CN113876774B - Application of neoliensinine in preparing medicine for treating leukemia - Google Patents

Abstract

The invention discloses an application of neoliensinine in preparation of a medicament for preventing and/or treating leukemia, wherein the structure of the neoliensinine is shown as a formula (1). The neoliensinine has the capability of inhibiting the activity of AML cells, CML cells, T cell malignant tumors and DLBCL cells of human beings and inducing the apoptosis of the cells, and shows the application prospect of the neoliensinine in the preparation of the drugs for treating leukemia.

Description

Application of neoliensinine in preparing medicine for treating leukemia

Technical Field

The invention relates to application of a compound, in particular to application of neoliensinine and pharmaceutically acceptable salts thereof in preparing a medicament for treating leukemia.

Background

Lotus plumule is a commonly used Chinese medicine, also called Nelumbo Nucifera Gaertn, coix lachryma-jobi, and Coix lacryma-jobi, and is derived from young leaf and radicle of dried seed of Nelumbonaceuca Gaertn, nelumbo nucifera, nelumbo of Nymphaeaceae. The theory of traditional Chinese medicine holds that the lotus plumule has the effects of clearing away heart-fire, removing heat, stopping bleeding and arresting seminal emission, and is mainly used for treating symptoms such as vexation, thirst, hematemesis, spermatorrhea, conjunctival congestion, swelling and pain, hypertension and the like. The traditional Chinese medicine lotus plumule is also used for treating leukemia. A Chinese medicinal preparation for resisting leukemia and reversing drug resistance is prepared from plumula Nelumbinis and radix astragali, and has effects of resisting leukemia and reversing drug resistance, enhancing immunity, and improving antitumor effect [ Xie Zhaoxia. Chinese medicinal preparation for resisting leukemia and reversing drug resistance: CN99115402.9.1999.06.02. The alkaloid contained in the lotus plumule is mainly isoquinoline compound [ Zhang Guang, pan Yang. Research summary of alkaloid component in plant lotus, nanjing university of traditional Chinese medicine (Nature science edition), 2002, 18 (6): 382 to 384; yang Guangming, pan Yang research progress of Lianchen isoquinoline alkaloid and its action of relaxing smooth muscle, chinese traditional medicine J, 2019,44 (18): 3924-3934, only neferine (neferine) is currently found to have an Effect against human Chronic myelogenous Leukemia In Leukemia therapy [ Zhang YL, xiao YH, dong QX, ouyang WJ, qin Q.Neferine In the Lotus plumule patents of the anticancer Effect of Imatinib In Primary cardiac myoid Leukemedia In vitro of journal of Food Science,2019,84 (4): 904-910.].

4325 Zxft 4325, etc. found that the plumula nelumbinis contains new structural type compound plumula nelumbinis alkaloid (English name: neolinine, abbreviation: neoL), and has proved that the plumula nelumbinis alkaloid has the function of inhibiting non-small cell lung cancer invasion and metastasis in the previous period [ Zhang Wanting, wang Xinzhu, mi Yuhui, yang Guangming, pan Yang, the plumula nelumbinis alkaloid has the function of inhibiting H1299 cell invasion and migration induced by transforming growth factor beta 1 and the mechanism thereof. 1459-1467.]. Yang GM et al found the vasodilatory action of neoliensinine on mesenteric vascular smooth muscle [ Yang GM, sun J, pan Y, zhang JL, xiao M, zhu MS.isolation and identification of a tribenzoisoquinoline alkali from Nelumbo reticulata Gaertn, a novel potential smooth muscle tissue.Fitoteapia, 2018, 124:58-65.]. Zhang Junli and the like have studied the inhibitory effect and molecular mechanism of liensinine on the contraction of mesenteric vascular smooth muscle [ Zhang Junli, xiamin, wang Peng, zhangxuan, pan Yang, yang Guangming, the inhibitory effect and molecular mechanism of liensinine on the contraction of mesenteric vascular smooth muscle, nanjing university of traditional Chinese medicine (Nature science edition), 2019,35 (3): 313-318.].

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of the neoliensinine or pharmaceutically acceptable salt in preparing a medicament for treating leukemia.

The technical scheme is as follows: the invention relates to an application of the neoliensinine and the pharmaceutically acceptable salt thereof in preparing the drugs for preventing and/or treating leukemia.

The application is that the neoliensinine has a structure shown in formula (1):

the application of the compound can be used for treating leukemia, including acute myelogenous leukemia, chronic granulocytic leukemia, T-cell malignant tumor and diffuse large B-cell lymphoma.

The application prevents and/or treats leukemia through activity inhibition and apoptosis induction of human AML cells, human CML cells, human T cell malignant tumors and human DLBCL cells.

The application of the neoliensinine and the pharmaceutically acceptable salts thereof in preparing the medicines for preventing and/or treating the acute myeloid leukemia.

The application of the neoliensinine and the pharmaceutically acceptable salts thereof in preparing the medicines for preventing and/or treating chronic granulocytic leukemia.

The application of the neoliensinine and the pharmaceutically acceptable salts thereof in preparing the medicines for preventing and/or treating the T cell malignant tumor.

Application of liensinine and pharmaceutically acceptable salts thereof in preparation of medicines for preventing and/or treating diffuse large B cell lymphoma.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages: through MTT cell activity detection experiments and Annexin V-FITC/PI cell apoptosis detection experiments, the research discovers that the new structural type compound of the tribenzylisoquinoline phenylether type neoliensinine in the lotus plumule has the capability of inhibiting the activity of AML cells, CML cells, T cell malignant tumors and DLBCL cells of a human and inducing the apoptosis of the cells, verifies that the neoliensinine has the function of resisting leukemia, and shows the application prospect of the neoliensine in the preparation of the medicine for treating the leukemia.

Drawings

FIG. 1 is a graph of the effect of 0-32 μ M liensinine on the viability level of the AML cell line THP1 cells at 6h and 12 h;

FIG. 2 is the effect of 0-32 μ M of liensinine on the viability level of AML cell line MV411 cells at 6h and 12 h;

FIG. 3 is a graph of the effect of 0-32 μ M of liensinine on the viability level of the T cell malignant cell line Jurkat cells at 6h and 12 h;

FIG. 4 is a graph of the effect of 0-32 μ M liensinine on the viability level of the T cell malignant tumor cell line Molt4 cells at 6h and 12 h;

FIG. 5 is a graph of the effect of 0-32 μ M liensinine on the viability level of the T cell malignant tumor cell line Hut102 cells at 6h and 12 h;

FIG. 6 is the effect of 0-32 μ M liensinine on the viability level of CML cell line K562 cells at 6 h;

FIG. 7 shows the effect of 0-32. Mu.M liensinine on the viability level of DLBCL cell line OCILY3 cells at 6h and 12 h;

FIG. 8 is the effect of 0-32 μ M liensinine on viability levels of DLBCL cell line R1.1 cells at 6h and 12 h;

FIG. 9 is the effect of 0-32 μ M liensinine on the viability level of the DLBCL cell line Sudhl4 cells at 6h and 12 h;

FIG. 10 is a graph of the effect of 0-32 μ M liensinine on the viability level of the DLBCL cell line Sudhl8 cells at 6h and 12 h;

FIG. 11 is a measurement of apoptosis-inducing ability of 8. Mu.M liensinine on AML cell lines THP1, MV411, U937 and ME1 cells at 12 h;

FIG. 12 is a graph of the measurement of apoptosis-inducing ability of 8. Mu.M liensinine on CML cell line K562 cells at 4 h;

FIG. 13 is a graph of the apoptosis-inducing ability of 8 μ M liensinine at 12h on T cell malignant cell lines Jurkat, hut102 and Molt4 cells;

FIG. 14 is a measurement of apoptosis-inducing ability of 8. Mu.M liensinine on DLBCL cell lines Sudhl8, sudhl4, R1.1 and OCILY3 cells at 12 h.

Detailed Description

Example 1

Effect of plumula Nelumbinis neoline on cell viability of human leukemia cell line

1.1 Experimental materials

1.1.1 reagents and materials

(1) Compound (I)

Alternanthemic plumula Nelumbinis neoline (C) ₆₃ H ₆₉ N ₃ O ₁₀ Molecular weight: 1027.49 Is provided by Nanjing university of traditional Chinese medicine, medicinal fungus and traditional Chinese medicine biotechnology institute, and is light yellow powder with purity more than 90%. Before use, the compound powder is prepared into a stock solution with the concentration of 0.01M by using dimethyl sulfoxide and stored at the temperature of minus 80 ℃. When in use, the corresponding culture medium for cell culture is adopted to dilute to the required concentration.

(2) Cell lines

Human AML cell lines include THP1 and MV411 cells, human CML cell lines include K562 cells, human T cell malignancies include Jurkat, hut102 and Molt4 cells, and human DLBCL cells include Sudhl4, sudhl8, R1.1 and OCILY3 cells, all purchased from shanghai national academy of sciences cells.

(3) Cell culture reagent

RPMI-1640 medium (GIBCO, carlsbad, CA, USA): 5.2g of RPMI-1640 powder and 1.0g of NaHCO were taken ₃ Co-dissolved in 0.5L ddH ₂ And (4) in O. Filtering with sterile cylindrical filter, sterilizing, packaging, and storing at 4 deg.C. Before use, 100U/mL penicillin (Chenxin pharmaceutical Co., ltd., jining, china) and 100mg/L streptomycin (Chenxin pharmaceutical Co., ltd.) were added.

Fetal bovine serum (GIBCO) was stored at-20 deg.C, inactivated in 56 deg.C water bath for 35min before use, and then stored at 4 deg.C for a week. When in use, the fetal calf serum and the culture medium are mixed according to the ratio of 1: 10.

(4) Relevant index detection kit

MTT solution: the procedure was performed in the dark, and 0.05g of MTT (Hippocampus Biotech, shanghai, china) powder was dissolved in 10mL of PBS buffer, and the solution was prepared to a concentration of 5mg/mL by sonication for 20 min. Filtering with 0.22 μm sterile filter head, packaging, and storing at-80 deg.C. It is dissolved at room temperature in advance.

1.1.2 Experimental instruments

Model YJ-875 medical decontamination bench (decontamination plant, suzhou, china); 3111 type water jacket CO ₂ Incubators (ThermoFisher Scientific, waltham, MA, USA); 702 type ultra-low temperature refrigerator (ThermoFisher)Scientific); electronic balances (sidoris instruments systems limited, beijing, china); QIUJING blood cell counting plate (seminal biochemical instruments ltd., shanghai, china); LD4-2 common centrifuges (medical centrifuge factory, shanghai, china); model 5417R desk refrigerated high speed centrifuge (Eppendorf AG, hamburg, germany); a Research type single channel adjustable pipette (Eppendorf AG); THZ-312 type desk type constant temperature oscillator (fine macro test equipment limited, shanghai, china); varioskan full-wavelength microplate reader (ThermoFisher Scientific).

1.2 Experimental methods

(1) Cell viability assay

The principle is as follows: MTT can be reduced by intracellular mitochondrial dehydrogenase to generate blue-purple crystalline nail tympany (Formazan), and the Optical Density (OD) values are detected at the wavelength of 570nm and compared, so that the relative level of cell viability can be reflected. The operation process comprises the following steps: 100 μ L of cells (5000 cells/well) were uniformly cultured in a 96-well plate, 100 μ L of neoliensinine was added at a certain concentration, and the cell content was 5% CO at 37 ℃% ₂ Culturing in an incubator; after the drug effect was completed, 15. Mu.L of MTT solution was added to each well. After incubation in the incubator for 3h, the OD at 570nM was determined using a microplate reader. Inhibition% = (1-mean absorbance of drug-administered cells/mean absorbance of control cells) × 100%; graphpad Prism 8.0 software half maximal inhibitory concentration (50% inhibition concentration, IC) ₅₀ )。

1.3, results of the experiment

The MTT method is used for detecting the activity inhibition effect of natural compound neoliensinine extracted from the traditional Chinese medicine lotus plumule on different leukemia cells under different concentrations. The result shows that the 0-32 mu M of the neoliensinine has obvious activity inhibition effect on the cells after acting on the leukemia cells for 6 hours or 12 hours, and the time and the concentration of the neoliensine are dependent. IC of liensinine on THP1 in 6h and 12h ₅₀ 18.15 μ M and 4.90 μ M, respectively (FIG. 1); IC of liensinine to MV411 at 6h and 12h ₅₀ 3.00. Mu.M and 1.99. Mu.M, respectively (FIG. 2); IC of liensinine on Jurkat at 6h and 12h ₅₀ 3.32. Mu.M and 2.17. Mu.M, respectively (FIG. 3); IC of liensinine to Molt4 at 6h and 12h ₅₀ Respectively 18.15. Mu.M and 4.89. Mu.M (FIG. 1)4) (ii) a IC of Alternanthensine on Hut102 at 6h and 12h ₅₀ 8.19. Mu.M and 2.91. Mu.M, respectively (FIG. 5); IC of liensinine to K562 in 6h ₅₀ 5.49 μ M (FIG. 6); IC of liensinine on OCILY3 at 6h and 12h ₅₀ 6.88. Mu.M and 1.98. Mu.M, respectively (FIG. 7); IC of liensinine to R1.1 in 6h and 12h ₅₀ 4.48 μ M and 2.98 μ M, respectively (FIG. 8); IC of liensinine on Sudhl4 at 6h and 12h ₅₀ 9.42 μ M and 4.98 μ M, respectively (FIG. 9); IC of liensinine on Sudhl8 at 6h and 12h ₅₀ 4.00. Mu.M and 1.23. Mu.M, respectively (FIG. 10). The results show that the neoliensinine has obvious activity inhibition effect on human AML cells, human CML cells, human T cell malignant tumor cells and human DLBCL cells. μ M means μmol/L.

Example 2

Effect of plumula Nelumbinis neoline on apoptosis induction of human leukemia cell line

2.1 Experimental materials

2.1.1 reagents and materials

(1) Compound (I)

Same as 1.1.1.

(2) Cell lines

Human AML cell lines include THP1, MV411, U937 and ME1 cells, human CML cell lines include K562 cells, human T cell malignancies include Jurkat, hut102 and Molt4 cells, and human DLBCL cells include Sudhl4, sudhl8, R1.1 and OCILY3 cells, all purchased from shanghai national academy of sciences.

(3) Cell culture reagent

The same as 1.1.1.

(4) Relevant index detection kit

The AnnexinV/PI apoptosis detection kit (Novozan Biotechnology, nanjing, china) is used for detecting apoptosis.

2.1.2 Experimental instruments

Model YJ-875 medical decontamination workstation (decontamination plant, suzhou, china); 3111 type water jacket CO ₂ Incubators (ThermoFisher Scientific, waltham, MA, USA); model 702 ultra-low temperature refrigerator (ThermoFisher Scientific); electronic balances (sidoris instruments systems limited, beijing, china); QIUJING bloodCell counting plates (refinement biochemical instruments ltd., shanghai, china); LD4-2 common centrifuges (medical centrifuge factory, shanghai, china); model 5417R desk refrigerated high speed centrifuge (Eppendorf AG, hamburg, germany); research type single channel adjustable pipettors (Eppendorf AG); flow cytometry (Becton-Dickinson and Company, NJ, USA).

2.2 Experimental methods

(1) Apoptosis detection

The principle is as follows: the binding of Annexin V to cellular Phosphatidylserine (PS) was used as a standard for apoptosis testing. Apoptosis is usually accompanied by the destruction of the cell membrane. When early apoptosis (early apoptosis) occurs, PS turns outwards from the inner side of a lipid bilayer of a cell membrane so as to be combined with Annexin V-FITC dye, and FL-1 enhanced signals can be detected by a flow cytometer; after entering late apoptosis (late apoptosis), due to increased cell membrane permeability, propidium Iodide (PI) -PerCP dye binds to cellular nucleic acids, and thus FL-3 enhancement signal can be detected. Therefore, by utilizing Annexin V/PI double-staining experiments, the proportion of apoptotic cells is counted, and early-withering/late-withering cells can be distinguished. The operation process comprises the following steps: collecting and centrifuging the cells treated by the neonicotinoid, washing the cells for 2 times by phosphate-buffered saline (PBS), and then resuspending the cells by using 40 mu L binding Buffer; add 1.7 u L Annexin V and 1.7 u L PI dye room temperature shading incubation for 10 minutes (minutes, min), supplement 250 u L binding Buffer heavy suspension, can use the flow cytometer to detect. The apoptotic cells were Annexin V-FITC positive cells.

2.3, results of the experiment

The apoptosis induction effect of the neoliensinine on human leukemia cells is detected by an Annexin V/PI apoptosis detection method. The results show that the apoptosis rates of 4 cells of human AML cell lines U937, THP1, MV411 and ME1 after 8 μ M of neonicotinoid is acted for 12h are respectively 87.7%,71.2%,97.1% and 70.2% (FIG. 11); after the human CML cell line K562 cells act for 4 hours at 8 mu M of neoliensinine, the apoptosis rate is 55.5 percent (figure 12); after the human T cell malignant tumor cell lines Jurkat, hut102 and Molt4 cells act for 12 hours at 8 mu M of neonicotinoid of lotus plumule, the apoptosis rates of the 3 cells are 83.5 percent, 43.2 percent and 86.6 percent respectively (figure 13); after the human DLBCL cell lines Sudhl8, sudhl4, R1.1 and OCILY3 cells are acted on 8 mu M neoliensine for 12 hours, the apoptosis rates of the 4 cells are respectively 88.2%,70.8%,65.4% and 93.9% (FIG. 14). The result shows that the neoliensinine has the apoptosis induction effect on human AML cells, human CML cells, human T cell malignant tumor cells and human DLBCL cells, and the effect of the neoliensinine on leukemia resistance is verified.

Claims (5)

1. The application of the neoliensinine and the pharmaceutically acceptable salt thereof in preparing the medicine for preventing and/or treating leukemia is characterized in that the leukemia is acute myelogenous leukemia and chronic granulocytic leukemia.

2. The use of claim 1, wherein the neoliensinine has the structure of formula (1):

formula (1).

3. Use according to claim 1, characterized in that leukemia is prevented and/or treated by inhibition of viability and induction of apoptosis of human AML cells, human CML cells.

4. The application of the neoliensinine and the pharmaceutically acceptable salts thereof in preparing the medicines for preventing and/or treating the T cell malignant tumor.

5. The application of the liensinine and the pharmaceutically acceptable salt thereof in preparing the medicine for preventing and/or treating diffuse large B cell lymphoma.

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