Preparation of solanum nigrum root extract and application of solanum nigrum root extract in treatment of acute myelogenous leukemia
By using sunflower root extract to inhibit the proliferation of malignant cells, the problem of difficulty in effectively treating acute myeloid leukemia in the prior art is solved, and the effect of prolonging survival and improving the condition is achieved.
Patent Information
- Application Number
- CN202510061033.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is difficult to effectively treat acute myeloid leukemia, especially in inhibiting malignant cell proliferation and improving disease status.
Sunflower root extract is used as a drug component to treat acute myeloid leukemia by inhibiting malignant cell proliferation. The specific method includes crushing the extract of the sunflower root, adding 50% ethanol at a feed-liquid ratio of 1:10, ultrasonic assisted extraction, and lyophilization to obtain the dried extract by vacuum freeze drying.
Sunflower root extract can effectively inhibit the proliferation of acute myeloid leukemia cells, prolong the survival of mice, improve organ lesions, and reduce the expression of Ki67, a malignant cell proliferation-related protein in the spleen.
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Figure CN119868453A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of plant extracts, and in particular to application of a nightshade root extract in the preparation of a plant extract for treating acute myeloid leukemia. Background Art
[0002] Solanum nigrum L. is an annual herb of the Solanaceae family. It is rich in triterpenes, organic acids, phenols, flavonoids, steroids and other ingredients. It has the effects of dispersing blood stasis and swelling, clearing away heat and detoxifying.
[0003] Acute myeloid leukemia (AML) is a blood disease of malignant clones of hematopoietic stem cells. AML is characterized by rapid onset, critical condition, many complications and high mortality. Its main symptoms include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc., and are often accompanied by yin deficiency and internal heat manifestations such as five-heart irritability, thirst, preference for cold drinks, pale tongue with little fluid, and thin pulse. According to its clinical symptoms, it is classified into the categories of "acute labor", "heat labor", "blood syndrome", "deficiency labor", "symptom accumulation", "scrofula" and so on in traditional Chinese medicine. Therefore, the present invention uses the Chinese medicine Solanum nigrum as the research object to explore the medicinal parts of Solanum nigrum that can inhibit AML cell proliferation and improve the state of AML mice. Summary of the invention
[0004] The purpose of the present invention is to provide a use of a Solanum nigrum root extract in preparing a drug for treating acute myeloid leukemia in view of the shortcomings and deficiencies of the prior art.
[0005] The present invention uses a mouse acute myeloid leukemia model to explore for the first time the therapeutic effect of Solanum nigrum root extract in acute myeloid leukemia. The study found that the therapeutic effect of Solanum nigrum root extract is presumably achieved by inhibiting the proliferation of malignant cells.
[0006] In order to achieve the above object, the present invention adopts the following technical scheme:
[0007] The invention relates to an application of the Solanum nigrum extract in preparing a medicine for treating acute myeloid leukemia.
[0008] The medicine is a medicine capable of inhibiting the proliferation of acute myeloid leukemia cells.
[0009] Preferably, the drug is a Solanum nigrum root extract having the best pharmacological effect, and the root is superior to other medicinal parts.
[0010] The medicine is a medicine capable of prolonging the survival period of acute myeloid leukemia mice and reducing the peripheral blood leukocytes of acute myeloid leukemia mice.
[0011] The medicine is a medicine capable of improving leukocyte infiltration in the liver, spleen and bones.
[0012] The drug is a drug capable of inhibiting proteins related to the proliferation of malignant cells in the spleen.
[0013] Preferably, the malignant cell proliferation-related protein is Ki67 protein.
[0014] The preparation method of the Solanum nigrum root extract comprises:
[0015] The Solanum nigrum root extract was crushed, and 50% ethanol was added at a solid-liquid ratio of 1:10 (w / v), and ultrasonic-assisted extraction was performed for 30 minutes, and the extract was soaked overnight. The next day, the filtrate was filtered and combined with the extract, and vacuum freeze-dried to obtain a 50% ethanol extract of dried Solanum nigrum root.
[0016] A pharmaceutical composition for treating acute myeloid leukemia comprises the Solanum nigrum root extract and a pharmaceutically acceptable auxiliary material or carrier.
[0017] Compared with the prior art, the present invention has the following effective effects:
[0018] The present invention reports for the first time that Solanum nigrum root extract can inhibit the in vitro proliferation of acute myeloid leukemia cells. In addition, the present invention has found that Solanum nigrum root extract can prolong the survival of leukemia mice and can effectively improve the organ lesions of leukemia mice, and the root is superior to other medicinal parts. The above findings can provide a therapeutic drug for acute myeloid leukemia drugs, which provides a new idea for further studying the onset of acute myeloid leukemia; at the same time, Solanum nigrum root extract can be used in new anti-acute myeloid leukemia drug screening and testing, and provides guidance for the development of new drugs for the treatment of acute myeloid leukemia. In addition, the extraction method of Solanum nigrum root of the present invention is simple, the process is controllable, and the preparation cycle is short, which is suitable for industrial production and regulation. Therefore, Solanum nigrum root extract has broad application prospects in the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1 This is a graph showing the results of the activity test of Solanum nigrum root, stem and leaf extracts on acute myeloid leukemia cells. Instructions attached Figure 2 The activity and IC of Solanum nigrum root extracts at different concentrations on acute myeloid leukemia cells 50 The results of the measurement are shown in the figure. Figure 3 This is a statistical graph of the effects of Solanum nigrum root extract on the body weight change curve, survival time curve and organ-to-body ratio of acute myeloid leukemia mice. Instructions attached Figure 4 This is the statistical result of the effect of Solanum nigrum root extract on peripheral blood leukocytes in mice with acute myeloid leukemia. Instructions attached Figure 5 This is the H&E staining result showing the effect of Solanum nigrum root extract on the organs of mice with acute myeloid leukemia. Instructions attached Figure 6 This is the immunohistochemistry diagram and statistical analysis diagram of the effect of Solanum nigrum root extract on Ki67 protein expression in the tissues of acute myeloid leukemia mice. DETAILED DESCRIPTION
[0019] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.
[0020] Cell culture:
[0021] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in a 37°C incubator containing 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL). Acute myeloid leukemia cells HL-60 and Molm-13 were donated by the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and the Children's Hospital Affiliated to Capital Medical University, respectively, and were cultured in a 37°C incubator containing 5% CO2 using 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL).
[0022] Example 1: Effects of different parts of Solanum nigrum on acute myeloid leukemia cells
[0023] 1 Experimental methods
[0024] 1.1 Extraction of different medicinal parts of Solanum nigrum
[0025] The roots, stems and leaves of Solanum nigrum were crushed and added with 50% ethanol solution at a mass volume ratio (w / v) of 1:10, ultrasonicated for 30 minutes, and soaked overnight. The next day, the extracts of different parts of Solanum nigrum were filtered and freeze-dried to obtain 50% ethanol extracts of different parts of Solanum nigrum.
[0026] 1.2 Cell proliferation assay
[0027] The CCK-8 method was used to determine the effects of different parts of Solanum nigrum extract on the proliferation of acute myeloid leukemia cells C1498, and the optimal medicinal part of Solanum nigrum was selected. Acute myeloid leukemia cells C1498 were prepared into 1×10 5 / mL cell suspension, mix well, and dispense into 1.5mL centrifuge tubes, and add extracts from different parts of Solanum nigrum at a concentration of 40mg / mL to make a final concentration of 40μg / mL. The drug-containing cell suspension was inoculated into a 96-well plate, 200μL per well, and incubated in an incubator at 37°C with 5% CO2 for 48h, and then 20μL of CCK-8 solution was added to each well for a total incubation of 2h. The absorbance at 450nm was measured by an enzyme reader and the cell survival rate was determined.
[0028] 1.3 Statistical analysis
[0029] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant.
[0030] 2 Experimental results
[0031] The cell viability study was conducted using 50% ethanol extract freeze-dried powder from the roots, stems and leaves of Solanum nigrum. The results of the CCK8 experiment showed that the extracts of the roots (p < 0.0001), stems (p < 0.0001) and leaves (p < 0.0001) of Solanum nigrum can effectively inhibit the proliferation of acute myeloid leukemia cells; the extract of the root of Solanum nigrum has the strongest effect (see the instructions for use). Figure 1 A, B). Therefore, in the later stage of this study, Solanum nigrum root will be used to continue to explore its effect on acute myeloid leukemia.
[0032] Example 2: Effect of Solanum nigrum root extract on acute myeloid leukemia cells
[0033] 1 Experimental methods
[0034] 1.1 Cell proliferation assay
[0035] Solanum nigrum root was dissolved in DMSO to obtain an extract solution with a concentration of 40 mg / mL, and gradient dilution was used to obtain drug solutions of 20, 10, 5, 2.5, and 1.25 mg / mL, respectively. The drug solutions were added to the culture medium containing acute myeloid leukemia cells (C1498, HL-60, and Molm-13), so that the final concentrations of the drugs were 40, 20, 10, 5, 2.5, and 1.25 μg / mL, respectively. The culture medium containing the drug and cells was inoculated in a 96-well plate, 200 μL per well, and incubated in an incubator at 37°C and 37% CO2 for 48 hours. 20 μL of CCK-8 solution was added to each well and incubated for 2 hours. The absorbance at 450 nm was measured by an enzyme reader, and the cell survival rate was determined, and the IC 50 value.
[0036] 1.2 Statistical analysis
[0037] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant.
[0038] 2 Experimental results
[0039] The results of the CCK8 experiment showed (attached to the instruction manual) Figure 2 ), IC of Solanum nigrum root extract on C1498, HL-60 and Molm-13 cells 50 They were 5.32±0.09μg / mL, 12.17±1.56μg / mL and 9.90±1.41μg / mL respectively.
[0040] Example 3: Effect of Solanum nigrum root extract on acute myeloid leukemia mice
[0041] 1 Experimental methods
[0042] 1.1 Effects of Solanum nigrum root extract on acute myeloid leukemia mice
[0043] Acute myeloid leukemia C1498 cells with good growth and logarithmic growth were taken at 2×10 6 The cells were injected into the tail vein of C57BL / 6 mice. One day after the tail vein injection of cells, the mice were randomly divided into a model group, a cytarabine group (positive drug group, 25 mg / kg), a low-dose Solanum nigrum root extract (SNLR-L, 25 mg / kg), a medium-dose Solanum nigrum root extract group (SNLR-M, 50 mg / kg) and a high-dose Solanum nigrum root extract group (SNLR-H, 100 mg / kg). C57BL / 6 mice injected with an equal amount of saline by tail vein were used as a blank group. The positive drug group was continuously administered by tail vein for 10 days, and each Chinese medicine group was continuously gavaged until the end of the experiment. Blood was collected at 4°C, centrifuged at 3000rpm for 10 minutes, and the supernatant was taken. Part of the liver, spleen and bones were fixed for subsequent tissue sections. The remaining liver, spleen, lung, kidney and other organs were collected and stored at -80°C for later use.
[0044] 1.2 H&E staining
[0045] Place tissue sections in xylene for 20 minutes to remove the paraffin on the sections, and then place the sections in anhydrous ethanol for 5 minutes, 75% ethanol for 5 minutes, and finally in distilled water for 10 minutes for hydration. Place the hydrated sections in a hematoxylin aqueous solution for staining for 3-8 minutes, and immediately rinse the stain with distilled water. Prepare 1% hydrochloric acid ethanol differentiation solution, and after each section is placed in the differentiation solution for a few seconds, immediately rinse with distilled water. Place the sections in 85% ethanol and 95% ethanol solutions for 10 minutes each for dehydration, then place them in alcohol eosin staining solution for 2-3 minutes, and rinse with distilled water. Place the sections in anhydrous ethanol for 15 minutes, then transparentize them in xylene for 20 minutes, and seal them with neutral resin. Microscopic examination, image acquisition and analysis.
[0046] 1.4 Immunohistochemistry
[0047] Place tissue sections in xylene for 20 minutes for dewaxing, and then place the sections in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, rinse with distilled water for 1 minute, and place in PBS buffer. Place the sections in the prepared antigen retrieval solution, 95°C for 30 minutes, cool to room temperature, and then rinse with PBS buffer for 1 minute. Add an appropriate amount of endogenous peroxidase blocker (completely cover the sections), incubate at room temperature for 10 minutes, and rinse with PBS buffer for 10 minutes. Prepare anti-Ki67 primary antibody solution (1:500), add an appropriate amount of primary antibody solution, and incubate at 2-8°C overnight. The next day, rinse with PBS buffer for 10 minutes. Add an appropriate amount of reaction enhancement solution, incubate at 37°C for 20 minutes, and rinse with PBS buffer for 10 minutes. Add an appropriate amount of enhanced enzyme-labeled goat anti-rabbit IgG polymer, incubate at 37°C for 30 minutes, and rinse with PBS buffer for 10 minutes. Add an appropriate amount of freshly prepared DAB colorimetric solution, incubate at room temperature for 5-15 minutes, and rinse with tap water. Stain with hematoxylin for 10-20 seconds, rinse with tap water to turn blue. Put the slices into anhydrous ethanol for 15 minutes, xylene for 15 minutes to make it transparent, and seal with neutral resin. Observe under a microscope and collect and analyze images.
[0048] 1.5 Statistical analysis
[0049] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant. Immunohistochemical images were statistically analyzed using Image J software.
[0050] 2 Experimental results
[0051] Instructions attached Figure 3 As shown in the weight change graph (A), there was no significant difference in the weight of mice in each group. Figure 3As shown in the survival curve of (B), compared with the model group, low and high concentrations of Solanum nigrum root extracts can effectively prolong the survival time of acute myeloid leukemia mice (p < 0.05). Figure 3 As shown in (C), compared with the blank group, the liver-to-body ratio of the model group mice increased significantly (p < 0.01); compared with the model group, the liver-to-body ratio of the Ara-C group (positive drug) group decreased significantly (p < 0.01), and the liver-to-body ratio of the high-concentration Solanum nigrum root extract group decreased significantly (p < 0.05). Figure 3 As shown in (D), compared with the blank group, the spleen-body ratio of the model group mice increased significantly (p < 0.0001); compared with the model group, the spleen-body ratio of the Ara-C group (positive drug) group decreased significantly (p < 0.001), and the spleen-body ratio of the high-concentration Solanum nigrum root extract group decreased significantly (p < 0.05). Figure 4 The peripheral blood leukocyte statistics shown in the figure show that compared with the blank group, the number of leukocytes in the peripheral blood of the model group mice increased significantly (p < 0.01); compared with the model group, Ara-C (positive drug) (p < 0.01) and Solanum nigrum root extract (p < 0.05) can effectively reduce the proportion of leukocytes in the peripheral blood of mice. Figure 5 The H&E staining results shown in the figure show that compared with the blank group, there are a large number of leukocyte infiltrations in the liver, spleen, and bone marrow of the model group mice, while the Solanum nigrum root extract group can effectively reduce the leukocyte infiltration in the organ parts and improve the damage to the organ structure. Figure 6 The immunohistochemistry results shown in the figure showed that compared with the blank group, the expression of Ki67 protein in the spleen of the model group mice was significantly increased (p < 0.05), while the Ara-C (positive drug) group (p < 0.05) and the Solanum nigrum root extract group (p < 0.01) could effectively reduce the expression of Ki67 protein in the spleen.
Claims
1. A method for preparing a Solanum nigrum root extract, characterized in that: The following steps are involved: The Solanum nigrum root was crushed, and a 50% ethanol solution was added at a solid-liquid ratio of 1:10, and ultrasonic-assisted extraction was performed, and the mixture was soaked overnight. The next day, the extracts were filtered and combined, and vacuum freeze-dried to obtain 50% ethanol extracts of the dried Solanum nigrum root, stem, and leaf.
2. The use according to claim 1, characterized in that The medicine is an extract of the root part of Solanum nigrum with the best medicinal effect, and the root part is superior to other medicinal parts.
3. A Solanum nigrum root extract, characterized in that The preparation method according to claim 1 is adopted.
4. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The invention comprises the Solanum nigrum root extract as claimed in claim 2 and a pharmaceutically acceptable auxiliary material or carrier.
5. Use of the Solanum nigrum root extract according to claim 2 in the preparation of a drug for treating acute myeloid leukemia.
6. The use according to claim 4, characterized in that: The medicine is a medicine capable of inhibiting the proliferation of acute myeloid leukemia cells C1498, HL-60 and Molm-13.
7. The use according to claim 4, characterized in that: The drug can prolong the survival period of acute myeloid leukemia mice, reduce leukocyte infiltration in mouse tissues, or inhibit the proliferation of malignant cells in the body of acute myeloid leukemia mice, and improve the living conditions of mice.