Application of herba lysimachiae in preparation of medicine for treating leukemia

Abstract

The invention provides application of jinyu in preparation of a medicine for treating leukemia, and belongs to the technical field of biological pharmacy. The herba lysimachiae is capsules and tablets and mainly comprises five medicinal materials, namely, gaultheria yunnanensis, schefflera kwangsiensis, sargentgloryvine stem, alangium platanifolium and psammosilene tunicoides The invention proves that the Jingulian capsule can inhibit proliferation of human erythroleukemia cells HEL, and is dose-dependent and time-dependent; the cell cycle of human erythroleukemia cells HEL is retarded in the S period; the apoptosis of human erythroleukemia cells HEL can be obviously induced. Furthermore, in-vivo experiments show that low, medium and high dosages of the herba lysimachiae have no influence on liver and kidney functions of mice; the Jingulian capsule can reduce the weight of the spleen of a mouse with leukemia, increase the heme-to-solution ratio, improve the spleen structure, reduce the expression of a CD71 + cell population, promote the spleen and red blood cells with immature bone marrow to differentiate to a Ter119 + cell population and recover the spleen structure of the mouse. At present, Jingulian capsules and tablets are clinically used Miao medicines, can effectively shorten the clinical prophase research, and have good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and medicine, and particularly relates to the application of golden bone lotus in preparing medicine for treating leukemia. Background Art

[0002] Jingulian capsules can dispel wind and dampness, reduce swelling and relieve pain. They are used for joint swelling and pain caused by rheumatism and paralysis, and poor flexion and extension. The main ingredients include five medicinal materials: fennel, peach leaves, spatholobi, maple, and golden iron lock. Hard capsules, the contents are yellow-brown to reddish-brown powder; slight odor, slightly bitter taste.

[0003] Leukemia is a type of malignant clonal disease of hematopoietic stem cells. It is mainly divided into acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). At present, the main treatment options for leukemia are chemotherapy, targeted therapy, immunotherapy and stem cell transplantation, but the treatment effect is far from optimistic. It is urgent to improve the early diagnosis rate and explore high-efficiency and low-toxic therapeutic drugs, which are the key ways to treat and improve the survival rate of leukemia patients and improve the prognosis and quality of life of patients.

[0004] Invention patent CN112107609A discloses the use of golden bone lotus preparations in the preparation of drugs for treating chalazion, CN118059146A discloses the use of golden bone lotus in the preparation of products for preventing and treating liver cancer, CN115105537A discloses the use of golden bone lotus preparations in the preparation of drugs for treating or preventing lung injury, and CN111840358A discloses the use of golden bone lotus capsules in the preparation of anti-breast cancer drugs, but no reports on its anti-leukemia effect and its mechanism have been found. Summary of the invention

[0005] The invention aims to provide a new use for the golden bone lotus.

[0006] The present invention provides the application of golden bone lotus in the preparation of a drug for treating leukemia. The application aims at solving the difficulties in treating leukemia, finds new high-efficiency and low-toxic natural drug resources, improves the survival rate of leukemia patients and improves the prognosis and quality of life of patients. At the same time, it embodies the new efficacy of golden bone lotus and the important role of new use of old drugs.

[0007] The above-mentioned drugs for treating leukemia include drugs for inhibiting leukemia cell activity, drugs for inhibiting leukemia cell proliferation, and drugs for inducing leukemia cell apoptosis. Among them, in the drugs for inhibiting leukemia cell activity, leukemia cells include human erythroleukemia cells HEL and K562, acute lymphoblastic leukemia cells CEM-C1, Jurkat and CEM-C7H2, and mouse leukemia cells CB3; in the drugs for inducing leukemia cell apoptosis, leukemia cells are mainly human erythroleukemia cells HEL.

[0008] In the present invention, the half inhibitory concentration (IC 2 ) of Jingulian capsule on different leukemia cells HEL, K562, CB3, Jurkat, CEM-C1 and CEM-C7H2 was detected by MTT method in vitro. 50 ), screen out IC 50 The lowest cells; in vitro, the inhibition rate of human erythroleukemia cells HEL after 24h, 48h, and 72h of Jingulian capsule was detected by MTT method; the morphological changes of human erythroleukemia cells HEL after 24h and 48h of Jingulian capsule were observed by inverted microscope; the cycle arrest changes of human erythroleukemia cells HEL after 24h of Jingulian capsule were detected by flow cytometry, and the apoptosis rate of human erythroleukemia cells HEL after 24h of Jingulian capsule was detected. The results showed that Jingulian capsule could inhibit the proliferation of human erythroleukemia cells HEL, induce apoptosis of human erythroleukemia cells HEL, and block the cycle of human erythroleukemia cells HEL in the S phase. It was proved that Jingulian capsule had a good effect on human erythroleukemia cells HEL. In vivo experiments further showed that Jingulian capsule could improve the spleen structure of leukemia mice, reduce the burden of spleen, increase blood volume ratio, and reduce CD71 + The expression of cell populations promotes immature cells to Ter119 + The cell group differentiation and subsequent effective treatment of leukemia mice demonstrated that Jingulian Capsule has important potential in preventing and treating leukemia.

[0009] The above-mentioned golden bone lotus includes golden bone lotus capsules and golden bone lotus tablets, and the main ingredients include five medicinal materials including radix ophiopogonis, phragmites australis, radix spatholobi, octagonal maple, and radix scutellariae. The present invention can directly prepare golden bone lotus into a drug for treating leukemia, or combine it with other drugs for treating leukemia to prepare a compound preparation, or prepare it with a pharmaceutically acceptable carrier to prepare it into a drug for treating leukemia. The drug adopts an oral dosage form or an injection dosage form allowed in pharmacy, or a dosage form made by modern technology.

[0010] The beneficial effects of the present invention are as follows:

[0011] 1. Compared with the existing uses of Golden Bone Lotus, the present invention has discovered a new efficacy of Golden Bone Lotus in preventing and treating leukemia, and it has the potential to become a drug for preventing and treating leukemia.

[0012] 2. Golden Bone Lotus has been used in clinical practice. It has low toxicity, can effectively shorten preclinical research, and has good application prospects.

[0013] 3. Golden Bone Lotus can inhibit the vitality of leukemia cells, inhibit the proliferation of leukemia cells, block the leukemia cell cycle, induce leukemia cell apoptosis and effectively treat leukemia mice. It has an important prospect for improving the survival rate of leukemia patients and improving the prognosis and quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A is the 72h half-inhibitory concentration (IC50) of different concentrations of Jingulian capsule on different leukemia cells HEL, K562, CB3, Jurkat, CEM-C1, CEM-C7H2 and human normal liver cells HL-7702 detected by MTT method. 50 ); B is the inhibition rate of human erythroleukemia cells HEL after Jingulian capsule was applied for 24h, 48h and 72h; C is the morphological changes of human erythroleukemia cells HEL after Jingulian capsule was applied for 24h and 48h (magnification 200 times, scale bar 100μm).

[0015] Figure 2 A in the middle is the detection of cell cycle arrest of human erythroleukemia cells HEL treated with different concentrations of Jingulian capsule for 24 hours using flow cytometry; B is a cell cycle statistics chart.

[0016] Figure 3 A is the flow cytometry test of the apoptosis rate of human erythroleukemia cells HEL24h and 48h after treatment with different concentrations of Jingulian capsule; B is the 24h apoptosis rate statistical graph; C is the 48h apoptosis rate statistical graph.

[0017] Figure 4 The liver and kidney function tests of each group of mice. A is the alanine aminotransferase (ALT) of liver function; B is the aspartate aminotransferase (AST) of liver function; C is the blood urea nitrogen (BUN) of kidney function; and D is the creatinine (CREA) of kidney function.

[0018] Figure 5 A is a comparison of spleens of mice in each group; B is a statistical analysis of spleen weights of mice in each group; C is a statistical analysis of blood volume ratios of mice in each group; D, E, F, and G are comparisons of the weights of the heart, liver, lungs, and kidneys of mice, respectively.

[0019] Figure 6 The spleen CD71 + and Ter119 + Changes in cell population expression. A is flow cytometry detection of CD71 in spleen of leukemia mice after Jingulian capsule treatment + and Ter119 + Expression of cell populations; B and C are spleen CD71+ and Ter119 + Statistical plots of cell population expression.

[0020] Figure 7 The bone marrow CD71 + and Ter119 + Changes in cell population expression. A is flow cytometry detection of bone marrow CD71 in leukemia mice after Jingulian capsule treatment + and Ter119 + Expression of cell populations; B and C are bone marrow CD71 + and Ter119 + Statistical plots of cell population expression.

[0021] Figure 8 HE staining images of spleens of mice in each group (magnification 200 times, scale bar 100 μm). DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the embodiments and drawings, but the protection scope of the present invention is not limited to these embodiments.

[0023] Example 1

[0024] Experimental materials: RPMI-1640 and DMEM culture medium, fetal bovine serum (FBS) were purchased from Gibco (USA); penicillin-streptomycin mixture (100×) was purchased from Hyclone (USA); apoptosis kit was purchased from BD (USA); PI (propidium iodide) was purchased from Bio-Time (China); MTT and Triton X-100 were purchased from Beijing Solebow (China); RNase A was purchased from Dalian Bao Bio (China); Jingulian capsule was purchased from Guizhou Yibai Pharmaceutical Co., Ltd.; SD male rats and Balb / c mice were purchased from Chongqing Tengxin Huafu Laboratory Animal Sales Co., Ltd.; vincristine was purchased from Shenzhen Wanle Pharmaceutical Co., Ltd.; NIH3T3 cells (expressing F-MuLV clone 57 vector) were kindly donated by Professor Ben-David Yaacov of Guizhou Natural Products Research Center; Anti-Mouse Ter119 (FITC) was purchased from BD (USA); Anti-Mouse CD71 (APC) was purchased from Thermo Fisher (USA); human erythroleukemia cells HEL and K562, acute lymphoblastic leukemia cells Jurkat and CEM-C7H2 and human normal liver cells HL-7702 were purchased from the American Type Culture Collection (ATCC); acute lymphoblastic leukemia cell line CEM-C1 was purchased from Shanghai Fantai Biotechnology Co., Ltd.

[0025] Preparation of Jingulian Capsule sample mother solution: take the contents of Jingulian Capsule, dissolve in 60% ethanol aqueous solution by volume, shake on a shaker for 1 hour, ultrasonicate for 40 minutes, filter twice with absorbent cotton, take the filtrate and use a rotary evaporator to remove ethanol, store at -80℃ overnight, use a freeze dryer to remove moisture, and obtain a powdered sample; use physiological saline to prepare the powdered sample into 20mg / mL, 25mg / mL, and 30mg / mL mother solutions, and store at -20℃.

[0026] Preparation of 5% MTT solution: weigh 0.5 g of MTT powder, add 100 mL of sterilized PBS buffer, dissolve at 60°C, filter through a 0.22 μm microporous filter to sterilize, and store at -20°C.

[0027] Preparation of triple solution: Weigh 10 g of SDS, add less than 95 mL of double distilled water to fully dissolve it, then add 5 mL of isopropanol and make up to 100 mL with double distilled water, and finally add 100 μL of hydrochloric acid.

[0028] Cell culture: The above cells were taken out, and the HEL, K562, Jurkat, CEM-C1, CEM-C7H2, and CB3 cells were revived and cultured in RPMI-1640 medium containing 10% FBS in a 37°C, 5% CO2 incubator. The HL-7702 cells were revived and cultured in DMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator.

[0029] 1. Cell viability detection

[0030] Different leukemia cells HEL, K562, CB3, Jurkat, CEM-C1, CEM-C7H2 in the logarithmic growth phase were diluted with RPMI-1640 medium containing 10% FBS, and human normal liver cells HL-7702 were diluted with DMEM medium containing 10% FBS, and seeded in a 96-well plate at a density of 8000 cells / well, 90 μL per well, 100 μL of sterilized double distilled water was added to each well for four weeks, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for culture; after 4 hours, Jin Gu Lian capsule solution was added to each well as a treatment group, with concentrations of 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL (the mother solution was diluted with RPMI-1640 serum-free medium at different concentrations), 10 μL per well; a control group (Control) was set up, 10 μL of each well was added RPMI-1640 serum-free medium; 3 replicate wells were set up in each group and cultured in a 37°C, 5% CO2 incubator; after 72 hours, 10 μL of 5% MTT solution was added to each well and cultured in a 37°C, 5% CO2 incubator; after 4 hours, 100 μL of triple solution was added to each well and the 96-well plate was placed in a 37°C, 5% CO2 incubator overnight; the next morning, the absorbance (OD) value of each well at a wavelength of 570 nm was measured by an ELISA instrument to calculate the half-inhibitory concentration. Inhibition rate = (OD value of the control group - OD value of the treatment group) / OD value of the control group.

[0031] The results are as follows Figure 1 As shown in A, different concentrations of Jingulian capsule acted on different leukemia cell lines for 72 hours, and the half-inhibitory concentration for human erythroleukemia cells HEL was the lowest (150.92±3.69μg / mL). Therefore, human erythroleukemia cells HEL were selected as the main cells for the study. The half-inhibitory concentration for human normal liver cells HL-7702 was greater than 300μg / mL.

[0032] 2. MTT method was used to determine the inhibition rate of different concentrations of Jingulian capsule on human erythroleukemia cells HEL

[0033] Human erythroleukemia cells HEL in the logarithmic proliferation phase were diluted with RPMI 1640 medium containing 10% FBS, and seeded in a 96-well plate at a density of 8000 cells / well, 90 μL per well, 100 μL of sterilized double distilled water was added to each well for four weeks, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for culture; 4 hours later, each well was added with Jin Gu Lian capsule solution as a treatment group, with concentrations of 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL, 10 μL per well; a control group (Control) was set up, and 10 μL of RPMI-1640 serum-free medium was added to each well; 3 replicates were set up in each group and cultured in a 37°C, 5% CO2 incubator; the cells were treated for 24h, 48h, and 72h, and 10 μL of 5% was added to each well. MTT solution was added and cultured in a 37°C, 5% CO2 incubator. After 4 hours, 100 μL of triple solution was added to each well and cultured in a 37°C, 5% CO2 incubator overnight. The next morning, the absorbance (OD) value of each well at a wavelength of 570 nm was measured with an enzyme-labeled instrument to calculate the inhibition rate.

[0034] The results are as follows Figure 1 As shown in B, Jingulian capsule significantly inhibited the proliferation of human erythroleukemia cells HEL, and had a time- and concentration-dependent effect. When the drug concentration of Jingulian capsule on human erythroleukemia cells HEL was 300 μg / mL, the inhibition rate was 48.35% at 24h, 72.52% at 48h, and 83.18% at 72h, showing a time-dependent effect.

[0035] 3. Cell morphology observation

[0036] Human erythroleukemia HEL cells in logarithmic growth phase were diluted with RPMI 1640 medium containing 10% FBS to a concentration of 3×10 5 The cells were seeded at a density of 10 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 4 hours, different concentrations of Jin Gu Lian capsule solution (150μg / mL, 200μg / mL, 250μg / mL) were added, 20μL per well; a control group (Control) was set up, and 20μL RPMI-1640 serum-free culture medium was added to each well; then each well was supplemented with RPMI-1640 serum-free culture medium to 2mL to make the volume of each group consistent, and cultured in a 37°C, 5% CO2 incubator; cell morphology changes were observed at 24h and 48h.

[0037] The results are as follows Figure 1 As shown in C, compared with the Control group, after 24h and 48h of treatment with Jingulian Capsule on human erythroleukemia cells HEL, the number of cells decreased, and the cells were broken and shrunken as the drug concentration increased.

[0038] 4. Flow cytometry detection of cell cycle

[0039] Human erythroleukemia HEL cells in logarithmic growth phase were diluted with RPMI 1640 medium containing 10% FBS to a concentration of 3×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 4 hours, different concentrations of Jin Gu Lian capsule solution (150 μg / mL, 200 μg / mL, 250 μg / mL) were added, 20 μL per well; a control group (Control) was set up, and 20 μL RPMI-1640 serum-free culture medium was added to each well; then each well was supplemented to 2 mL with RPMI-1640 serum-free culture medium to make the volume of each group consistent, and cultured in a 37°C, 5% CO2 incubator; 24 hours after the cells were collected, fixed with a volume concentration of 70% ethanol aqueous solution, -20°C overnight, 500 μL dye (mixed and prepared by 6 mL PBS buffer, 300 μLPI (propidium iodide), 4 μL Triton X-100, and 30 μL RNase A) was added to each sample, stained in the dark for 30 minutes, and the cell cycle was detected by flow cytometry.

[0040] The results are as follows Figure 2 As shown, Jingulian Capsule blocks the HEL cycle of human erythroleukemia cells at the S phase in a dose-dependent manner.

[0041] 5. Detection of cell apoptosis by flow cytometry

[0042] Human erythroleukemia HEL cells in logarithmic growth phase were diluted with RPMI 1640 medium containing 10% FBS to a concentration of 3×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 4 hours, different concentrations of Jin Gu Lian capsule solution (150μg / mL, 200μg / mL, 250μg / mL) were added, 20μL per well. A control group (Control) was set up, and 20μL RPMI-1640 serum-free medium was added to each well. Then each well was supplemented with RPMI-1640 serum-free medium to 2mL to make the volume of each group consistent, and cultured in a 37°C, 5% CO2 incubator. The cells were collected at 24h and 48h, 50μL 1×Binding Buffer was added to each tube, and gently blown to mix, 1.5μL PI and 1.5μL Annexin V were added to each tube, and after staining on ice for 15min in the dark, cell apoptosis was detected by flow cytometry.

[0043] The results are as follows Figure 3As shown in the results, compared with the Control group, Jingulian capsule could significantly induce apoptosis of human erythroleukemia cells HEL in a time- and dose-dependent manner.

[0044] In the above experiments, each experiment was repeated three times independently, and the experimental data were expressed as mean ± standard deviation (n = 3, compared with the Control group, *P < 0.05, **P < 0.01, ***P < 0.001).

[0045] 6. The therapeutic effect of Jingulian capsule on leukemia mice

[0046] Leukemia mouse model induced by Friend murine leukemia virus (F-MuLV): Balb / c mice were injected intraperitoneally with F-MuLV within 48 hours after birth, 120μL / mouse. At 4 weeks, they were randomly divided into Model group, vincristine group (VCR group, positive drug control group), low-dose Jin Gu Lian capsule group (JGLC-L group), medium-dose Jin Gu Lian capsule group (JGLC-M group) and high-dose Jin Gu Lian capsule group (JGLC-H group). Normal Balb / c mice (Normal Control group, abbreviated as NC group) were set as negative control. Each group had 10 mice, half male and half female. Drugs were administered starting at 6 weeks, and samples were collected 4 weeks after gavage. The blood volume ratio of mice was detected, the weight of heart, liver, spleen, lung and kidney was weighed, serum was separated to detect liver and kidney function, and spleen tissue was retained for pathological section examination. Administration: JGLC-L, JGLC-M and JGLC-H groups were administered by gavage once a day for 4 consecutive weeks; VCR group was injected intraperitoneally once a week for 4 consecutive weeks; Control group was gavaged with normal saline. The dosage for JGLC group was converted to the dosage for mice according to the human dosage in the instruction manual, i.e. JGLC-L group: 225.25 mg / kg / d, JGLC-M group: 675.75 mg / kg / d, JGLC-H group: 1126.25 mg / kg / d, and 200 μL was gavaged per 20 g mouse. The dosage for VCR group was converted to the dosage for mice according to the human dosage in the instruction manual, i.e. VCR group: 0.5 mg / kg, and 100 μL was injected intraperitoneally per 20 g mouse.

[0047] Figure 4 The results showed that compared with the NC group, the liver and kidney indicators of the mice in the Model group were abnormal, and the low, medium and high doses of Jingulian capsule had no effect on the liver and kidney function of the mice. Compared with the Model group, Jingulian capsule reduced the spleen weight of leukemia mice (see Figure 5 A, B), thereby reducing the burden on the spleen; compared with the Model group, Jingulian capsule increased the blood volume ratio of leukemia mice ( Figure 5 C); Compared with the NC group, the liver weight of the mice in the Model group increased ( Figure 5E) In the spleen and bone marrow of mice, Jingulian capsule reduced CD71 compared with the Model group. + The expression of cell populations promotes immature cells to Ter119 + Cell population differentiation, improving anemia symptoms in mice (see Figure 6 and Figure 7 Compared with the Model group, Jingulian capsule reduced the infiltration of leukemia cells in the spleen of mice and restored the structure of the spleen of mice (see Figure 8 ). In conclusion, Jingulian capsule can effectively treat leukemia mice.

[0048] From the above results, it can be seen that Jingulian has the potential value to be developed into the preparation of drugs for the prevention and treatment of leukemia.

Claims

1. Application of Golden Bone Lotus in the preparation of drugs for the treatment of leukemia.

2. The use of the golden bone lotus according to claim 1 in preparing a drug for treating leukemia, characterized in that: The drugs include drugs for inhibiting leukemia cell activity, drugs for inhibiting leukemia cell proliferation, and drugs for inducing leukemia cell apoptosis.

3. The use of the golden bone lotus according to claim 2 in the preparation of a drug for treating leukemia, characterized in that: In the drug for inhibiting the activity of leukemia cells, the leukemia cells include human erythroleukemia cells HEL and K562, acute lymphoblastic leukemia cells CEM-C1, Jurkat and CEM-C7H2, and mouse leukemia cells CB3.

4. The use of the golden bone lotus in the preparation of a drug for treating leukemia according to claim 2, characterized in that: In the drug for inducing leukemia cell apoptosis, the leukemia cells are mainly human erythroleukemia cells HEL.

5. The use of the golden bone lotus according to any one of claims 1 to 4 in the preparation of a drug for treating leukemia, characterized in that: The golden bone lotus includes golden bone lotus capsules and golden bone lotus tablets.

6. The use of the golden bone lotus according to any one of claims 1 to 4 in the preparation of a drug for treating leukemia, characterized in that: The medicine is prepared by directly preparing Jin Gu Lian into a medicine for treating leukemia, or preparing a compound preparation by combining Jin Gu Lian with other medicines for treating leukemia, or preparing a medicine for treating leukemia by combining Jin Gu Lian with a pharmaceutically acceptable carrier.

7. The use of Jin Gu Lian in preparing a drug for treating leukemia according to claim 6, characterized in that: The medicine is in the form of oral preparation or injection permitted in pharmaceutical science, or in the form prepared by modern technology.

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