Application of duchesnea indica leaf and nightshade root combined medicine in treating acute myelogenous leukemia

Abstract

The invention relates to the field of plant extracts, and particularly discloses application of a duchesnea indica leaf and nightshade root extract combined medicine in the aspect of treating acute myelogenous leukemia. The invention aims to provide a novel combined medicine for treating acute myelogenous leukemia. It is found for the first time that the duchesnea indica leaf and nightshade root extract combined effect can effectively inhibit cell proliferation of acute myelogenous leukemia, prolong the survival time of mice with acute myelogenous leukemia, inhibit proliferation of malignant cells in the mice and improve the survival state of the mice. The invention provides a new thought for further researching the development of the acute myelogenous leukemia medicine.

Description

Technical Field

[0001] The invention relates to the field of plant extracts, and in particular to application of a combined drug of snakeberry leaf and nightshade root extracts in treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a blood disease caused by malignant clones of hematopoietic stem cells. The pathogenesis of leukemia is related to multiple factors such as infection, radiation, chemical agents, lifestyle, and genetics. Ancient Chinese medical books record: "Deficiency of qi and blood, self-generation of internal toxins, invasion of external evils, combined internal and external toxins, damage to internal organs, attack the bone marrow, disrupt qi and blood, and imbalance of yin and yang." At present, the treatment of AML mainly includes intensive induction therapy, non-intensive therapy, hematopoietic stem cell inhibition and new immunotherapy. In recent years, traditional Chinese medicine has made remarkable achievements in the treatment of acute myeloid leukemia AML through syndrome differentiation and the use of effective ingredients of traditional Chinese medicine. Single Chinese medicine, Chinese medicine monomers and Chinese medicine compound can change the occurrence and development of tumors, and demonstrate their effectiveness through multiple mechanisms such as inducing cell differentiation, inhibiting cell proliferation, and reversing cell resistance.

[0003] Duchesnea indica (Andr.) Focke is a perennial herbaceous plant of the genus Duchesnea in the Rosaceae family, which has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and dispersing blood stasis and reducing swelling. Solanum nigrum L. is an annual herbaceous plant of the Solanaceae family, which has the effects of clearing away heat and detoxifying, reducing swelling and dispersing knots, and reducing inflammation and diuresis. Due to the functional characteristics of the two, the two Chinese medicines of Duchesnea indica and Solanum nigrum are widely used in the treatment of various tumor diseases, such as Longshe Yangquan Decoction, Tenglong Buzhong Decoction, Chinese medicine compound injection and other Chinese medicine compound. Therefore, the present invention starts from the combination of Chinese medicines, and through in vitro and in vivo experimental evaluation, explores the effect of the combined action of Duchesnea indica and Solanum nigrum on acute myeloid leukemia. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a combined action of extracts of snakeberry leaves and nightshade roots in the preparation of 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 effects of snakeberry leaf and nightshade root extracts in acute myeloid leukemia. The study found that the therapeutic effects of snakeberry leaf and nightshade root extracts are 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 discloses an application of the snakeberry leaf and nightshade root extract in preparing a medicine for treating acute myeloid leukemia.

[0008] The drug combination is a drug capable of inhibiting the proliferation of acute myeloid leukemia cells.

[0009] Preferably, the drug combination is a drug that can synergistically inhibit the proliferation of acute myeloid leukemia cells.

[0010] The drug combination is a drug capable of prolonging the survival period of acute myeloid leukemia mice and reducing the peripheral blood leukocytes of acute myeloid leukemia mice.

[0011] The drug combination is a drug capable of improving leukocyte infiltration in the liver, spleen and bones.

[0012] The drug combination 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] A pharmaceutical composition for treating acute myeloid leukemia comprises the Solanum nigrum root extract and a pharmaceutically acceptable auxiliary material or carrier.

[0015] Compared with the prior art, the present invention has the following effective effects:

[0016] The present invention reports for the first time that extracts of snakeberry leaves and nightshade roots can synergistically inhibit the in vitro proliferation of acute myeloid leukemia cells. In addition, the present invention has found that extracts of snakeberry leaves and nightshade roots 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 drugs, providing new ideas for further research on the onset of acute myeloid leukemia; at the same time, extracts of snakeberry leaves and nightshade roots can be used in 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. Therefore, the combined use of snakeberry leaves and nightshade root extracts has broad application prospects in the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a graph showing the results of the combined activity test of snakeberry leaf and nightshade root extracts on acute myeloid leukemia cells. Figure 2 This is a graph showing the results of the activity assay of rat drug-containing plasma on acute myeloid leukemia cells C1498. Figure 3 This is a statistical graph showing the body weight change curve, survival time curve and organ-to-body ratio of the combined effect of snakeberry leaf and nightshade root extracts on acute myeloid leukemia mice. Figure 4 This is the statistical result of the combined effect of snakeberry leaf and nightshade root extract on peripheral blood leukocytes in mice with acute myeloid leukemia. Figure 5This is the H&E staining result of the combined effect of snakeberry leaf and Solanum nigrum root extract on the organs of mice with acute myeloid leukemia. Figure 6 This is the immunohistochemistry image and statistical analysis of protein expression of the combined effect of snakeberry leaf and Solanum nigrum root extract on Ki67 protein expression in the tissues of acute myeloid leukemia mice. DETAILED DESCRIPTION

[0017] 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.

[0018] Cell culture:

[0019] 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).

[0020] Example 1: Effect of the combined action of snakeberry leaf and nightshade root extract on acute myeloid leukemia cells

[0021] 1 Experimental methods

[0022] 1.1 Cell proliferation assay

[0023] The CCK-8 method was used to determine the effect of the combined effect of extracts of snakeberry leaf (DIFL) and nightshade root (SNLR) (SD) on the proliferation of acute myeloid leukemia C1498 cells. Acute myeloid leukemia C1498 cells were prepared into 1×10 5 The cell suspension of 100 μg / mL was mixed and dispensed into 1.5 mL centrifuge tubes, and the corresponding drug concentrations were added according to Table 1. The drug-containing cell suspension was inoculated into a 96-well plate, 200 μL per well, and incubated in an incubator at 37°C with 5% CO2 for 48 h. After that, 20 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance at 450 nm was measured by a microplate reader and the cell survival rate was determined. Table 1 Concentration ratio of extracts from snakeberry leaves and Solanum nigrum root for combined action on C1498 cells

[0024] 1.2 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] In order to evaluate the inhibitory effect of the combined action of snakeberry leaf and nightshade root extract on the proliferation of AML cell lines, C1498 cells were treated with nightshade root extract and snakeberry leaf extract at different concentrations and ratios for 48 hours, and the cell activity was determined by CCK8. The results of the CCK8 experiment showed (see the instruction manual) Figure 1 A), compared with the single drug effect, the combined effect of Solanum nigrum root and Snakeberry leaf on the proliferation inhibition of AML cells is enhanced. CompuSyn software calculation analysis shows that CI <1, indicating that the combined effect of Snakeberry leaf and Solanum nigrum root can synergistically inhibit the proliferation of AML cells (attached to the instruction manual Figure 1 B).

[0028] Example 2: Effects of drug-containing plasma in rats on acute myeloid leukemia cells

[0029] 1 Experimental methods

[0030] 1.1 In vitro efficacy analysis of drug-containing plasma in rats

[0031] C1498 cells were diluted to 1×10 5 The cell suspension of 500 μg / mL was dispensed into 1.5 mL EP tubes, and different concentrations (2% drug-containing plasma + 8% FBS, 5% drug-containing plasma + 5% FBS, 10% drug-containing plasma) of drug-containing plasma were added to treat the cells. The mixture of cell suspension and drug-containing plasma was inoculated into a 96-well plate, 200 μL per well. After incubation for 48 h, the CCK8 method was used to determine the effect of drug-containing plasma on the proliferation of C1498 cells.

[0032] 1.2 Statistical analysis

[0033] 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.

[0034] 2 Experimental results

[0035] The results of the effect of drug-containing plasma on AML cell viability in rats showed (attached to the instruction manual) Figure 2), the higher the concentration ratio of drug-containing plasma, the more significant the inhibitory effect on AML cell activity. Figure 2 (BC) showed that compared with the control group, 5% plasma containing snakeberry leaf extract (p < 0.05) and snakeberry leaf and nightshade root extract (p < 0.01) could effectively inhibit the proliferation of C1498 cells, and 10% plasma containing nightshade root extract (p < 0.001) and snakeberry leaf and nightshade root extract (p < 0.001) could effectively inhibit the proliferation of AML cells. In addition, compared with single drug-containing plasma, the inhibitory effect of the combined plasma of the two drugs on AML cells was more obvious (p < 0.05).

[0036] Example 3: Effect of the combined action of snakeberry leaf and nightshade root extract on acute myeloid leukemia mice

[0037] 1 Experimental methods

[0038] 1.1 Effects of Solanum nigrum root extract on acute myeloid leukemia mice

[0039] 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) and a combined action group of snakeberry leaves and nightshade roots (SD group, 50 mg / kg nightshade roots + 100 mg / kg snakeberry leaves). 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.

[0040] 1.2 H&E staining

[0041] 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.

[0042] 1.3 Immunohistochemistry

[0043] 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.

[0044] 1.4 Statistical analysis

[0045] 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.

[0046] 2 Experimental results

[0047] 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 3 As shown in the survival curve of (B), compared with the model group, the combination of snakeberry leaf and nightshade root extract 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 combined action group of snakeberry leaf and nightshade root extract decreased significantly (p < 0.05). Figure 3As 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 combined action group of snakeberry leaf and nightshade root extract decreased significantly (p < 0.01). 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) and snakeberry leaf extract can effectively reduce the proportion of leukocytes in the peripheral blood of mice (p < 0.01). 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 combined action group of snakeberry leaf and nightshade root extract 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 and the combined action group of snakeberry leaf and nightshade root extracts could effectively reduce the expression of Ki67 protein in the spleen (p < 0.05).

Claims

1. Application of a combination of snakeberry leaf and nightshade root extracts in the treatment of acute myeloid leukemia.

2. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The drug mass ratio of snakeberry leaf and nightshade root is 1:2-1:

5.

3. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The invention comprises the combined use of the extract of snakeberry leaf and nightshade root as claimed in claim 2 and a pharmaceutically acceptable auxiliary material or carrier.

4. The use according to claims 1 and 2, characterized in that The drug is a drug that can synergistically inhibit the proliferation of acute myeloid leukemia cells C1498.

5. The use according to claim 3, 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.

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