells in the logarithmic growth phase with trypsin and terminate digestion with serum-containing medium; 2. Count after trypan blue staining and adjust the cell density to 5×10 4 cells / mL; 3. Add 2 mL of the cell suspension to each well of a 6-well plate and culture in a cell culture incubator until the cell density is greater than 80%; 4. Treat the cells according to the following groups: The control group was pretreated with serum-free medium for 12 h; The Moringa oleifera seed polysaccharide C group was pretreated with 50, 100, 200 μg / mL of Moringa oleifera seed polysaccharide C for 12 h; The sorafenib group was pretreated with 5 μM sorafenib for 12 h; The combined groups a, b, and c were pretreated with 50, 100, 200 μg / mL of Moringa oleifera seed polysaccharide C combined with 5 μM sorafenib for 12 h respectively; Replace with fresh serum-free medium and continue to culture for 24 h, then centrifuge to collect the supernatant; 5. Resuspend human umbilical vein endothelial cells (2×105 cells / mL), 100 μL of the cell suspension was inoculated onto Matrigel matrix gel and incubated in a cell culture incubator for 4 hours. Ten fields of view were randomly selected to calculate the number of tube formations, and the results obtained were as Figure 3 shown in Table 6.

[0048] Table 6 Inhibitory effect of Moringa oleifera seed polysaccharide C combined with sorafenib on angiogenesis induced by liver cancer cells detected in vitro

[0049] According to the Bliss independence model, the expected effect of the combination group was calculated by the following formula for the expected inhibitory effects of combination group a, combination group b, and combination group c: E combination = E A + E B - (E A × E B) The calculated expected inhibitory effects are shown in Table 6.

[0050] As can be seen from the results in Table 6, when 50, 100, and 200 μg / mL of Moringa oleifera seed polysaccharide C were combined with sorafenib respectively, they all had a significant inhibitory effect on angiogenesis induced by liver cancer cells. At the same time, according to the results of the Bliss independence model, the actual inhibitory effects of combination group a, combination group b, and combination group c were all significantly higher than the expected inhibitory effects, indicating that when Moringa oleifera seed polysaccharide C and sorafenib were combined, they could synergistically inhibit angiogenesis induced by liver cancer cells.

[0051] Example 5: Further verification of the synergistic inhibition of angiogenesis induced by liver cancer cells by Moringa oleifera seed polysaccharide C combined with sorafenib through in vivo experiments (chorioallantoic membrane angiogenesis experiment in chicken embryos) 1. Obtain fresh hatching eggs, set up 4 groups with 5 eggs in each group, and incubate them in an incubator at 37 °C and 60% humidity for 7 days, turning them 3 times a day; 2. On the 8th day, clean the eggshell with 75% alcohol, make a window on the egg air chamber with sterile forceps and sterile scissors, with a diameter of about 2 cm, carefully remove the eggshell and its inner shell membrane to expose the chorioallantoic membrane of the chicken embryo; 3. Place a medium-speed qualitative filter paper with a diameter of 5 mm at the opening of the air chamber. In the control group, add 50 μL of the culture medium supernatant obtained from the control group in Example 4. In the Moringa oleifera seed polysaccharide C group, add 50 μL of the culture medium supernatant obtained from the 100 μg / mL Moringa oleifera seed polysaccharide C group in Example 4. In the sorafenib group, add the culture medium supernatant obtained from the sorafenib group in Example 4. In the combination group, add the culture medium supernatant obtained from combination group b in Example 4; 4. Use a breathable dressing to seal the opening of the air chamber, put the treated hatching eggs into the incubator, and add the culture medium supernatant once every 8 h, 50 μL each time; Treat continuously for 3 days, then place in an incubator. On the 11th day, observe the angiogenesis situation, randomly select 5 fields of view, and calculate the vascular coverage rate.

[0052] Table 7 Inhibitory effect of Moringa oleifera seed polysaccharide C combined with sorafenib on angiogenesis induced by hepatocellular carcinoma cells detected in in vivo experiments

[0053] From Table 7 and Figure 4 (a is the control group, b is the Moringa oleifera seed polysaccharide C group, c is the sorafenib group, d is the combined group) The results of the chicken embryo chorioallantoic membrane angiogenesis experiment showed that compared with the Moringa oleifera seed polysaccharide C group and the sorafenib group, the vascular coverage rate of the combined group was significantly reduced, and the inhibition rate (68.27%) was significantly higher than the expected value of the Bliss model (51.15%), further verifying that the combination of Moringa oleifera seed polysaccharide C and sorafenib has a significant synergistic inhibitory effect on angiogenesis induced by hepatocellular carcinoma cells.

Claims

1. A regulatory drug for inhibiting angiogenesis in liver cancer, characterized in that, The drug contains an effective dose of moringa seed polysaccharide as an active ingredient, and the molecular weight range of the moringa seed polysaccharide is 5 - 10 kDa; The moringa seed polysaccharide is prepared by the following preparation method: (1) After defatting the dried moringa seed powder, ultrasonic-assisted water extraction is used to extract the crude moringa seed polysaccharide; (2) Remove proteins with Sevage reagent and dialyze to remove small molecule impurities; (3) Use ultrafiltration membrane for fractional purification, collect the fraction with a molecular weight of 5 - 10 kDa, and freeze-dry to obtain moringa seed polysaccharide.

2. The regulatory drug according to claim 1, wherein In the said preparation method: The solid-liquid ratio of the ultrasonic-assisted water extraction method is 1:20; the parameters of the ultrasonic-assisted water extraction method are frequency 40 kHz, power 200 W, temperature 40°C, and time 30 minutes; The molecular weight cut-off for dialysis is 3.5 kDa, and the dialysis time is 48 h; The ultrafiltration membrane fractional purification is to sequentially filter with ultrafiltration membranes with molecular weight cut-offs of 50 kDa, 10 kDa, and 5 kDa, and collect the 5 - 10 kDa retentate.

3. The regulatory drug according to claim 2, wherein In the said regulated drug, the effective dose of the moringa seed polysaccharide is 50 - 200 μg / mL.

4. Use of Moringa oleifera seed polysaccharide in the preparation of a drug for treating liver cancer, characterized in that, The moringa seed polysaccharide is prepared by the preparation method described in claim 1 or 2.

5. According to the application described in claim 4, in the liver cancer treatment drug, the effective dose of the moringa seed polysaccharide is 50 - 200 μg / mL; The liver cancer treatment drug is used to inhibit the proliferation of liver cancer cells and the angiogenesis induced by liver cancer cells.

6. A pharmaceutical composition for synergistically inhibiting the growth of liver cancer, characterized in that, The pharmaceutical composition consists of moringa seed polysaccharide and sorafenib; In the said pharmaceutical composition, the moringa seed polysaccharide is prepared by the preparation method described in claim 1 or 2; the concentration of the moringa seed polysaccharide is 50 - 200 μg / mL; The concentration of sorafenib is ≥5 μM.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutical composition inhibits liver cancer growth by inhibiting the angiogenesis induced by liver cancer cells.

8. Use of a pharmaceutical composition in the preparation of a drug for synergistically inhibiting the growth of liver cancer, characterized in that, The pharmaceutical composition consists of moringa seed polysaccharide and sorafenib; The moringa seed polysaccharide is prepared by the preparation method described in claim 1 or 2; In the said pharmaceutical composition, the concentration of the moringa seed polysaccharide is 50 - 200 μg / mL; The concentration of sorafenib is ≥5 μM.

9. The application according to claim 8, characterized in that, The pharmaceutical composition achieves the effect of inhibiting liver cancer growth by synergistically inhibiting the angiogenesis induced by liver cancer cells.

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