Application of the Traditional Chinese Medicine Monomer Rhamnosin in the Preparation of Drugs for the Treatment of Leukemia

Abstract

The present invention provides the use of a traditional Chinese medicine monomer, rhamnosine, or a solvate, hydrate, or salt thereof, in the preparation of a drug for treating or alleviating leukemia. The present invention explores the anti-tumor activity of rhamnosine from multiple levels, including in vitro and in vivo levels. Experiments have shown that the traditional Chinese medicine monomer, rhamnosine, can inhibit the proliferation of leukemia cell lines, induce cell apoptosis, and enhance the effect of cytarabine in inhibiting leukemia; in vivo, rhamnosine can inhibit the growth of leukemia subcutaneous transplanted tumors and prolong the survival of tumor-bearing mice. Further experiments revealed the biological mechanism by which rhamnosine inhibits leukemia proliferation and promotes apoptosis by regulating the PI3K‑AKT signaling pathway, indicating that it has certain prospects in the treatment of leukemia.

Description

Technical Field

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

[0002] Leukemia is a malignant blood disease that poses a serious threat to human health, but its etiology remains largely unexplained. In my country, approximately 40,000 new leukemia patients are diagnosed each year, 60% of whom are adults and 40% are children. The peak incidence of leukemia in children is between the ages of 2 and 7. The incidence ratio of acute leukemia to chronic leukemia is 5.5:1, with acute myeloid leukemia (AML) having the highest incidence, followed by acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). Chronic lymphocyte leukemia (CLL) is the least common clinically. Currently, chemotherapy and hematopoietic stem cell transplantation are the most common treatments for leukemia. In recent years, the use of combination therapy and precision targeted drugs has played an irreplaceable role in the treatment of leukemia. Although clinical treatment methods and efficacy 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 minimal toxic side effects has become a hot topic in leukemia research.

[0003] In recent years, multidisciplinary treatment approaches, including Traditional Chinese Medicine (TCM), have been developed to alleviate leukemia patients' clinical symptoms, reduce tumor recurrence and metastasis, minimize the toxic side effects of chemotherapy drugs, and enhance their efficacy. Traditional Chinese medicine, a traditional medicine with thousands of years of development in China, is known for its milder effects and limited inhibitory effects on cancer cells, making it a hot topic in cancer chemotherapy drug research and development. 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 rhamnosin 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 object, the present invention provides a use of a traditional 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 the 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 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 results of the experiments showed that rhamnosin or its hydrate or pharmaceutically acceptable salt significantly inhibited the growth of various leukemia cells both in vitro and in vivo. Furthermore, it inhibited the proliferation of different leukemia cell types through the PI3K-AKT signaling pathway and induced apoptosis in leukemia cells and the expression of proteins in the apoptosis-related signaling pathway. Among them, rhamnosin had 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 Chinese herbal medicine monomer rhamnosin can inhibit the in vitro growth of the leukemia cell line HL-60 at a relatively low concentration (9 uM).

[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 growth of leukemia cells HL-60 in vivo.

[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 irrigation, eye drops, nasal spray, oral spray, and can also be applied locally on the skin surface or systemically through one or more of the following 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 rhamnosine 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 a solvate, hydrate or salt thereof and cytarabine.

[0019] Optionally, the above 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 forms of the pharmaceutical composition include, but are not limited to, injectable fluids, aerosols, creams, gels, pills, capsules, syrups, and transdermal patches.

[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 a traditional Chinese medicine monomer rhamnosin or its solvate, hydrate or salt in the preparation of a drug for treating or alleviating leukemia.

[0027] This study explored the anti-tumor activity of rhamnosin at multiple levels, both in vitro and in vivo. Experiments demonstrated that the traditional Chinese medicine monomer rhamnosin inhibited the proliferation of leukemia cell lines, induced apoptosis, and enhanced the anti-leukemia effect of cytarabine. In vivo, rhamnosin inhibited the growth of subcutaneous leukemia tumors and prolonged the survival of tumor-bearing mice. Further experiments revealed the biological mechanism by which rhamnosin inhibits leukemia proliferation and promotes apoptosis by regulating the PI3K-AKT signaling pathway, suggesting its promising potential in leukemia treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing rhamnosin inhibiting leukemia cell proliferation;

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

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

[0031] Figure 4 Figure 2 shows 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 figure shows the inhibitory effect of rhamnosin on the subcutaneous tumor growth model of mouse leukemia. DETAILED DESCRIPTION

[0034] To further illustrate the present invention, the following describes in detail the use of the traditional Chinese medicine monomer rhamnosin in the preparation of a leukemia treatment drug, in conjunction with examples. However, it should be understood that these descriptions are intended only to further illustrate the features and advantages of the present invention and are not intended to limit the scope of the claims.

[0035] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well 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 obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The cells were cultured in a 37°C incubator (95% humidity, 5% CO2) in IMDM supplemented with 10% fetal bovine serum. Cell viability was determined using the MTS assay. Leukemia cells HL-60, K562, and CCRF-CEM were seeded into 96-well plates and treated with varying concentrations of the present monomeric compound. An equal amount of DMSO was added to the control group. Each group was plated in triplicate. After 48 and 72 hours of culture, 20 μl of MTS was added to each well and incubated at 37°C for 2-3 hours. The absorbance at 490 nm was measured using a microplate reader. The experiment was repeated three times. Cell viability (%) = OD value of the drug-treated cells / OD value of the control group * 100%.

[0039] Experimental results:

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

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

[0042]

[0043] Example 2: Effect of the combined use of cytarabine and rhamnosine on leukemia cell proliferation

[0044] Experimental methods:

[0045] Cell viability was determined using the MTS assay. Leukemia cells HL-60, K562, and CCRF-CEM were seeded into 96-well plates and treated with varying concentrations of cytarabine and the Chinese medicinal monomer rhamnosine. An equal amount of DMSO was added to the control group. Each group was plated in triplicate. After 48 and 72 hours of culture, 20 μl of MTS was added to each well and incubated at 37°C for 2-3 hours. The absorbance at 490 nm was measured using a microplate reader. Experiments were repeated three times. Cell viability (%) = OD value of drug treatment / OD value of control group × 100%.

[0046] Experimental results:

[0047] The leukemia cells were treated with cytarabine and rhamnosine together. Figure 2 As shown in Figures A, B, and C, the combined use of cytarabine and rhamnosine exhibited significantly greater inhibitory effects on leukemia cells (HL-60 and K562) than either drug alone at low doses, demonstrating an additive inhibitory effect. However, the combined use exhibited an antagonistic effect on CCRF-CEM cells, leading to subsequent analysis focusing on K562 and HL-60 cells. ZIP synergy scores were all within the range of [-10, 10], indicating an additive effect between rhamnosine and cytarabine.

[0048] Example 3: Cell morphology of rhamnosin-induced apoptosis in 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. Fluorescent staining experiment with Hochest 33342 staining solution

[0053] Leukemia cells were treated with different concentrations of the Chinese medicine monomer compound rhamnosin for 24 hours, and then 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 with a micropipette, and each well was washed twice with 500 μL PBS for 3 minutes each time (a horizontal shaker should be used for washing, and washing for 3 minutes at low speed mode is the best effect), and finally the PBS was aspirated; 500 μL Hoechst 33342 staining solution was added to each well and stained for 5 minutes. For better staining effect, horizontal shaking can also be used for 5 minutes at low speed mode on a shaker; the Hoechst 33342 staining solution was removed with a micropipette, and each well was washed twice with 500 μL PBS for 3 minutes each time (a horizontal shaker should be used for washing, and washing for 3 minutes at low speed mode is the best effect), and finally the PBS was 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 48 hours, 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). Hoechst 33342 staining experiments revealed that compared with the solvent control group, the number of blue-bright cells (i.e., apoptotic cells stained with Hoechst 33342 due to chromatin condensation) in the leukemia cells treated with 20μM and 40μM rhamnosin increased in a concentration-dependent manner ( Figure 3 (Figure 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 seeded in 6-well plates and allowed to stabilize for 24 hours. Complete culture medium containing various concentrations of the traditional Chinese medicine monomer rhamnosin was then added. After 24 hours, apoptosis was detected using an apoptosis detection kit. For flow cytometry apoptosis analysis, cells were divided into unstained groups, PI-single-stained groups, Annexin V-single-stained groups, and PI and Annexin V-double-stained groups. The culture supernatant was collected and the cells were digested. The digestion was terminated with the collected culture supernatant, and the cells were collected into corresponding centrifuge tubes and centrifuged at 1000 rpm for 3 minutes, after which the supernatant was discarded. The cells were washed twice with PBS and centrifuged again. After aspirating the PBS, add 250 μL of 1× binding buffer to each tube and gently pipette to resuspend the cells. For the unstained group, omit PI and Annexin V. For the single-PI staining group, add 2.5 μL of PI staining solution; for the single-Annexin V staining group, add 2.5 μL of Annexin V staining solution. For the double-Annexin-V-PI staining group, add 2.5 μL of PI and Annexin V each and mix thoroughly. Incubate at room temperature in the dark for 15 minutes. Transfer the cells to a 5 mL flow cytometer and analyze them on a flow cytometer. The resulting image has four quadrants, representing different cell populations: the upper left quadrant, the lower left quadrant, the upper right quadrant, and the 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 seeded in 6-well plates and allowed to stabilize for 24 hours. After treatment with various concentrations of the traditional Chinese medicine monomer rhamnosine for 24 hours, the cells were harvested and fixed with 70% cold ethanol overnight at 4°C. The cells were centrifuged at 800g for 15 minutes, washed twice with cold PBS, and then resuspended in 500mL of PBS containing 50μg / mL PI and 100μg / mL RNase-free buffer for analysis by flow cytometry. 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. After boiling and denaturation, the protein samples were separated by polyacrylamide gel PAGE electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with corresponding protein antibodies and then incubated with fluorescently labeled secondary antibodies. The expression levels of the proteins were then detected using an Odyssey membrane 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 the apoptosis marker protein Bax increased with the increase of rhamnosin concentration, the expression of the anti-apoptotic protein Bcl-2 decreased with the increase of rhamnosin concentration, the expression of the cell cycle protein CDK4 decreased with the increase of rhamnosin concentration, and the expression of p-PI3K, p-AKT, and p-mTOR proteins also decreased with the increase of rhamnosin concentration, indicating that rhamnosin can inhibit the activation of the PI3K-AKT signaling pathway, thereby playing a role in treating leukemia ( Figure 4 (Figure C in the middle).

[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 treated in 24-well plates, with 12 normal zebrafish embryos per well in 1 ml of embryo culture medium. A blank control group, 1% DMSO, and six rhamnosin concentration gradients were established. Embryonic development was observed under a stereomicroscope 24, 48, 72, and 96 hours after administration. Hatching rate and malformation rate were calculated and analyzed at different time points (hatching rate = hatched embryos / total number of embryos * 100%; malformation rate = number of malformed embryos / number of surviving embryos * 100%).

[0069] Experimental results

[0070] Embryos incubated with 0.1% DMSO showed no significant difference compared to untreated embryos. During the observation period, there was no death or developmental deformity in untreated embryos. After 96 hours of exposure, the developing zebrafish embryos in the embryo groups exposed to rhamnosin at 32 μM and 64 μM showed that the compound was well tolerated by the developing zebrafish embryos and did not cause 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 may 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 a mouse leukemia model

[0072] Experimental methods:

[0073] 1×10 6Human leukemia HL-60 cells were subcutaneously injected into the backs of immunodeficient female mice (BLAB / c-nude). When the subcutaneous tumors grew to approximately 80-100 mm, the mice were divided into a negative control group and a drug treatment group, with six mice in each group. The drug treatment group received daily intraperitoneal injections of 10 mg / kg of rhamnosin dissolved in DMSO, while the negative control group received daily intraperitoneal injections of the same concentration of saline. After that, the mice were observed every day. The first administration was set as day 0, and then the weight and tumor size of the mice were measured every other day. The measurements were recorded continuously for 14 days. On the 14th day, the mice were killed and the tumor tissues were removed 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 removed and placed in the corresponding 50ml centrifuge tubes filled with formalin. They were fixed in a 4°C refrigerator for 24 hours, then embedded in paraffin, deparaffinized and sectioned, and after hematoxylin-eosin (H&E) staining, an Olympus-VS200 slide scanning microscope was used to analyze the tumor cell apoptosis and the effects of the drug 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 rhamnosin could effectively inhibit the growth of tumors ( Figure 6 Figures 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 mice's major organs such as the heart, liver, spleen, lungs, and kidneys ( Figure 6 These in vivo studies collectively demonstrated high antitumor efficacy with minimal side effects.

[0076] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such 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 salt in combination with cytarabine in the preparation of a drug for treating or alleviating leukemia; The rhamnosin has the structure shown in Formula I: The leukemia is human chronic myeloid leukemia.

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

3. The use according to claim 1, 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.

4. A pharmaceutical composition for treating or alleviating leukemia, comprising rhamnosine or a salt thereof and cytarabine.

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