A curcumin compound preparation and its application in treating MLL-r leukemia

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a curcumin compound preparation and its application in the treatment of MLL-r leukemia. The curcumin compound preparation includes the following raw materials: curcumin, fatty acid-aniline-hexoside compound, stabilizing promoter, and mixed solvent. Fatty acid-aniline-hexoside compound can load curcumin, effectively improve the solubility of curcumin, and improve bioavailability. The stabilizing promoter can effectively improve the stability of curcumin and improve drug efficacy. The mixed solvent used in the present invention is a mixture of multiple solvents in a reasonable ratio, which can effectively promote the compounding of fatty acid-aniline-hexoside compound and curcumin and improve bioavailability. The curcumin prepared by the present invention has high solubility, high bioavailability, strong stability, high safety, and synergistic synergy of each component, which can effectively inhibit MV-4-11 cell proliferation and can be used to prepare drugs for the treatment of MLL-r leukemia.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a curcumin compound preparation and application thereof in treating MLL-r leukemia. Background Art

[0002] Acute leukemia is the most common malignant tumor in children. In recent years, with the development of allogeneic bone marrow cell transplantation (allo-HSCT), the prognosis of childhood leukemia has been greatly improved. The 5-year disease-free survival rate (DFS) of childhood acute lymphoblastic leukemia (ALL) reaches 80-90%, and that of childhood acute myeloid leukemia (AML) reaches 60-70%. However, the 5-year survival rate of MLL rearrangement (MLL-r) leukemia is only 30-40%. Childhood MLL-r leukemia accounts for about 75% of infantile acute leukemia, and has the characteristics of young onset age, high malignancy, and poor chemotherapy effect. Therefore, it is of great significance to explore the biological mechanism of MLL-r leukemia and find new treatment methods. Current studies have shown that small molecule metabolites may be important regulatory molecules for the malignant transformation of normal cells and tumorigenesis. The applicant's previous study found that arachidonic acid (AA) transported by exosomes is a key factor in carcinogenesis. The targeted metabolome mass spectrometry test of serum of children with MLL-r was conducted, and it was found that the level of arachidonic acid was significantly higher than that of ordinary children, indicating that arachidonic acid metabolism is activated in the occurrence and development of MLL-r leukemia. The use of ASB14780 (cPLA2 inhibitor, a key rate-limiting enzyme that catalyzes the production of AA from cell membrane phospholipids) can effectively inhibit the proliferation of MV-4-11 (human myelomonocytic leukemia cells); exogenous AA supplementation can promote the malignant proliferation of MV-4-11 cells, indicating that arachidonic acid can induce the malignant proliferation of MLL-r leukemia cells. Many studies have shown that curcumin (cur) can inhibit the AA-mediated cPLA2, COX-2 and 5-LOX pathways. Curcumin may intervene in the occurrence and development of MLL-r leukemia by targeting and inhibiting arachidonic acid, and can be used in the treatment of MLL-r leukemia. However, curcumin has disadvantages such as insolubility in water, poor photostability and low bioavailability, which limit its use. Therefore, how to develop a curcumin compound preparation with high solubility, strong stability and high bioavailability for the treatment of MLL-r leukemia is a technical problem that needs to be solved urgently. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention proposes a curcumin compound preparation and its application in the treatment of MLL-r leukemia. The present invention prepares a fatty acid-aniline-hexoside compound, which can load curcumin, effectively improve the solubility of curcumin, improve bioavailability, prolong the drug action time, and improve drug efficacy. The present invention prepares a stabilizing promoter, which can effectively improve the stability of curcumin, and the combined use with curcumin can play a good synergistic effect, improve drug efficacy, and enhance high efficiency and low toxicity. The mixed solvent used in the present invention can effectively promote the compounding of fatty acid-aniline-hexoside compounds and curcumin, and improve bioavailability. The curcumin compound preparation prepared by the present invention has high solubility, high bioavailability, strong stability, high biosafety and low toxicity, and the components synergize to effectively inhibit the proliferation of MV-4-11 cells induced by arachidonic acid, and the tumor inhibition rate is improved.

[0004] The present invention is achieved through the following technical solutions:

[0005] A curcumin compound preparation comprises the following raw materials: curcumin, a fatty acid-aniline-hexoside compound, a stabilizing promoter and a mixed solvent.

[0006] Furthermore, the preparation method of the fatty acid-aniline-hexoside compound comprises the following steps:

[0007] (1) 2-Hydroxy-4-trifluoromethylaniline and fatty acid were mixed in a molar ratio of 1:1 and added to a reaction kettle. Deionized water was added and the pH was adjusted to 7.4 with sodium bicarbonate. The temperature was maintained at 50-60° C. and stirred at 180 rpm for 20-30 min to obtain an intermediate product A.

[0008] (2) Add glucuronolactone to the intermediate product A obtained in step (1), place in an ice-water bath at 0°C, add NaOH, stir at 200 rpm for 2 h, and heat at 50°C, 10 -3 MPa was concentrated to dryness to obtain intermediate product B;

[0009] (3) The intermediate product B obtained in step (2) was mixed with ethyl acetate, stirred at 200 rpm for 1 h, and then filtered. The filtrate was heated at 50°C and 10 -3 MPa and concentrated to 20% of the original volume, and spray-dried to obtain a fatty acid-aniline-hexoside compound.

[0010] Furthermore, the mass ratio of the 2-hydroxy-4-trifluoromethylaniline to deionized water in step (1) is 1:20.

[0011] Furthermore, the fatty acid in step (1) is at least one of linoleic acid and α-linolenic acid.

[0012] Furthermore, in step (2), the ratio of the amount of glucuronide to the intermediate product A is 1 g:40 mL.

[0013] Furthermore, in step (2), the mass ratio of NaOH to glucuron-lactone is 1:150.

[0014] Furthermore, in step (3), the mass ratio of the intermediate product B to ethyl acetate is 1:3.

[0015] Furthermore, the preparation method of the stabilizing promoter comprises the following steps:

[0016] (a) Inulin was added to deionized water, heated to 95°C, stirred at 200 rpm for 30-40 min, succinic acid was added, triethylamine was added dropwise, and stirring was continued for 1-2 h to obtain reaction mixture A;

[0017] (b) Add ellagic acid to the reaction mixture A of step (a), adjust the pH to 3.2-3.6 with 1 mol / L hydrochloric acid solution, heat at 100° C. for 2-3 h, freeze-dry, wash with deionized water, and dry at 80° C. to obtain a stabilizing promoter.

[0018] Furthermore, in step (a), the dosage ratio of inulin, deionized water, succinamic acid and triethylamine is 1 g:50 mL:0.5 g:50 mg.

[0019] Furthermore, in step (b), the ratio of the reaction mixture A to ellagic acid is 50 mL:0.8 g.

[0020] Furthermore, the mixed solvent is toluene, carbon tetrachloride and ethyl acetate mixed in a volume ratio of 1:1:0.8.

[0021] The present invention also provides a method for preparing the curcumin composite preparation, comprising the following steps:

[0022] S1: Add fatty acid-aniline-hexoside compound and curcumin into a mixed solvent, stir at 40°C and 200 rpm for 1 hour to obtain a mixed solution A; mix the stabilizing agent and anhydrous ethanol evenly to obtain a mixed solution B;

[0023] S2: Mix the mixed solution A obtained in step S1 with the mixed solution B, heat at 55° C. for 1 h, and vacuum dry at 50° C. and -0.09 MPa to obtain a curcumin composite preparation.

[0024] Furthermore, in step S1, the usage ratio of the fatty acid-aniline-hexoside compound, curcumin and the mixed solvent is 5 g:2 g:100 mL.

[0025] Furthermore, in step S1, the ratio of the stabilizing agent to anhydrous ethanol is 1 g:15 mL.

[0026] Furthermore, in step S2, the volume ratio of the mixed solution A to the mixed solution B is 10:1.

[0027] The present invention also provides application of the curcumin compound preparation in preparing a medicine for treating MLL-r leukemia.

[0028] Furthermore, the dosage form of the drug includes capsules, tablets, pills, injections and granules; the drug may also include pharmaceutically acceptable excipients.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention prepares a fatty acid-aniline-hexoside compound, which can load curcumin, effectively improve the solubility of curcumin, has good dispersibility, is more easily absorbed and utilized, improves bioavailability, prolongs drug action time, increases pharmacological action and efficacy, reduces adverse drug reactions, and helps to enhance the stability of curcumin, has a synergistic effect, and improves drug efficacy. The present invention prepares a stabilizing promoter, which can effectively improve the stability of curcumin, improve the photostability of curcumin, and can play a good synergistic effect with the combined use of curcumin, improve drug efficacy, and enhance high efficiency and low toxicity. The mixed solvent used in the present invention is a reasonable ratio of toluene, carbon tetrachloride and ethyl acetate, which can effectively promote the compounding of fatty acid-aniline-hexoside compounds and curcumin to improve bioavailability. The curcumin compound preparation prepared by the present invention has high solubility, high bioavailability, strong stability, high biosafety and low toxicity. The components synergize to enhance the efficacy, can effectively inhibit arachidonic acid-induced MV-4-11 cell proliferation, improve the tumor inhibition rate, can be used to prepare drugs for treating MLL-r leukemia, and has clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 is the nuclear magnetic resonance spectrum of the fatty acid-aniline-hexoside compound described in Example 1 of the present invention, wherein A is 1 H figure, B is 13 Figure C;

[0033] Figure 2This is a schematic diagram of the chemical structure of the fatty acid-aniline-hexoside compound described in Example 1 of the present invention;

[0034] Figure 3 The figure is a detection diagram of serum arachidonic acid level in children with MLL-r leukemia by targeted mass spectrometry, where A is a hot spot map, the redder the color, the higher the expression of the metabolite, and the bluer the color, the lower the expression of the metabolite. B is the expression of arachidonic acid in the healthy control group and children with MLL-r leukemia;

[0035] Figure 4 This is the effect diagram of arachidonic acid promoting the proliferation of MV-4-11 cells;

[0036] Figure 5 This is the effect diagram of curcumin inhibiting arachidonic acid-induced MV-4-11 cell proliferation at 0 d;

[0037] Figure 6 This is the effect diagram of curcumin inhibiting arachidonic acid-induced MV-4-11 cell proliferation for 1 day;

[0038] Figure 7 This is a 3-day effect diagram of curcumin inhibiting arachidonic acid-induced MV-4-11 cell proliferation;

[0039] Figure 8 This is the effect diagram of curcumin inhibiting arachidonic acid-induced MV-4-11 cell proliferation for 5 days;

[0040] Fig. 9 This is a diagram showing the effect of curcumin in inhibiting the expression of arachidonic acid;

[0041] Fig.10 This is a test of the tumor inhibition rate of the curcumin compound preparations described in Example 1 and Comparative Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0043] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0044] Example 1: This example provides a curcumin compound preparation, comprising the following raw materials: curcumin, fatty acid-aniline-hexoside compound, a stabilizing agent, and a mixed solvent.

[0045] The preparation method of the fatty acid-aniline-hexoside compound comprises the following steps:

[0046] (1) 2-hydroxy-4-trifluoromethylaniline and linoleic acid were mixed in a molar ratio of 1:1 and added to a reaction kettle, deionized water was added, and the mass ratio of 2-hydroxy-4-trifluoromethylaniline to deionized water was 1:20. The pH was adjusted to 7.4 with sodium bicarbonate, the temperature was maintained at 60° C., and the stirring was continued at 180 rpm for 30 min to obtain an intermediate product A;

[0047] (2) Add glucuron lactone to the intermediate product A obtained in step (1), the ratio of glucuron lactone to intermediate product A is 1 g:40 mL, add NaOH in an ice-water bath at 0°C, the mass ratio of NaOH to glucuron lactone is 1:150, stir at 200 rpm for 2 h, and heat at 50°C, 10 -3 MPa was concentrated to dryness to obtain intermediate product B;

[0048] (3) The intermediate product B obtained in step (2) was mixed with ethyl acetate in a mass ratio of 1:3, stirred at 200 rpm for 1 h, and then filtered. The filtrate was heated at 50°C, 10 -3 MPa was concentrated to 20% of the original volume and spray dried to obtain a fatty acid-aniline-hexoside compound, the NMR spectrum of which is shown in Figure 1 The chemical structure diagram is shown in Figure 2 shown.

[0049] The preparation method of the stabilizing accelerator comprises the following steps:

[0050] (a) Inulin was added to deionized water, heated to 95°C, stirred at 200 rpm for 40 min, succinic acid was added, and triethylamine was added dropwise. The amount ratio of inulin, deionized water, succinic acid and triethylamine was 1 g:50 mL:0.5 g:50 mg. Stirring was continued for 2 h to obtain reaction mixture A.

[0051] (b) Adding ellagic acid to the reaction mixture A of step (a), the ratio of reaction mixture A to ellagic acid is 50 mL:0.8 g, adjusting the pH to 3.6 with 1 mol / L hydrochloric acid solution, heating at 100° C. for 3 h, freeze-drying, washing with deionized water, and drying at 80° C. to obtain a stabilizing promoter.

[0052] The mixed solvent is toluene, carbon tetrachloride and ethyl acetate mixed in a volume ratio of 1:1:0.8.

[0053] This embodiment also provides a method for preparing the curcumin composite preparation, comprising the following steps:

[0054] S1: Add fatty acid-aniline-hexoside compound and curcumin to a mixed solvent, the amount ratio of fatty acid-aniline-hexoside compound, curcumin and mixed solvent is 5 g:2 g:100 mL, stir at 40°C and 200 rpm for 1 h to obtain a mixed solution A; mix the stabilizing promoter and anhydrous ethanol evenly, the amount ratio of the stabilizing promoter to anhydrous ethanol is 1 g:15 mL, to obtain a mixed solution B;

[0055] S2: Mix the mixed solution A obtained in step S1 with the mixed solution B, wherein the volume ratio of the mixed solution A to the mixed solution B is 10:1, heat at 55° C. for 1 h, and vacuum dry at 50° C. and vacuum at -0.09 MPa to obtain a curcumin composite preparation.

[0056] Example 2: This example provides a curcumin compound preparation, comprising the following raw materials: curcumin, a fatty acid-aniline-hexoside compound, a stabilizing agent, and a mixed solvent.

[0057] The preparation method of the fatty acid-aniline-hexoside compound comprises the following steps:

[0058] (1) 2-hydroxy-4-trifluoromethylaniline and α-linolenic acid were mixed in a molar ratio of 1:1 and added to a reaction kettle, and deionized water was added, the mass ratio of 2-hydroxy-4-trifluoromethylaniline to deionized water was 1:20, the pH was adjusted to 7.4 with sodium bicarbonate, the temperature was maintained at 50° C., and the stirring was continued at 180 rpm for 20 min to obtain an intermediate product A;

[0059] (2) Add glucuron lactone to the intermediate product A obtained in step (1), the ratio of glucuron lactone to intermediate product A is 1 g:40 mL, add NaOH in an ice-water bath at 0°C, the mass ratio of NaOH to glucuron lactone is 1:150, stir at 200 rpm for 2 h, and heat at 50°C, 10 -3 MPa was concentrated to dryness to obtain intermediate product B;

[0060] (3) The intermediate product B obtained in step (2) was mixed with ethyl acetate in a mass ratio of 1:3, stirred at 200 rpm for 1 h, and then filtered. The filtrate was heated at 50°C, 10 -3 MPa and concentrated to 20% of the original volume, and spray-dried to obtain a fatty acid-aniline-hexoside compound.

[0061] The preparation method of the stabilizing accelerator comprises the following steps:

[0062] (a) Inulin was added to deionized water, heated to 95°C, stirred at 200 rpm for 30 min, succinic acid was added, and triethylamine was added dropwise. The amount ratio of inulin, deionized water, succinic acid and triethylamine was 1 g:50 mL:0.5 g:50 mg. Stirring was continued for 1 h to obtain reaction mixture A.

[0063] (b) Adding ellagic acid to the reaction mixture A of step (a), the ratio of reaction mixture A to ellagic acid is 50 mL:0.8 g, adjusting the pH to 3.2 with 1 mol / L hydrochloric acid solution, heating at 100° C. for 2 h, freeze-drying, washing with deionized water, and drying at 80° C. to obtain a stabilizing promoter.

[0064] The mixed solvent is toluene, carbon tetrachloride and ethyl acetate mixed in a volume ratio of 1:1:0.8.

[0065] This embodiment also provides a method for preparing the curcumin composite preparation, comprising the following steps:

[0066] S1: Add fatty acid-aniline-hexoside compound and curcumin to a mixed solvent, the amount ratio of fatty acid-aniline-hexoside compound, curcumin and mixed solvent is 5 g:2 g:100 mL, stir at 40°C and 200 rpm for 1 h to obtain a mixed solution A; mix the stabilizing promoter and anhydrous ethanol evenly, the amount ratio of the stabilizing promoter to anhydrous ethanol is 1 g:15 mL, to obtain a mixed solution B;

[0067] S2: Mix the mixed solution A obtained in step S1 with the mixed solution B, wherein the volume ratio of the mixed solution A to the mixed solution B is 10:1, heat at 55° C. for 1 h, and vacuum dry at 50° C. and vacuum at -0.09 MPa to obtain a curcumin composite preparation.

[0068] Example 3: This example provides a curcumin compound preparation, comprising the following raw materials: curcumin, fatty acid-aniline-hexoside compound, a stabilizing agent, and a mixed solvent.

[0069] The preparation method of the fatty acid-aniline-hexoside compound comprises the following steps:

[0070] (1) 2-hydroxy-4-trifluoromethylaniline and linoleic acid were mixed in a molar ratio of 1:1 and added to a reaction kettle, deionized water was added, and the mass ratio of 2-hydroxy-4-trifluoromethylaniline to deionized water was 1:20. The pH was adjusted to 7.4 with sodium bicarbonate, the temperature was maintained at 55° C., and the stirring was continued at 180 rpm for 25 min to obtain an intermediate product A;

[0071] (2) Add glucuron lactone to the intermediate product A obtained in step (1), the ratio of glucuron lactone to intermediate product A is 1 g:40 mL, add NaOH in an ice-water bath at 0°C, the mass ratio of NaOH to glucuron lactone is 1:150, stir at 200 rpm for 2 h, and heat at 50°C, 10 -3 MPa was concentrated to dryness to obtain intermediate product B;

[0072] (3) The intermediate product B obtained in step (2) was mixed with ethyl acetate in a mass ratio of 1:3, stirred at 200 rpm for 1 h, and then filtered. The filtrate was heated at 50°C, 10 -3 MPa and concentrated to 20% of the original volume, and spray-dried to obtain a fatty acid-aniline-hexoside compound.

[0073] The preparation method of the stabilizing accelerator comprises the following steps:

[0074] (a) Inulin was added to deionized water, heated to 95°C, stirred at 200 rpm for 35 min, succinic acid was added, and triethylamine was added dropwise. The amount ratio of inulin, deionized water, succinic acid and triethylamine was 1 g:50 mL:0.5 g:50 mg. Stirring was continued for 1.5 h to obtain reaction mixture A.

[0075] (b) Adding ellagic acid to the reaction mixture A of step (a), the ratio of reaction mixture A to ellagic acid is 50 mL:0.8 g, adjusting the pH to 3.4 with 1 mol / L hydrochloric acid solution, heating at 100° C. for 2.5 h, freeze-drying, washing with deionized water, and drying at 80° C. to obtain a stabilizing promoter.

[0076] The mixed solvent is toluene, carbon tetrachloride and ethyl acetate mixed in a volume ratio of 1:1:0.8.

[0077] This embodiment also provides a method for preparing the curcumin composite preparation, comprising the following steps:

[0078] S1: Add fatty acid-aniline-hexoside compound and curcumin to a mixed solvent, the amount ratio of fatty acid-aniline-hexoside compound, curcumin and mixed solvent is 5 g:2 g:100 mL, stir at 40°C and 200 rpm for 1 h to obtain a mixed solution A; mix the stabilizing promoter and anhydrous ethanol evenly, the amount ratio of the stabilizing promoter to anhydrous ethanol is 1 g:15 mL, to obtain a mixed solution B;

[0079] S2: Mix the mixed solution A obtained in step S1 with the mixed solution B, wherein the volume ratio of the mixed solution A to the mixed solution B is 10:1, heat at 55° C. for 1 h, and vacuum dry at 50° C. and vacuum at -0.09 MPa to obtain a curcumin composite preparation.

[0080] The difference between Comparative Example 1 and Example 1 is that phospholipid is used instead of fatty acid-aniline-hexoside compound.

[0081] The only difference between Comparative Example 2 and Example 1 is that inulin is used instead of the stabilization promoter.

[0082] The only difference between Comparative Example 3 and Example 1 is that ellagic acid is used instead of the stability accelerator.

[0083] The difference between Comparative Example 4 and Example 1 is that ethyl acetate is used instead of the mixed solvent.

[0084] Experimental Example 1: Referring to the method of national standard GB1886.76-2015, a standard curve was drawn, 20 mg of the curcumin compound preparation prepared in Example 1 and Comparative Examples 1-4 were weighed in a 5 mL EP tube, 3 mL of ultrapure water was added, and the mixture was shaken in a constant temperature water bath oscillator at 100 rpm at 25°C for 24 h, 1 mL of sample was taken, and then centrifuged at 6000 rpm for 10 min. 100 μL of the supernatant was drawn and diluted to 10 mL with anhydrous ethanol, and curcumin was used to replace the curcumin compound preparation as a blank group. The amount of curcumin was the same as that of curcumin in the curcumin compound preparation used, and the absorbance value was measured at 425 nm. The curcumin solubility was calculated according to the standard curve, and the results are shown in Table 1.

[0085]

[0086] The results in Table 1 show that the solubility of Example 1 is significantly higher than that of Comparative Examples 1-4, and the solubility of Example 1 and Comparative Examples 1-4 is significantly higher than that of the blank group, indicating that the curcumin composite formulation of the present invention can significantly improve the solubility of curcumin, thereby improving the bioavailability and efficacy.

[0087] Experimental Example 2: The curcumin composite preparations of Example 2 and Comparative Examples 1-4 were added to 80 vol% ethanol solution to make the initial concentration of curcumin 100 μg / mL, placed in a colorless transparent container, and placed in an artificial climate box with a temperature of 37°C, a light of 20000 Lux, and a humidity of 75%. The curcumin concentration was detected at 5 h, 24 h, and 7 d, and the retention rate was calculated. The retention rate (%) = (curcumin detection concentration / curcumin initial concentration) × 100%. The results are shown in Table 2.

[0088]

[0089] The results in Table 2 show that the retention rate of Example 2 is significantly higher than that of Comparative Examples 1-4, indicating that the curcumin composite preparation of the present invention has strong stability, good light stability, strong light resistance, and is easy to store.

[0090] Experimental Example 3: Male SD rats (230-250 g) were randomly divided into 6 groups (5 rats in each group) and fasted for 12 h, but not water. Oral gavage of curcumin was used as the control group, and the dosage was 50 mg / kg. 0.3 mL of blood was collected from the orbital vein of the rats 1 h after administration and placed in a heparinized EP tube. After the blood sample was centrifuged at 6000 r / min for 10 min, the supernatant was taken and stored at -80°C for testing. Other groups were gavaged with the curcumin compound preparations of Example 3 and Comparative Examples 1-4, and the dosage was equivalent to 50 mg / kg of curcumin. 0.3 mL of blood was collected from the orbital vein of the rats 1 h after administration and placed in a heparinized EP tube. After the blood sample was centrifuged at 6000 r / min for 10 min, the supernatant was taken and stored at -80°C for testing. Take 100 μL of the above plasma, add 30 μL of 100 μg / mL NT, add 500 μL of methanol, vortex for 3 minutes, centrifuge at 12000 r / min for 10 min, blow dry with a nitrogen blow dryer, and finally re-dissolve with 100 μL of methanol, take 20 μL for injection, and use high performance liquid chromatography (HPLC) to determine the concentration of curcumin in plasma. The same method was used to determine the concentration of curcumin in the plasma of other groups of curcumin preparations, and the plasma curcumin concentration of each group was tested again after 60 hours. The results are shown in Table 3.

[0091]

[0092] The results in Table 3 show that the curcumin concentration of Example 2 is significantly higher than that of Comparative Examples 1-4, and that of Example 2 and Comparative Examples 1-4 are significantly higher than that of the control group, with prolonged action time and reduced clearance rate, indicating that the curcumin composite preparation of the present invention can significantly improve the bioavailability of curcumin, thereby improving the efficacy.

[0093] Experimental Example 4: Detection of serum arachidonic acid levels in children with MLL-r leukemia by targeted mass spectrometry. Figure 3 . MLL-r leukemia cell line (MV-4-11 cells) was cultured with 5% fatty acid-free fetal bovine serum. Phospholipase A2 (cPLA2) is the key rate-limiting enzyme that catalyzes the production of arachidonic acid. The cPLA2 inhibitor (ASB) was used for co-stimulation to eliminate endogenous arachidonic acid metabolism. Exogenous arachidonic acid (25 μM) was further given, and the proliferation level of MV-4-11 cells was observed by CSFE method after 5 days. DMSO was used as the control group. The results are shown in Figure 4 As shown,

[0094] Figure 3 The results showed that the serum arachidonic acid level in children with MLL-r leukemia was significantly increased. Figure 4 The results showed that the fluorescence intensity of the ASB+AA group was lower than that of the ASB group, indicating that arachidonic acid can significantly promote the proliferation of MV-4-11 cells.

[0095] Experimental Example 5: 5% fatty acid-free fetal bovine serum was used to culture the MLL-r leukemia cell line (MV-4-11 cells), and DMSO group, ASB group, ASB+25 μM arachidonic acid group, and ASB+25 μM arachidonic acid+20 μM curcumin group were set up. The proliferation level of MV-4-11 cells in each group on days 0, 1, 3, and 5 was detected by CSFE method. The results are shown in the figure. Figure 5-Figure 8 shown. Figure 5-Figure 8 The results showed that curcumin could effectively inhibit arachidonic acid-induced MV-4-11 cell proliferation.

[0096] Experimental Example 6: DMSO group, ASB group, ASB + 25 μM arachidonic acid group and 25 μM arachidonic acid + 20 μM curcumin group were set up to detect the arachidonic acid expression level of MV-4-11 cells in each group. The results are as follows: Fig. 9 shown.

[0097] Fig. 9 The results showed that compared with the DMSO group, the expression of arachidonic acid in the ASB group decreased, and the expression of arachidonic acid in the ASB+arachidonic acid group increased after arachidonic acid supplementation. After continued administration of curcumin, the expression of arachidonic acid decreased, indicating that curcumin can inhibit the expression of arachidonic acid, thereby inhibiting the malignant transformation of arachidonic acid-induced MLL-r leukemia.

[0098] Experimental Example 7: MV-4-11 cell line was inoculated subcutaneously in the right axilla of mice, with a cell inoculation volume of 1×10 7 Once the transplanted tumor is formed, the experiment can begin. Use a vernier caliper to measure the diameter of the transplanted tumor. When the tumor grows to 100-200 mm 3 (initial tumor volume), the mice were randomly divided into 6 groups, each group had 10 mice, and were given the curcumin compound preparations of Example 1 and Comparative Examples 1-4 (the dosage was equivalent to 30 mg / kg of curcumin) and curcumin (30 mg / kg) every day for 21 days. The tumor volume was measured regularly during the period, and the tumor inhibition rate was calculated. Tumor inhibition rate (%) = [(initial tumor volume - measured tumor volume) / initial tumor volume] × 100%. The results are shown in Fig.10 shown.

[0099] Fig.10 The results showed that the tumor inhibition rate of Example 1 was significantly higher than that of Comparative Examples 1-4, and the tumor inhibition rates of Example 1 and Comparative Examples 1-4 were significantly higher than those of the curcumin group, indicating that the curcumin compound preparation of the present invention has a significant inhibitory effect on tumor growth of MLL-r leukemia MV-4-11, and can be used in the preparation of drugs for treating MLL-r leukemia. In addition, no mouse death or other side effects were observed during the experiment, indicating that the curcumin compound preparation of the present invention is highly safe.

[0100] Experimental Example 8: 50 male Wistar rats, 7 weeks old, were randomly divided into 5 groups, 10 rats in each group, and each group was given the curcumin compound preparations of Example 2 and Comparative Examples 1-4, respectively, by oral gavage, the dosage was equivalent to 30 mg / kg of curcumin, and the administration was given every day for 90 consecutive days. At the same time, the control group was orally given an equal amount of normal saline during the administration process, and the state of the animals was observed after the administration every day. The samples were collected on the 90th day of administration. The animals were fasted for 16 hours before being killed. The animals were anesthetized, and the main organs of the animals were removed to calculate the organ index: organ index (mg / kg) = (organ weight / rat weight after fasting) × 100%. The results are shown in Table 4.

[0101]

[0102] The results in Table 4 show that compared with the control group, the organ indexes of Example 2 and Comparative Examples 1-4 did not change significantly, indicating that the curcumin composite preparation of the present invention has high biological safety, high efficiency and low toxicity, and can be used to prepare drugs for treating MLL-r leukemia.

[0103] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0104] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A curcumin compound preparation, characterized in that, The invention comprises the following raw materials: curcumin, fatty acid-aniline-hexoside compound, stabilizing promoter and mixed solvent; The preparation method of the fatty acid-aniline-hexoside compound comprises the following steps: (1) 2-hydroxy-4-trifluoromethylaniline and a fatty acid, wherein the fatty acid is at least one of linoleic acid and α-linolenic acid, are mixed in a molar ratio of 1:1 and added to a reaction kettle, deionized water is added, the pH is adjusted with sodium bicarbonate, the temperature is maintained at 50-60° C., and the mixture is stirred to obtain an intermediate product A; (2) adding glucuronolactone to the intermediate product A obtained in step (1), placing in an ice-water bath at 0° C., adding NaOH, stirring, and concentrating to dryness to obtain an intermediate product B; (3) mixing the intermediate product B obtained in step (2) with ethyl acetate, stirring and filtering, concentrating the filtrate to 20% of the original volume, and spray drying to obtain a fatty acid-aniline-hexoside compound; The preparation method of the stabilizing accelerator comprises the following steps: (a) Inulin was added to deionized water, heated to 95°C, stirred, succinamic acid was added, triethylamine was added dropwise, and stirring was continued to obtain a reaction mixture A; (b) adding ellagic acid to the reaction mixture A of step (a), adjusting the pH with hydrochloric acid solution, heating at 100° C., freeze-drying, washing with deionized water, and drying to obtain a stabilizing promoter; The mixed solvent is toluene, carbon tetrachloride and ethyl acetate mixed in a volume ratio of 1:1:0.8; The preparation method of the curcumin composite preparation comprises the following steps: S1: adding fatty acid-aniline-hexoside compound and curcumin into a mixed solvent and stirring to obtain a mixed solution A; mixing a stabilizing agent and anhydrous ethanol evenly to obtain a mixed solution B; S2: Mix the mixed solution A obtained in step S1 with the mixed solution B, heat, and vacuum dry to obtain a curcumin composite preparation.

2. The curcumin compound formulation according to claim 1, characterized in that The mass ratio of the 2-hydroxy-4-trifluoromethylaniline to deionized water in step (1) is 1:

20.

3. The curcumin compound formulation according to claim 2, characterized in that The dosage ratio of the glucuron lactone to the intermediate product A in step (2) is 1 g:40 mL; the mass ratio of the NaOH to glucuron lactone in step (2) is 1:

150.

4. The curcumin compound formulation according to claim 3, characterized in that The mass ratio of the intermediate product B to ethyl acetate in step (3) is 1:

3.

5. The curcumin compound formulation according to claim 4, characterized in that The dosage ratio of inulin, deionized water, succinamic acid and triethylamine in step (a) is 1 g:50 mL:0.5 g:50 mg.

6. The curcumin compound formulation according to claim 5, characterized in that In step (b), the ratio of the reaction mixture A to ellagic acid is 50 mL:0.8 g.

7. The curcumin compound formulation according to claim 6, characterized in that The amount ratio of the fatty acid-aniline-hexoside compound, curcumin and the mixed solvent in step S1 is 5 g:2 g:100 mL; the amount ratio of the stabilizing promoter and anhydrous ethanol in step S1 is 1 g:15 mL; the volume ratio of the mixed solution A and the mixed solution B in step S2 is 10:

1.

8. The use of the curcumin compound formulation according to any one of claims 1 to 7, characterized in that: The curcumin compound preparation is used in the preparation of a medicine for treating MLL-r leukemia; the dosage form of the medicine includes capsules, tablets, pills, injections and granules; the medicine also includes pharmaceutically acceptable excipients.

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