CN114832072A

CN114832072A - Application of stranguria-treating capsule or preparation containing same in preparation of medicament for preventing and treating leukemia

Info

Publication number: CN114832072A
Application number: CN202210491183.6A
Authority: CN
Inventors: 秦川霞; 李艳梅; 黎中恒; 黄磊; 饶青; 宋晶睿
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-08-02

Abstract

The invention relates to the technical field of medicines, in particular to application of a stranguria clearing capsule or a preparation containing the components in preparing a medicament for preventing and treating leukemia. The components of the invention comprise: herba Polygoni Capitati, cortex Phellodendri, herba Oxalidis Corniculatae, herba et Gemma Agrimoniae, lalang grass rhizome and herba plantaginis. The stranguria clearing capsule or the preparation containing the components can effectively inhibit the proliferation of leukemia cells, and particularly has good inhibition effect on acute lymphocytic leukemia cells Jurkat, erythroleukemia cells HEL and chronic myelogenous leukemia cells K562; and the stranguria-treating and stranguria-relieving capsule is a Guizhou national medicine which is already on the market, has low toxic and side effects, can be directly applied to a human body, can effectively shorten the preliminary clinical research, and has good application prospect.

Description

Application of stranguria-treating and stranguria-treating capsule or preparation containing stranguria-treating and stranguria-treating capsules in preparation of leukemia preventing and treating medicines

Technical Field

The invention relates to the technical field of medicines, in particular to application of a stranguria clearing capsule or a preparation containing the components in preparing a medicament for preventing and treating leukemia.

Background

Leukemia (leukemia) is a malignant clonal disease of hematopoietic stem progenitor cells. Its cloned leukemia cells lose the ability to differentiate further and mature and arrest at different stages of cell development. In bone marrow and other hematopoietic tissues, leukemia cells proliferate and accumulate in large quantities and infiltrate other organs and tissues, while normal hematopoiesis is inhibited, with symptoms of anemia, hemorrhage, infection and infiltration of various organs.

The main current treatment means for leukemia in western medicine is chemotherapy. The chemotherapy drugs are almost all cytotoxic drugs, and have certain toxic and side effects on normal cells of a human body while killing tumor cells, particularly on cells with rapid division and proliferation, such as bone marrow hematopoietic cells, gastrointestinal mucosal epithelial cells and the like.

Disclosure of Invention

In order to solve the problems, the invention provides an application of a stranguria clearing capsule or a preparation containing the components in preparing a medicament for preventing and treating leukemia. The stranguria clearing capsule or the preparation containing the components can effectively inhibit the proliferation of leukemia cells, has a remarkable treatment effect on leukemia, and is low in toxic and side effects.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of a stranguria clearing capsule or a preparation containing the components in preparing a medicament for preventing and treating leukemia, wherein the components comprise: herba Polygoni Capitati, cortex Phellodendri, herba Oxalidis Corniculatae, herba et Gemma Agrimoniae, lalang grass rhizome and herba plantaginis.

Preferably, the components comprise the following components in parts by mass: 42 parts of four-season red, 30 parts of golden cypress, 30 parts of creeping oxalis, 16 parts of hairyvein agrimony, 24 parts of lalang grass rhizome and 20 parts of plantain herb.

Preferably, the stranguria clearing capsule or the preparation containing the components is directly prepared into a medicament for preventing and treating leukemia or is prepared into a medicament for preventing and treating leukemia together with other preparations for preventing and treating leukemia.

Preferably, the leukemia includes one or more of acute lymphocytic leukemia, acute myelocytic leukemia and chronic myelogenous leukemia.

Preferably, the acute myeloid leukemia comprises erythroleukemia.

Preferably, the prevention of leukemia comprises inhibiting leukemia cell proliferation or promoting leukemia cell apoptosis.

Preferably, the leukemia cells comprise one or more of acute lymphocytic leukemia cells Jurkat, erythroleukemia cells HEL and chronic myelogenous leukemia cells K562.

Preferably, the dosage form of the medicament comprises a dosage form prepared by modern technology.

Preferably, the dosage form of the medicament comprises an oral dosage form or an injection dosage form.

Has the advantages that:

the invention provides an application of a stranguria clearing capsule or a preparation containing the components in preparing a medicament for preventing and treating leukemia, wherein the components comprise: herba Polygoni Capitati, cortex Phellodendri, herba Oxalidis Corniculatae, herba et Gemma Agrimoniae, lalang grass rhizome and herba plantaginis. The stranguria clearing capsule or the preparation containing the components can effectively inhibit the proliferation of leukemia cells, and particularly has good inhibition effect on acute lymphocytic leukemia cells Jurkat, erythroleukemia cells HEL and chronic myelogenous leukemia cells K562; and the stranguria-treating and stranguria-relieving capsule is a Guizhou national medicine on the market, has low toxic and side effects, can be directly applied to a human body, can effectively shorten the preliminary clinical research, and has good application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 is a graph showing the effect of different concentrations of MLQ (urinary clear capsule core component) on the growth and proliferation of human acute lymphoblastic leukemia cells Jurkat (growth curve);

FIG. 2 shows the survival rate of human acute lymphoblastic leukemia cell Jurkat different MLQ concentrations (Neilclear Capsule core component) and at different times;

FIG. 3-1 shows the effect of MLQ (capsule core component of stranguria and Clean) of different concentrations on apoptosis of human acute lymphocytic leukemia cells Jurkat after 24h of culture (flow cytometry detection result);

FIG. 3-2 shows the effect of MLQ (capsule core component of stranguria and Clean) of 48h in culturing on apoptosis of human acute lymphocytic leukemia cells Jurkat (flow cytometry results);

FIG. 4 is a graph of the effect of different concentrations of MLQ (urinary clear capsule core component) on apoptosis (apoptosis rate) of human acute lymphocytic leukemia cells Jurkat;

FIG. 5 shows the survival rate of HEL (human erythroleukemia cell) after 48h of MLQ (core component of Milinqing Capsule) at different concentrations;

FIG. 6 shows the survival rate of human chronic myelogenous leukemia cell K562 at different concentrations of MLQ (Nelin Qing Capsule core component) for 48 h;

different treatment groups relative to the control group, wherein x represents P < 0.05; p < 0.01; p < 0.001.

Detailed Description

In the present invention, the components preferably include the following components in parts by mass: 42 parts of four-season red, 30 parts of golden cypress, 30 parts of creeping oxalis, 16 parts of hairyvein agrimony, 24 parts of lalang grass rhizome and 20 parts of plantain herb.

In the present invention, the stranguria clearing capsule or the preparation containing the components is preferably directly prepared into a leukemia prevention and treatment drug or is prepared into a leukemia prevention and treatment drug together with other leukemia prevention and treatment preparations.

In the present invention, the leukemia includes one or more of acute lymphocytic leukemia, acute myelocytic leukemia and chronic myelogenous leukemia.

In the present invention, the prevention of leukemia preferably includes inhibition of leukemia cell proliferation or promotion of leukemia cell apoptosis; the leukemia cells preferably comprise one or more of acute lymphocytic leukemia cells Jurkat, erythroleukemia cells HEL and chronic myelogenous leukemia cells K562. The invention determines that the stranguria clearing capsule and the core component thereof have obvious leukemia inhibiting effect through in-vitro leukemia cell resisting activity test, and flow cytometry proves that the medicine or the core component has the effect of promoting leukemia cell apoptosis and inhibiting leukemia cell growth, thus representing the important potential of being used as an anti-leukemia medicine.

In the present invention, the dosage form of the drug includes a dosage form prepared by modern technology, and more preferably includes an oral dosage form or an injection dosage form. The invention can adopt any oral preparation formulation allowed in pharmaceutics or any injection formulation allowed in pharmaceutics, and is convenient to apply.

In order to further illustrate the present invention, the application of the stranguria clearing capsule or the preparation containing the components provided by the present invention in the preparation of drugs for preventing and treating leukemia is described in detail with reference to the following examples, but the application of the stranguria clearing capsule or the preparation containing the components is not to be construed as limiting the protection scope of the present invention.

The% in the following examples are not specified as mass percent (wt.%), all in volume percent.

Example 1

In vitro anti-leukemic cell Activity assay

1. Experimental materials

Preparation of a tested sample solution: weighing a proper amount of stranguria clearing capsule medicine (after capsule shells are removed) according to the requirement, and mixing the raw materials according to a material-liquid ratio of 1 g: 20mL of the solution was dissolved in 60% ethanol (another 40% was ddH) ₂ O), fully and uniformly mixing, shaking for 1h by a shaking table, performing ultrasonic treatment for 40min, filtering absorbent cotton for 2 times, performing rotary evaporation by using a rotary evaporator to remove ethanol, standing overnight at the temperature of-80 ℃, and finally removing water by using a freeze dryer to obtain a powdery sample. The resulting powdery sample was weighed and prepared into a 100mg/ml mother liquor (hereinafter referred to as MLQ, solvent: 10% DMSO solution, and further 90% ddH solution) ₂ O) for standby.

Preparing an MTT solution: 0.5g of MTT powder was weighed, 100mL of sterilized PBS was added, dissolved at 60 ℃, filtered and sterilized with a 0.22 μm filter membrane, and stored at 4 ℃ for further use.

Cell line: jurkat is a key laboratory of chemical preservation of natural products of Chinese academy of sciences of Guizhou province. Recovering Jurkat from liquid nitrogen tank at 37 deg.C under 5% CO ₂ The culture was carried out in an incubator using RPMI-1640 medium (containing 5% FBS).

RPMI-1640 medium was purchased from Hycone, Fetal Bovine Serum (FBS) was purchased from Hangzhou Biotech Co., Ltd., Zhejiang, and Tetramethyl azo Lily salt (MTT), dimethyl sulfoxide (DMSO) and penicillin mixed solution were purchased from Beijing Soilebao Tech Co., Ltd.

2. Experimental methods

MTT method for determining inhibition effect of MLQ with different concentrations on leukemia cell proliferation

Human leukemia cells in logarithmic proliferation stage Jurkat (the seeding density of Jurkat is 1X 10) ⁴ cells/well) were seeded in 4 96-well plates, each 96-well plate was treated as follows: after 4h of incubation in 90. mu.l of RPMI-1640 medium (containing 5% FBS) per well, 10. mu.l of MLQ was added to give final concentrations of 62.5. mu.g/ml, 125. mu.g/ml and 250. mu.g/ml, 4 duplicate wells (designated as experimental group) and 1 experimental blank well (experimental blank well with medium only and drug at the corresponding concentration, no Jurkat cells inoculated, for subtracting the effect of the drug's own color), and a control group (10. mu.l of 10% DMSO solution added) with 4 duplicate wells and 1 control blank well (control blank well with medium only and 10. mu.l of 10% DMSO solution, no Jurkat cells inoculated).

The cells are placed in an incubator at 37 ℃ for timing, 4 96-well plates are respectively cultured for 24h, 48h, 72h and 96h, 10 mu l of MTT solution is added into each well of the 96-well plates at different time points, after continuous culture is carried out for 4h, a triple solution is added (the preparation of the triple solution is that 10g of SDS, 5ml of isopropanol and 100 mu l of hydrochloric acid are mixed, ddH is added ₂ The final volume is 100ml to obtain the triple solution) 100 mul/hole, culturing overnight at 37 ℃, measuring the absorbance (OD value) of each hole with the wavelength of 570nm by a microplate reader, calculating the survival rate, and the results are shown in tables 1-4, figure 1 and figure2。

The survival rate (experimental OD value-corresponding concentration experimental blank OD value)/(control average OD value-control blank OD value) is 100%.

TABLE 1 Absorbance of incubation at different times for the control group and the 62.5. mu.g/ml concentration-treated group

TABLE 2125. mu.g/ml and absorbance at different times for the 250. mu.g/ml concentration treatment groups

TABLE 362.5 survival (%)

TABLE 4125. mu.g/ml and 500. mu.g/ml concentrations survival (%)

Note: tables 1-4 show 4 multiple holes for 1, 2, 3, and 4.

From the above results, it was found that the average survival rate of Jurkat cells was only about 14.15% at a drug concentration of 250. mu.g/ml, and 27.28% at a concentration of 125. mu.g/ml. At different time points, when the drug concentration is 62.5 mug/ml, 125 mug/ml and 250 mug/ml, the survival rate is obviously different; therefore, the core component of the stranguria clearing capsule can effectively inhibit the growth of leukemia cell Jurkat and has obvious time and concentration dependence.

Example 2

Flow cytometry to determine apoptosis

The experimental materials were as in example 1, and the experimental methods were as follows:

the human leukemia cells Jurkat in logarithmic proliferation phase are added at 5X 10 ⁵ cells/dish were inoculated at a density of 60mm round dishes, 2000. mu.l of RPMI-1640 medium (containing 5% FBS) was added to each dish, and MLQ was added at different concentrations after 4 hours to give final concentrations of 150. mu.g/ml, 300. mu.g/ml, and 600. mu.g/ml, respectively. Control group was added with equal amount of solvent (10% DMSO solution) in 5% CO ₂ Culturing in 37 deg.C incubator, collecting 24h and 48h cells, staining with PI propidium iodide and annexin V-FITC for 15min, measuring apoptosis on flow cytometer, and counting apoptosis rate. The results are shown in FIG. 3-1, FIG. 3-2, FIG. 4 and Table 5.

TABLE 5 apoptosis rates (%)

Group of	24h	48h
			Control group	5.94	7.12
150μg/ml	7.94	12.68
			300μg/ml	29.18	48.28
600μg/ml	73.26	80.55

From the results, the core component of the stranguria clearing capsule has good apoptosis promoting effect on leukemia cell Jurkat and has obvious time and concentration dependence.

Example 3

A method similar to example 1, except that:

1) cell line: the HEL cells are stored in a chemical key laboratory of natural products of Chinese academy of sciences of Guizhou province. Recovering HEL from liquid nitrogen tank, and recovering at 37 deg.C with 5% CO ₂ Culturing in RPMI-1640 medium (containing 5% FBS) in an incubator;

2) HEL cells in a logarithmic proliferation phase (the seeding density of the HEL cells is 8000 cells/well) are seeded in a 96-well plate, 90. mu.l of RPMI-1640 medium (containing 5% FBS) is added to each well, 10. mu.l of MLQ with different concentrations are added to each well after 4h of culture to make the final concentrations of 125. mu.g/ml, 250. mu.g/ml and 500. mu.g/m, 4 duplicate wells (marked as experimental group) and 1 experimental blank well (experimental blank well has only medium and corresponding concentration of drug, no HEL cells are inoculated and is used to subtract the effect of the color of the drug itself), a control group (10. mu.l of 10% DMSO solution is added) is additionally set, and 4 duplicate wells and 1 control blank well (control blank well has only medium and 10. mu.l of 10% DMSO solution, no HEL cells are inoculated).

The cells are placed in an incubator at 37 ℃ for timing, the cells are taken out after 48h of culture, 10 mu l of MTT solution is added into each well, after 4h of culture is continued, a triple liquid is added (the preparation of the triple liquid is that 10g of SDS, 5ml of isopropanol and 100 mu l of hydrochloric acid are mixed, ddH is added ₂ The final volume is 100ml to obtain the triple solution), 100 mul/hole, overnight at 37 ℃ in an incubator, measuring the absorbance (OD value) of each hole with the wavelength of 570nm by an enzyme-labeling instrument, calculating the survival rate, and the experimental results are shown in tables 6-9 and figure 5.

Survival rate (experimental OD value-corresponding concentration experimental blank OD value)/(control average OD value-control blank OD value) × 100%.

TABLE 6 Absorbance of control group and 125. mu.g/ml concentration-treated group cultured for 48 hours

TABLE 7250. mu.g/ml and absorbance of 500. mu.g/ml concentration treated groups cultured for 48h

TABLE 8125 μ g/ml survival (%)

TABLE 9250. mu.g/ml survival (%)

Note: tables 6-9 show that 1, 2, 3, and 4 are 4 multiple holes.

From the above results, the average survival rate of HEL is only 6.79% at a drug concentration of 500 μ g/ml, and 60.79% at a drug concentration of 250 μ g/ml, which indicates that the core component of the stranguria-treating and pain-relieving capsule can effectively inhibit the growth of leukemia cells HEL and has significant concentration dependence.

Example 4

A method similar to example 1, except that:

1) cell line: the K562 cells are stored in a chemical key laboratory of natural products of Chinese academy of sciences of Guizhou province. Recovering K562 from liquid nitrogen tank at 37 deg.C and 5% CO ₂ Culturing in RPMI-1640 medium (containing 5% FBS) in an incubator;

2) k562 cells (K562 cells were seeded at 8000 cells/well) in a logarithmic proliferation phase in 96-well plates at 90. mu.l RPMI-1640 medium (containing 5% FBS) per well, after 4h of incubation, 10. mu.l of MLQ at different concentrations were added to each well to give final concentrations of 62.5. mu.g/ml, 125. mu.g/ml, 250. mu.g/ml and 500. mu.g/ml, 4 duplicate wells (identified as experimental) and 1 experimental blank well (experimental blank well with medium only and drug at the corresponding concentration, no K562 cells inoculated for subtraction of the effect of the drug's own color) were set, and a control (10. mu.l of 10% DMSO solution was added) was set with 4 duplicate wells and 1 control blank well (control blank well with medium only and 10. mu.l of 10% DMSO solution, no K562 cells inoculated).

The cells are placed in an incubator at 37 ℃ for timing, the cells are taken out after 48h of culture, 10 mu l of MTT solution is added into each well, after 4h of culture is continued, a triple liquid is added (the preparation of the triple liquid is that 10g of SDS, 5ml of isopropanol and 100 mu l of hydrochloric acid are mixed, ddH is added ₂ The final volume is 100ml to obtain the triple solution), 100 mul/well, overnight at 37 ℃, measuring the absorbance (OD value) of each well with the wavelength of 570nm by a microplate reader, calculating the survival rate, and the experimental results are shown in tables 10-14 and fig. 6.

TABLE 10 Absorbance of control group cultured for 48h

Table 1162.5. mu.g/ml and 125. mu.g/ml absorbance for 48h of the treated groups

TABLE 12250. mu.g/ml and 500. mu.g/ml Absorbance of the treated groups at 48h incubation

TABLE 1362.5 μ g/ml survival (%)

TABLE 14250 μ g/ml survival (%)

Note: tables 10 to 14 show that 1, 2, 3 and 4 are 4 multiple holes.

From the above results, the average survival rate of K562 is only 6.52% at a drug concentration of 500 μ g/ml, and the average survival rate is only 31.37% at a drug concentration of 250 μ g/ml, so that the core component of the stranguria clearing capsule can effectively inhibit the growth of leukemia cells K562 and has significant concentration dependence.

In conclusion, the core component of the stranguria clearing capsule or the stranguria clearing capsule can effectively inhibit the proliferation of leukemia cells, and particularly has good effects of inhibiting and promoting the apoptosis of leukemia cells of acute lymphocytic leukemia cells Jurkat, erythroleukemia cells HEL and chronic myelogenous leukemia cells K562; has potential value in preparing leukemia cell proliferation inhibitor or medicine for preventing and treating leukemia.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the stranguria clearing capsule or the preparation containing the components in preparing the medicament for preventing and treating the leukemia is characterized in that the components comprise: herba Polygoni Capitati, cortex Phellodendri, herba Oxalidis Corniculatae, herba et Gemma Agrimoniae, lalang grass rhizome and herba plantaginis.

2. The use according to claim 1, wherein the ingredients comprise the following components in parts by mass: 42 parts of four-season red, 30 parts of golden cypress, 30 parts of creeping oxalis, 16 parts of hairyvein agrimony, 24 parts of lalang grass rhizome and 20 parts of plantain herb.

3. The use according to claim 1 or 2, wherein the stranguria clearing capsule or the preparation containing the components is directly used for preparing the medicament for preventing and treating leukemia or is used for preparing the medicament for preventing and treating leukemia together with other preparations for preventing and treating leukemia.

4. The use according to claim 1 or 2, wherein the leukemia comprises one or more of acute lymphocytic leukemia, acute myeloid leukemia and chronic myeloid leukemia.

5. The use of claim 4, wherein the acute myeloid leukemia comprises erythroleukemia.

6. The use of claim 1 or 2, wherein the prevention of leukemia comprises inhibiting leukemia cell proliferation or promoting leukemia cell apoptosis.

7. The use of claim 6, wherein said leukemia cells comprise one or more of acute lymphocytic leukemia cells Jurkat, erythroleukemic cells HEL, and chronic myelogenous leukemia cells K562.

8. Use according to claim 1 or 2, wherein the pharmaceutical dosage form comprises a dosage form made using modern technology.

9. The use of claim 8, wherein the pharmaceutical dosage form comprises an oral dosage form or an injectable dosage form.