Application of traditional Chinese medicine monomer rhamnatin in preparation of medicine for treating leukemia

Abstract

The invention provides an application of a traditional Chinese medicine monomer rhamnatin or a solvate, hydrate or salt thereof in preparation of a medicine for treating or relieving leukemia. The antitumor activity of rhamnatin is discussed from multiple aspects such as in-vitro and in-vivo levels. Experiments show that the traditional Chinese medicine monomer rhamnatin can inhibit proliferation of leukemia cell strains, induce cell apoptosis and enhance the leukemia inhibition effect of cytarabine; in vivo, rhamnatin can inhibit the growth of leukemia subcutaneous transplantation tumors and prolong the lifetime of tumor-bearing mice. Further experiments reveal that rhamnatin realizes a biological mechanism of inhibiting leukemia proliferation and promoting apoptosis by regulating a PI3K-AKT signal channel, and shows that rhamnatin has a certain prospect in treatment of leukemia.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of a traditional Chinese medicine monomer rhamnosine in preparing a medicine for treating leukemia. Background Art

[0002] Leukemia is a malignant disease of the blood system that seriously threatens human life and health, but its etiology has not been fully elucidated so far. In my country, there are about 40,000 new leukemia patients each year, of which 60% are adults and 40% are children, and the high incidence period of childhood leukemia is 2-7 years old. Among the incidence rates of leukemia, the ratio of acute leukemia to chronic leukemia is 5.5:1, among which acute myeloid leukemia (AML) has the highest incidence rate, followed by acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML), while chronic lymphocyte leukemia (CLL) is the rarest clinically. At present, chemotherapy and hematopoietic stem cell transplantation are the most common treatments for leukemia in clinical practice. In recent years, the application of combined therapy and precision targeted drugs has occupied an irreplaceable position in the treatment of leukemia. Although the clinical treatment methods and effects of leukemia have been greatly expanded and improved, a considerable number of patients have resistance to chemotherapy and targeted drug therapy, resulting in refractory leukemia and relapse. Based on this, finding new chemotherapy drugs with high safety and less toxic side effects has become one of the hot spots in leukemia research.

[0003] In recent years, in order to alleviate the clinical symptoms of leukemia patients, reduce the recurrence rate and metastasis of tumors, reduce the toxic side effects of chemotherapy drugs and enhance the efficacy, multidisciplinary comprehensive treatment methods including traditional Chinese medicine have been developed. As a traditional medicine, traditional Chinese medicine has a history of thousands of years in China. Traditional Chinese medicine has a relatively mild effect and can also have a certain inhibitory effect on cancer cells. Therefore, it has become a hot spot in the research and development of cancer chemotherapy drugs. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a use of a traditional Chinese medicine monomer rhamnosine in the preparation of a drug for treating leukemia, thereby providing a new approach for treating leukemia with traditional Chinese medicine.

[0005] To achieve the above-mentioned purpose, the present invention provides a use of a Chinese medicine monomer rhamnosin or its solvate, hydrate or salt in the preparation of a drug for treating or alleviating leukemia;

[0006] The rhamnosin has a structure shown in Formula I:

[0007]

[0008] The above-mentioned salts are pharmaceutically acceptable salts well known to those skilled in the art, including but not limited to one or more of salts of inorganic acids and organic acids, and the acids include but are not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid (toluenesulfonate), 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid or mandelic acid, etc.

[0009] The leukemia includes, but is not limited to, one or more of human chronic myeloid leukemia, human acute myeloid leukemia and human acute lymphoblastic leukemia.

[0010] The dosage form of the drug includes, but is not limited to, an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup or a transdermal patch.

[0011] The test results show that the above-mentioned rhamnosin or its hydrate or pharmaceutically acceptable salt can significantly inhibit the growth of various leukemia cells in vitro and in vivo, and can inhibit the proliferation of different types of leukemia cells and induce apoptosis of leukemia cells and the expression of apoptosis-related signaling pathway proteins through the PI3K-AKT signaling pathway. Among them, rhamnosin has the strongest inhibitory activity against acute myeloid leukemia and the weakest inhibitory activity against acute T-lymphocytic leukemia.

[0012] In a specific embodiment of the present invention, the above-mentioned Chinese herbal medicine monomer rhamnosine can inhibit the in vitro growth of leukemia cell line HL-60 at a relatively low concentration (9uM).

[0013] In a specific embodiment of the present invention, in a mouse animal model, administration of 10 mg / kg / day of rhamnosin can effectively inhibit the in vivo growth of leukemia cells HL-60.

[0014] The administration methods of the above-mentioned rhamnosin include but are not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal infusion, eye drops, nasal spray, oral spray, and can also be applied locally on the skin surface or systemically through one or more of the above-mentioned methods.

[0015] The above-mentioned rhamnosin can be used alone or in combination with other drugs.

[0016] Based on this, the present invention provides the use of the above-mentioned traditional Chinese medicine monomer rhamnosine or its solvate, hydrate or salt in combination with cytarabine in the preparation of a drug for treating or alleviating leukemia.

[0017] The experimental results show that rhamnosin can enhance the effect of cytarabine in inhibiting leukemia, especially acute myeloid leukemia cells, and the two have a synergistic effect.

[0018] In another aspect, the present invention provides a pharmaceutical composition for treating or alleviating leukemia, comprising rhamnosine or its solvate, hydrate or salt and cytarabine.

[0019] Optionally, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] The leukemia includes, but is not limited to, one or more of human chronic myeloid leukemia, human acute myeloid leukemia and human acute lymphoblastic leukemia cells, preferably acute myeloid leukemia.

[0021] The dosage form of the pharmaceutical composition includes, but is not limited to, injectable fluid, aerosol, cream, gel, pill, capsule, syrup, and transdermal patch.

[0022] The present invention provides a method for treating or alleviating leukemia, comprising:

[0023] The rhamnosin or the pharmaceutical composition is administered to a subject, wherein the subject is a human or a mammal, preferably a human.

[0024] Preferably, the administered dose does not exceed the effective dose.

[0025] The mammals include, but are not limited to, one or more of rodents, cats, canines, primates, and the like.

[0026] Compared with the prior art, the present invention provides the use of the traditional Chinese medicine monomer rhamnosin or its solvate, hydrate or salt in the preparation of drugs for treating or alleviating leukemia.

[0027] The present invention explores the anti-tumor activity of rhamnosin from multiple levels, including in vitro and in vivo levels. Experiments show that the Chinese medicine monomer rhamnosin can inhibit the proliferation of leukemia cell lines, induce cell apoptosis, and enhance the effect of cytarabine in inhibiting leukemia; in vivo, rhamnosin can inhibit the growth of leukemia subcutaneous transplanted tumors and prolong the survival of tumor-bearing mice. Further experiments revealed that rhamnosin achieves its biological mechanism of inhibiting leukemia proliferation and promoting apoptosis by regulating the PI3K-AKT signaling pathway, indicating that it has certain prospects in the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing that rhamnosin inhibits the proliferation of leukemia cells;

[0029] Figure 2 This is a graph showing the effect of rhamnosine combined with cytarabine on the proliferation of leukemia cells;

[0030] Figure 3 This is the cell morphology of rhamnosin-induced apoptosis of leukemia cells;

[0031] Figure 4 This is a diagram showing that rhamnosin inhibits proliferation and induces cell apoptosis by regulating the PI3K-AKT signaling pathway;

[0032] Figure 5 This is a diagram showing the effect of rhamnosin on the growth and development of zebrafish;

[0033] Figure 6 This is a graph showing the inhibitory effect of rhamnosin on the growth of subcutaneous leukemia tumors in mice. DETAILED DESCRIPTION

[0034] In order to further illustrate the present invention, the application of the Chinese medicine monomer rhamnosin provided by the present invention in the preparation of a drug for treating leukemia is described in detail below in conjunction with the examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0035] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0036] Example 1: Rhamnosin inhibits the proliferation of leukemia cells

[0037] Experimental methods:

[0038] The leukemia cells HL-60, K562 and CCRF-CEM used in this example were from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The cells were cultured in a 37°C constant temperature incubator (humidity 95%, CO2 concentration 5%), and the culture medium was IMDM containing 10% fetal bovine serum. The cell viability was determined by the MTS method. Leukemia cells HL-60, K562 and CCRF-CEM were inoculated into 96-well plates, and different concentrations of the monomer compound were added. The control group was added with an equal amount of DMSO. Each group was set up with 3 replicate wells. After continuing to culture for 48h and 72h, 20μ1 MTS was added to each well and incubated at 37°C for 2-3 hours. The absorbance value at 490nm was detected by an enzyme marker. The experiment was repeated 3 times independently. Cell viability (%) = drug added OD value / control group OD value * 100%.

[0039] Experimental results:

[0040] The toxic effects of rhamnosin on three leukemia cells are shown in Table 1. Figure 1As shown in Figures A and B, cytarabine and rhamnosine alone have a significant inhibitory effect on the proliferation of three leukemia cells (K562 cells, HL-60 cells, and CCRF-CEM cells), and the higher the concentration, the stronger the inhibitory effect. Among them, HL-60 cells are most sensitive to their effects.

[0041] Table 1 IC of rhamnosin against three leukemia cells at 48h 50 value

[0042]

[0043] Example 2: Effect of the combination of cytarabine and rhamnosine on the proliferation of leukemia cells

[0044] Experimental methods:

[0045] The cell survival rate was determined by MTS method. Leukemia cells HL-60, K562 and CCRF-CEM were inoculated into 96-well plates, and different concentrations of cytarabine and the Chinese medicine monomer compound rhamnosine were added. The control group was added with an equal amount of DMSO. Each group was set up with 3 replicate wells. After continuing to culture for 48h and 72h, 20μ1 MTS was added to each well and incubated at 37℃ for 2-3 hours. The absorbance value at 490nm was detected by an enzyme marker. The experiment was repeated 3 times independently. Cell survival rate (%) = drug added OD value / control group OD value * 100%.

[0046] Experimental results:

[0047] The results of treating leukemia cells with cytarabine and rhamnosine were as follows: Figure 2 As shown in Figures A, B, and C, within the low-dose concentration range, the inhibitory effect of cytarabine and rhamnosine combined on leukemia cells (HL-60, K562) was significantly higher than that of single-drug treatment, and the inhibitory effect was additive; but it was antagonistic to CCRF-CEM cells, so the subsequent analysis was mainly based on K562 and HL-60 cells. The ZIP synergy scores were all in [-10, 10], indicating that the combination of rhamnosine and cytarabine had an additive effect.

[0048] Example 3: Cell morphology of rhamnosin-induced apoptosis of leukemia cells

[0049] Experimental methods:

[0050] 1. Effect of rhamnosin on leukemia cell morphology

[0051] HL-60 cells were seeded in 6-well plates. After 24 hours of cell attachment, complete culture medium containing different concentrations of the Chinese medicine monomer compound rhamnosine was added. After 24 and 48 hours, the effect of rhamnosine on the morphology of leukemia cells was observed under an inverted microscope.

[0052] 2. Fluorescence staining experiment with Hochest 33342 staining solution

[0053] After treating leukemia cells with different concentrations of the Chinese medicine monomer compound rhamnosin for 24 hours, the culture medium was aspirated with a micropipette, 500 μL of fixative was added, and the cells were fixed for 10 minutes; the fixative was removed and aspirated with a micropipette, and each well was washed twice with 500 μL PBS for 3 minutes each time (a horizontal shaker was preferably used for washing, and washing for 3 minutes in low-speed mode was the best effect), and the PBS was finally aspirated; 500 μL Hoechst 33342 staining solution was added to each well, and the cells were stained for 5 minutes. In order to achieve a better staining effect, the cells were horizontally shaken for 5 minutes in low-speed mode of the shaker; the Hoechst 33342 staining solution was removed and aspirated with a micropipette, and each well was washed twice with 500 μL PBS for 3 minutes each time (a horizontal shaker was preferably used for washing, and washing for 3 minutes in low-speed mode was the best effect), and the PBS was finally aspirated, and the cells were wrapped with tin foil to avoid light; the cells were quickly placed under a fluorescence microscope for observation and photography (excitation wavelength of about 350 nm, emission wavelength of about 460 nm).

[0054] Experimental results:

[0055] After different concentrations of rhamnosin (20μM, 40μM) were applied to leukemia HL-60 cells for 48h, the cell morphology was observed under an inverted microscope. Compared with the control group, the number and volume of leukemia cells were significantly reduced, the cells were sparse, and the morphology was irregular ( Figure 3 Figure A in the middle). Through the Hoechst 33342 staining experiment, it was found that compared with the solvent control group, the number of blue bright cells (i.e. apoptotic cells stained by Hoechst 33342 due to chromatin condensation) in each group of leukemia cells treated with 20μM and 40μM rhamnosin increased in a concentration-dependent manner ( Figure 3 (Fig. B).

[0056] Example 4: Rhamnosin inhibits the growth of leukemia cells in vitro by inducing apoptosis

[0057] Experimental methods:

[0058] 1. Cell apoptosis experiment

[0059] HL-60 cells were inoculated in 6-well plates. After 24 hours of stabilization, complete medium containing different concentrations of the Chinese medicine monomer compound rhamnosine was added. After 24 hours, apoptosis detection was performed using an apoptosis detection kit. In the flow cytometry apoptosis detection, the cells were divided into unstained group, single PI staining group, single Annexin V staining group, and PI and Annexin V double staining group. The culture supernatant was collected, the cells were digested, and the cells were collected into the corresponding centrifuge tubes after terminating the digestion with the collected culture supernatant. The supernatant was discarded after centrifugation at 1000rpm for 3min. The centrifuged cells were washed twice with PBS and centrifuged again. After discarding PBS, add 250 μL of 1× binding buffer to each tube, and gently blow with a pipette to resuspend the cells. No PI and Annexin V were added to the unstained group, 2.5 μL of PI staining solution was added to the single PI staining group, 2.5 μL of Annexin V staining solution was added to the single PI staining group, and 2.5 μL of PI and Annexin V were added to the Annexin-V-PI double staining group and mixed. Incubate at room temperature in the dark for 15 minutes, transfer the cells to a 5 mL flow tube, and detect on a flow cytometer. After flow cytometry detection, there are 4 quadrants in the image obtained, and different quadrants represent different cell populations, among which the upper left quadrant, lower left quadrant, upper right quadrant, and lower right quadrant represent dead cells, normal non-apoptotic cells, late apoptotic cells, and early apoptotic cells, respectively.

[0060] 2. Cell cycle experiment

[0061] HL-60 cells were inoculated in 6-well plates. After 24 hours of stabilization, cells were treated with different concentrations of the Chinese medicine monomer compound rhamnosine for 24 hours and then collected. The cells were fixed with 70% cold ethanol at 4°C overnight. The cells were centrifuged at 800g for 15 minutes, washed twice with cold PBS, and then resuspended with 500mL of PBS containing PI (50g / mL) and RNase-free (100g / mL) and analyzed by flow cytometry. The data were analyzed using ModFit LT 3.2 software.

[0062] 3. Western Blot Experiment

[0063] After the cells were treated with different concentrations of rhamnosin for 48 hours, the cells were lysed with lysis buffer to extract proteins. The protein samples were separated by polyacrylamide gel PAGE electrophoresis after boiling and denaturation, transferred to nitrocellulose membrane, incubated with corresponding protein antibodies, and then incubated with fluorescently labeled secondary antibodies, and then the expression level of the protein was detected using the Odyssey scanner.

[0064] Experimental results:

[0065] Flow cytometry revealed that rhamnosin could also induce G1 arrest and apoptosis in HL-60 cells in a dose-dependent manner. Figure 4 At the same time, Western Blot experiments found that the expression of apoptosis marker protein Bax increased with the increase of rhamnosine concentration, the expression of anti-apoptotic protein Bcl-2 decreased with the increase of rhamnosine concentration, the expression of cell cycle protein CDK4 decreased with the increase of rhamnosine concentration, and the expression of p-PI3K, p-AKT, and p-mTOR proteins also decreased with the increase of rhamnosine concentration, indicating that rhamnosine can inhibit the activation of PI3K-AKT signaling pathway, thereby playing a role in treating leukemia ( Figure 4 (Figure C).

[0066] Example 5: Effect of rhamnosin on the growth and development of zebrafish

[0067] Experimental methods:

[0068] Zebrafish embryos were purchased from Shanghai Fish Biological Co., Ltd. and processed in 24-well plates, with 12 normal zebrafish embryos in each well and 1 ml of embryo culture medium. A blank control group, 1% DMSO and 6 gradient concentrations of rhamnosin groups were set up. Embryonic development was observed under a stereomicroscope at 24, 48, 72, and 96 hours after administration. The hatching rate and deformity rate at different time points were calculated and analyzed (hatching rate = hatched embryos / total number of embryos*100%; deformity rate = number of deformed embryos / number of surviving embryos*100%).

[0069] Experimental Results

[0070] Embryos incubated with DMSO 0.1% showed no significant difference compared to untreated embryos. During the observation period, untreated embryos did not show any mortality or developmental malformations. After 96 hours of exposure, in the embryo groups exposed to rhamnosin at 32 μM and 64 μM, the developing zebrafish embryos tolerated the compound well and did not induce obvious signs of toxicity ( Figure 5 At a dose of 64 μM, live zebrafish showed developmental abnormalities, including tail deformation, scoliosis, edema, hemorrhage, or delayed hatching, while at a dose of 32 μM, these malformations occurred less frequently. In summary, it is reasonable to assume that a relatively safe concentration for zebrafish embryos can be less than 16 μM ( Figure 5 (Figures B and C in the middle).

[0071] Example 6: Inhibitory effect of rhamnosin on subcutaneous tumor growth in mouse leukemia model

[0072] Experimental methods:

[0073] 1×10 6Human leukemia cells HL-60 were subcutaneously injected into the back of immunodeficient female mice (BLAB / c-nude). When the subcutaneous tumor grew to about 80-100 mm, the mice were divided into a negative control group and a drug treatment group, with 6 nude mice in each group. The mice in the drug treatment group were intraperitoneally injected with 10 mg / kg of rhamnosin dissolved in DMSO every day, and the negative control group was intraperitoneally injected with the same concentration of saline every day. After that, the mice were observed every day, and the first administration was set as day 0. The weight and tumor size of the mice were then measured every other day for 14 consecutive days. The mice were killed on the 14th day and the tumor tissues were taken out and photographed. On the 14th day of treatment, all mice were killed and the five organ tissues of tumor, heart, liver, spleen, lung and kidney were taken out and placed in the corresponding 50ml centrifuge tubes filled with formalin, and fixed in a 4°C refrigerator for 24h, then embedded in paraffin, deparaffinized and sectioned, and stained with hematoxylin-eosin (H&E) and analyzed using an Olympus-VS200 slide scanning microscope to analyze the apoptosis of tumor cells and the effects of drugs on various organ tissues.

[0074] Experimental results:

[0075] The results of the subcutaneous tumor-bearing experiment of leukemia cells showed that 10 mg / kg of rhamnosine could effectively inhibit the growth of tumors ( Figure 6 A and B in the middle). The body weight of nude mice after rhamnosine treatment did not change significantly ( Figure 6 C in the middle). Organ sections were taken from all mice 14 days later. Hematoxylin and eosin (H&E) staining showed that rhamnosin did not cause damage to the mouse's major organs, such as the heart, liver, spleen, lungs, and kidneys ( Figure 6 These in vivo studies collectively demonstrated that it has high antitumor efficacy with minimal side effects.

[0076] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of the Chinese medicine monomer rhamnosine or its solvate, hydrate or salt in the preparation of drugs for treating or alleviating leukemia; The rhamnosin has a structure shown in Formula I:

2. The use of the Chinese medicine monomer rhamnosine or its solvate, hydrate or salt in combination with cytarabine in the preparation of a drug for treating or alleviating leukemia; The rhamnosin has a structure shown in Formula I:

3. The use according to claim 1 or 2, characterized in that: The leukemia is one or more of human chronic myeloid leukemia, human acute myeloid leukemia and human acute lymphoblastic leukemia.

4. The use according to claim 1 or 2, characterized in that: The drug is in the form of an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup or a transdermal patch.

5. The use according to claim 1 or 2, characterized in that: The drug can be administered orally, intravenously, intramuscularly, subcutaneously, sublingually, rectally, by eye drops, nasal spray, oral spray, or topically or systemically through the skin.

6. A pharmaceutical composition for treating or alleviating leukemia, comprising rhamnosine or its solvate, hydrate or salt and cytarabine.

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