Preparation of duchesnea indica leaf extract and application of duchesnea indica leaf extract in treatment of acute myelogenous leukemia
By using snakeberry leaf extract, the problem of difficulty in effectively treating acute myeloid leukemia in the prior art was solved, inhibition of leukemia cells and improvement of leukemia mice was achieved, and new drug development directions were provided.
Patent Information
- Application Number
- CN202510061128.7
- 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 leukemia cell proliferation and improving patient status.
Snakeberry leaf extract was used to prepare 50% ethanol extract by ultrasonic assisted extraction, and through in vitro cell experiments and in vivo mouse model research, it investigated its inhibitory effect on acute myeloid leukemia cells and its effect on the prolongation of survival and improvement of organ lesions in leukemia mice.
Snakeberry leaf extract can effectively inhibit the proliferation of acute myeloid leukemia cells, prolong the survival of leukemia mice, and improve their organ lesions, providing a new drug idea for the treatment of acute myeloid leukemia.
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Figure CN119868440A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of plant extracts, and in particular to application of a snakeberry leaf extract in preparing a drug for treating acute myeloid leukemia. Background Art
[0002] Duchesnea indica (Andr.) Focke is a perennial herbaceous plant of the genus Duchesnea in the Rosaceae family. It is rich in triterpenes, organic acids, phenols, flavonoids, steroids and other chemical components. Duchesnea was first recorded in the "Famous Doctors' Records": "Duchesnea is used to treat severe heat in the chest and abdomen", and has the effects of clearing heat, cooling blood, reducing swelling and detoxifying.
[0003] Acute myeloid leukemia (AML) is a kind of acute leukemia, which is a blood disease of malignant clones of hematopoietic stem cells. The main sites of AML are in the bone marrow and blood. Traditional Chinese medicine believes that AML is mostly caused by deficiency of qi and blood, self-generation of endotoxins, and invasion of exogenous evils, which causes internal and external toxicity, damages the internal organs, attacks the bone marrow, disturbs qi and blood, and causes imbalance of yin and yang. Traditional Chinese medicine does not have the title of leukemia, and it belongs to the category of asthenia, blood deficiency, and malignant nucleus according to its clinical manifestations. Due to deficiency of healthy qi (yin deficiency), damp-heat toxic evil invades internally, burns Ying Yin, enters the blood and injures the marrow, resulting in the occurrence of acute leukemia. Therefore, the basic principles of traditional Chinese medicine for the treatment of leukemia are "clearing" and "replenishing". Therefore, the present invention uses Chinese medicine Solanum nigrum as the research object, and through in vitro and in vivo experimental evaluation, digs out the medicinal parts of Duchesnea sinensis that have the function of inhibiting the proliferation of acute myeloid leukemia cells and improving the state of acute myeloid leukemia mice. Summary of the invention
[0004] The purpose of the present invention is to provide a use of a snakeberry leaf extract in preparing a drug for treating acute myeloid leukemia in view of the shortcomings and deficiencies of the prior art.
[0005] In order to achieve the above object, the present invention adopts the following technical scheme:
[0006] The invention discloses a preparation method of a snakeberry leaf extract and application of the extract in the preparation and treatment of acute myeloid leukemia.
[0007] The principles and advantages of this technical solution are:
[0008] The inventors conducted a large number of experiments to verify the role of a snakeberry leaf extract in the treatment of acute myeloid leukemia. The leaves are superior to other medicinal parts, as follows:
[0009] (1) Preparation of a root, stem and leaf extract of snakeberry: crush the root, stem and leaf of snakeberry, add a 50% ethanol solution at a solid-liquid ratio of 1:10, perform ultrasonic-assisted extraction, and soak overnight. The next day, filter and combine the extracts, and vacuum freeze-dry to obtain a 50% ethanol extract of the root, stem and leaf of dried snakeberry.
[0010] (2) Effects of a root, stem, and leaf extract of snakeberry on acute myeloid leukemia cells: 40 μg / mL of a root, stem, and leaf extract of snakeberry was co-incubated with acute myeloid leukemia C1498 cells for 48 hours, and the cell activity was detected using a cell counting kit (CCK-8).
[0011] (3) Effect of a snakeberry leaf extract on different acute myeloid leukemia cells: Different concentrations of snakeberry leaf extract were co-incubated with acute myeloid leukemia cells C1498, HL-60 and Molm-13 for 48 h. The cell activity was determined by CCK-8 method and the IC 50 value.
[0012] (4) Effects of a snakeberry leaf extract on acute myeloid leukemia model mice: First, blood smears were used to observe the proportion of white blood cells and primitive cells in the blood of mice; the weight changes and survival of mice were counted. Secondly, the organ-body ratio of mice was calculated and H&E staining was used to observe the differences in mouse organs. Finally, protein immunohistochemistry of Ki67 was used to analyze the proliferation of malignant cells in the spleen of mice.
[0013] Compared with the prior art, the present invention has the following advantages and effective effects:
[0014] The present invention reports for the first time that snakeberry leaf extract can inhibit the in vitro proliferation of acute myeloid leukemia cells, and the leaves are superior to other medicinal parts. In addition, the present invention reports for the first time that snakeberry leaf extract can prolong the survival of leukemia mice and can effectively improve the organ lesions of leukemia mice. The above findings can provide a therapeutic drug for acute myeloid leukemia, providing new ideas for further research on the onset of acute myeloid leukemia; at the same time, snakeberry leaf extract can be applied to the screening and testing of new anti-acute myeloid leukemia drugs, providing guidance for the development of new drugs for the treatment of acute myeloid leukemia. In addition, the extraction method of snakeberry leaves of the present invention is simple, the process is controllable, and the preparation cycle is short, which is suitable for industrial production and regulation. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1 This is a graph showing the results of the activity test of the root, stem and leaf extracts of snakeberry on acute myeloid leukemia cells. Instructions attached Figure 2 The activity and IC of different concentrations of snakeberry leaf extract on acute myeloid leukemia cells 50The results of the measurement are shown in the figure. Figure 3 This is a statistical graph of the effects of snakeberry leaf 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 a graph showing the statistical results of the effect of snakeberry leaf 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 snakeberry leaf extract on the organs of acute myeloid leukemia mice. Instructions attached Figure 6 This is the immunohistochemistry graph and statistical analysis graph of protein expression of the effect of snakeberry leaf extract on Ki67 protein expression in the tissues of acute myeloid leukemia mice. DETAILED DESCRIPTION
[0015] 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.
[0016] Example 1: Effects of extracts from different parts of snakeberry on acute myeloid leukemia cells
[0017] 1 Experimental methods
[0018] 1.1 Extraction of different medicinal parts of snakeberry
[0019] The roots, stems and leaves of Duchesnea indica 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 Duchesnea indica were filtered and freeze-dried to obtain 50% ethanol extracts of different parts of Duchesnea indica.
[0020] 1.2 Cell culture
[0021] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2, with the medium changed 2-3 times a week.
[0022] 1.3 Cell proliferation assay
[0023] The CCK-8 method was used to determine the effects of different parts of snakeberry extract on the proliferation of acute myeloid leukemia cells C1498, and the optimal medicinal part of snakeberry was screened. 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 snakeberry at a concentration of 40mg / mL to make the 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 2h. The absorbance at 450nm was measured by an enzyme reader and the cell survival rate was determined.
[0024] 1.4 Statistical analysis
[0025] 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.
[0026] 2 Experimental results
[0027] The cell viability study was conducted using freeze-dried powder of 50% ethanol extracts from the roots, stems and leaves of snakeberry. 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 snakeberry could effectively inhibit the proliferation of acute myeloid leukemia cells; and the extract of snakeberry leaves had the strongest effect (see the instructions for use). Figure 1 A, B). Therefore, in the later stage of this study, snakeberry leaves will be used to continue to explore its effect on acute myeloid leukemia.
[0028] Example 2: Effect of snakeberry leaf extract on acute myeloid leukemia cells
[0029] 1 Experimental methods
[0030] 1.1 Cell culture
[0031] 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).
[0032] 1.2 Cell proliferation assay
[0033] The extract solution of snakeberry leaves was dissolved in DMSO to obtain an extract solution with a concentration of 80 mg / mL, and gradient dilution was used to obtain drug solutions of 40, 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 80, 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.
[0034] 1.3 Statistical analysis
[0035] 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.
[0036] 2 Experimental results
[0037] The results of the CCK8 experiment showed (attached to the instruction manual) Figure 2 ), IC of snakeberry leaf extract on C1498, HL-60 and Molm-13 cells 50 They were 24.54±2.22μg / mL, 31.23±2.01μg / mL and 58.74±3.64μg / mL respectively.
[0038] Example 3: Effect of snakeberry leaf extract on acute myeloid leukemia mice
[0039] 1 Experimental methods
[0040] 1.1 Cell culture
[0041] 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).
[0042] 1.2 Effects of snakeberry leaf extract on acute myeloid leukemia mice
[0043] Acute myeloid leukemia C1498 cells with good growth and logarithmic growth were taken at 2×10 6The 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 snakeberry leaf extract (DIFL-L, 50 mg / kg), a medium-dose snakeberry leaf extract group (DIFL-M, 100 mg / kg) and a high-dose snakeberry leaf extract group (DIFL-H, 200 mg / kg). C57BL / 6 mice injected with an equal amount of saline in the 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] 1.3 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). Immunohistochemical images were statistically analyzed using Image J software. 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.
[0050] 2 Experimental results
[0051] Instructions attached Figure 3 As shown in the survival curve of (A), compared with the model group, low, medium and high concentrations of snakeberry leaf extract can effectively prolong the survival time of acute myeloid leukemia mice (p < 0.05). Figure 3 As shown in the weight change curve of (B), there was no significant difference in the weight of mice in each group. 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 of snakeberry leaf 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 < 0.001); compared with the model group, the spleen-body ratio of the Ara-C group (positive drug) group decreased significantly (p < 0.01), and the spleen-body ratio of the high concentration of snakeberry leaf 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 snakeberry leaf 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 snakeberry leaf 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 < 0.01) and the snakeberry leaf extract group (p < 0.01) could effectively reduce the expression of Ki67 protein in the spleen.
Claims
1. A method for preparing a snakeberry leaf extract, characterized in that: The following steps are involved: The snakeberry leaves were 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 a 50% ethanol extract of dried snakeberry leaves.
2. The use according to claim 1, characterized in that The medicine is an extract of the snakeberry leaf part with the best medicinal effect.
3. A snakeberry leaf 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 snakeberry leaf extract according to claim 2 and a pharmaceutically acceptable auxiliary material or carrier.
5. Use of the snakeberry leaf 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.