A beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a preparation method thereof

Abstract

The present invention relates to a beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a preparation method thereof, wherein the composition comprises beauvericin and an indole compound, wherein the mass ratio of the beauvericin to the indole compound is 1:(0.2-0.5). The beauvericin and the indole compound in the composition have a significant inhibitory effect on the proliferation of human prostate cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceuticals, and in particular relates to a beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a preparation method thereof. Background Art

[0002] Prostate cancer is a common malignant tumor of the male reproductive system. Its incidence rate increases with age. It is a disease that often occurs in elderly men. The common clinical manifestations of prostate cancer are difficulty urinating, followed by symptoms such as urgency, frequency of urination, and hematuria. Urinating is also accompanied by pain or burning sensation in the lower back, upper thigh, or pelvis. Prostate cancer tumors have almost no clinical manifestations in the early stages and are difficult to detect. Therefore, it is difficult to diagnose early based on clinical symptoms alone. When discovered, it has generally developed into advanced prostate cancer, causing serious adverse consequences for patients.

[0003] The main treatments for prostate cancer include biological therapy, surgery, radiotherapy and chemotherapy, which not only cause great pain to patients, but also require huge treatment costs, causing great financial pressure on the patients' families. Therefore, it is of great significance to invent a pharmaceutical composition that can inhibit the proliferation of human prostate cancer cells. Summary of the invention

[0004] In view of the above-mentioned existing technical problems, the present invention provides a beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a preparation method thereof. The beauvericin composition for inhibiting the proliferation of human prostate cancer cells comprises beauvericin and indole compounds. Beauvericin and indole compounds have significant inhibitory effects on the proliferation of human prostate cancer cells.

[0005] The invention provides a beauvericin composition for inhibiting the proliferation of human prostate cancer cells. The composition comprises beauvericin and an indole compound, wherein the mass ratio of the beauvericin to the indole compound is 1:(0.2-0.5).

[0006] In some embodiments of the present invention, the structure of the indole compound is as follows:

[0007]

[0008] Wherein, R is selected from C1~C12 alkyl.

[0009] In some embodiments of the present invention, the structure of the indole compound is as follows:

[0010]

[0011] Wherein, R is selected from C4~C9 alkyl.

[0012] In some embodiments of the present invention, the structure of the indole compound is as follows:

[0013]

[0014] Wherein, R is selected from C6 alkyl.

[0015] In some embodiments of the present invention, the method for preparing the indole compound comprises the following steps:

[0016] The carboxylic acid compound RCOOH is chlorinated and then reacted with the indole compound Reaction to obtain indole compounds.

[0017] In some embodiments of the present invention, the chlorination reagent is SOCl2.

[0018] In some embodiments of the present invention, the temperature of the acyl chlorination is 80-100° C. and the time is 12-20 hours.

[0019] In some embodiments of the present invention, the mass ratio of the carboxylic acid compound to the indole compound is 2.1-2.5:1.

[0020] In some embodiments of the present invention, the reaction temperature is 110-130° C. and the reaction time is 12-18 hours.

[0021] In some embodiments of the present invention, the mass ratio of the carboxylic acid compound to SOCl2 is 1:1.2-1.5.

[0022] Another object of the present invention is to provide the use of the beauvericin composition for inhibiting the proliferation of human prostate cancer cells in drugs for inhibiting the proliferation of human prostate cancer cells.

[0023] In some embodiments of the present invention, the human prostate cancer cells include human prostate cancer cells LNCAP or human prostate cancer cells PC-3.

[0024] In some embodiments of the present invention, the drug includes an effective amount of a beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a pharmaceutically acceptable carrier.

[0025] In some embodiments of the present invention, the drug is administered orally or by injection.

[0026] In some embodiments of the present invention, the dosage form of the drug is a suspension, granules, capsules, powders, tablets, solutions, pellets and / or injections.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The indole compounds in the beauvericin composition for inhibiting the proliferation of human prostate cancer cells of the present invention can reduce the cytotoxicity of beauvericin and improve the application prospect of beauvericin in the treatment of human prostate cancer.

[0029] (2) The beauvericin composition for inhibiting the proliferation of human prostate cancer cells of the present invention has a good inhibitory effect on the migration and invasion of human prostate cancer cells LNCAP and human prostate cancer cells PC-3. DETAILED DESCRIPTION

[0030] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0031] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0034] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0035] Example 1 Preparation of Indole Compound A1

[0036] 10 parts by mass of heptanoic acid and 14 parts by mass of SOCl2 were added to a reaction bottle, 50 parts by mass of N,N-dimethylformamide was added, and the mixture was reacted at 95°C for 16 hours. Then 4.5 parts by mass of indole compound was added, and the mixture was stirred for 1 hour. 1 mL of triethylamine was added, and the mixture was reacted at 120°C for 15 hours to obtain indole compound A1 with a yield of 83.6%. The H NMR spectrum of the indole compound A1 is as follows:

[0037] 1H NMR (400MHz, CDCl3, ppm) δ7.68(s,1H,Ph),7.10(s,1H,NH),5.76(t,1H,CH),4.34(s,2H,CH2),3.65(s,3H,CH3),3.34(d,2H,C H2),2.51(t,4H,CH2),2.39-2.34(m,4H,CH2),1.80(s,3H,CH3),1.67(t,4H,CH2),1.30-1.34(m,12H,CH2),0.87(t,6H,CH3).

[0038] The structure of the indole compound A1 is shown below:

[0039]

[0040] Example 2 Cell growth assay

[0041] (1) Experimental subjects: normal human prostate epithelial cells RWPE-1, human prostate cancer cells LNCAP (androgen-sensitive), and human prostate cancer cells PC-3 (androgen-insensitive);

[0042] (2) Experimental drugs: beauvericin, beauvericin + indole compound A1 (mass ratio of 1:0.3), solvent DMSO;

[0043] (3) Experimental methods:

[0044] S1. Collect cells in the logarithmic phase, adjust the concentration of the cell suspension, add 90uL per well, about 2000 cells, and inoculate them in the culture plate;

[0045] S2. After the cells adhered to the wall, 10 μL of drugs with different concentrations were added to each well. Five replicate wells were set for each drug concentration and the cells were placed in a 5% CO2, 37°C incubator for incubation.

[0046] S3. After 48 hours of drug addition, the plates were collected and observed with naked eyes under an inverted microscope. Then, 10uL of MTT solution (5mg / mL) was added to each well. After incubation for 4 hours, the reaction was terminated. The culture medium in the wells was carefully aspirated, and 100μL of DMSO was added to each well. The wells were gently shaken on a shaker for 10 minutes to fully dissolve the crystal particles.

[0047] S4. Detect the absorbance OD of each well at a wavelength of 490 nm on an ELISA reader, and calculate the drug inhibition rate (%) of the cells according to formula (1);

[0048]

[0049] Calculate IC based on drug inhibition rate 50In this experiment, the culture medium without cells was set as the basic control group, and DMSO solution with the same dilution ratio as the experimental drug was added to each well as the blank control group. The drug experimental groups contained beauvericin and beauvericin + indole compound A1, respectively. The results are shown in Table 1.

[0050] Table 1. IC of drugs 50 (μm).

[0051]

[0052]

[0053] As shown in Table 1, the cytotoxicity test results of beauvericin alone on normal human prostate epithelial cells RWPE-1 and human prostate cancer cells LNCAP and PC-3 were not much different. 50 The cytotoxicity of beauvericin to normal human prostate epithelial cells RWPE-1 was about 3.0 μM to 3.2 μM. However, the combination of beauvericin and indole compound A1 significantly reduced the cytotoxicity to normal human prostate epithelial cells RWPE-1. 50 It was reduced to 13.8 μM, and its toxicity to human prostate cancer cells LNCAP and PC-3 was significantly reduced, indicating that at the same concentration, the combination of beauvericin and indole compound A1 can achieve a significant inhibitory effect on the growth of human prostate cancer cells, while having little toxicity to normal cells.

[0054] Example 2 Cell migration experiment

[0055] (1) Experimental subjects: human prostate cancer cells LNCAP (androgen-sensitive) and human prostate cancer cells PC-3 (androgen-insensitive);

[0056] (2) Experimental drugs: beauvericin, indole compound A1, beauvericin + indole compound A1 (mass ratio of 1:0.3), solvent DMSO;

[0057] (3) Experimental methods:

[0058] Add 2 mL of complete medium containing 10% FBS (fetal bovine serum) and 2 × 10 6The cells were plated in 3 wells and 3 replicate wells were set for each well. The cells were cultured for about 24 hours. The scratch test was performed when the number of cells could cover 95% of the surface after overnight attachment. The old culture medium was first sucked off, and the cell scratch line was formed by evenly moving the pipette tip up and down. The cells were rinsed 3 times with 2 mL of sterile 1×PBS. After removing the scratched cells, a complete culture medium containing 20% ​​FBS (fetal bovine serum) was added, and then the experimental drugs were added separately (the working concentrations of the experimental drugs were 10 μM and 20 μM). At the same concentration, the added volume of the experimental drugs in each group was the same. The solvent DMSO was used as the blank control group. The cells were cultured in a 37°C, 5% CO2 incubator. Samples were taken at 0h and 24h after drug addition, and the cell migration ratio was counted and the data were analyzed. The results are shown in Tables 2 to 3.

[0059] Table 2. Effects of drugs on the migration of human prostate cancer cells LNCAP ( n=3).

[0060]

[0061] Note: *P<0.05, **P<0.01, all compared with the blank control group.

[0062] As shown in Table 2, compared with the blank control group (DMSO), the scratch area of ​​human prostate cancer cell LNCAP treated with beauvericin or indole compound A1 was significantly larger than that of the blank control group, indicating that the migration speed of human prostate cancer cell LNCAP treated with beauvericin or indole compound A1 was reduced, and the ability to migrate to the central scratch area was weakened, which was statistically significant (P<0.05). The scratch area of ​​human prostate cancer cell LNCAP treated with beauvericin + indole compound A1 (mass ratio of 1:0.3) was significantly larger than the cell scratch after beauvericin or indole compound A1 treatment, indicating that the combination of beauvericin and indole compound A1 can significantly reduce the migration rate of human prostate cancer cell LNCAP, which was statistically significant (P<0.01).

[0063] Table 3. Effects of drugs on migration of human prostate cancer cells PC-3 ( n=3).

[0064]

[0065] Note: *P<0.05, **P<0.01, all compared with the blank control group.

[0066] As can be seen from Table 2, compared with the blank control group (DMSO), the scratch area of ​​human prostate cancer cell PC-3 treated with beauvericin or indole compound A1 was significantly larger than that of the blank control group, indicating that the migration speed of human prostate cancer cell PC-3 treated with beauvericin or indole compound A1 was reduced, and the ability to migrate to the central scratch area was weakened, which was statistically significant (P<0.05). The scratch area of ​​human prostate cancer cell PC-3 treated with beauvericin + indole compound A1 (mass ratio of 1:0.3) was significantly larger than the cell scratches treated with beauvericin or indole compound A1, indicating that the combination of beauvericin and indole compound A1 can significantly reduce the migration rate of human prostate cancer cell PC-3, which was statistically significant (P<0.01).

[0067] Example 3 Cell invasion assay

[0068] (1) Experimental subjects: human prostate cancer cells LNCAP (androgen-sensitive) and human prostate cancer cells PC-3 (androgen-insensitive);

[0069] (2) Experimental drugs: beauvericin, indole compound A1, beauvericin + indole compound A1 (mass ratio of 1:0.3), solvent DMSO;

[0070] (3) Experimental methods:

[0071] S1. Digest the cells, centrifuge and discard the culture medium after terminating the digestion, wash once with PBS (phosphate buffered saline), resuspend in serum-free medium, and adjust the cell density to 1×10 6 Pieces / mL;

[0072] S2, take 100 μL of cell suspension, add 10% BSA (bovine serum albumin) to a final concentration of 0.1%, then add drugs (set up blank control group (add DMSO with the same dilution ratio of drugs) and drug treatment group (drug working concentration is 10 μM, 20 μM), each group has 3 replicates), at the same concentration, the added volume of experimental drugs in each group is the same, and finally fill with culture medium containing 10% FBS (fetal bovine serum) to make the total volume of each well 200 μL, mix well, gently and evenly add to the upper chamber of Transwell covered with glue, immediately add 600 μL of culture medium containing 20% ​​FBS (fetal bovine serum) to the lower chamber of 24-well plate, and culture for 24 hours;

[0073] S3. Take out the Transwell chamber, discard the culture medium in the well, gently wipe off the unmigrated cells and gel in the upper chamber with a cotton swab, and put it into a clean 24-well plate, wash it once with 1×PBS, fix it with methanol for 30 minutes, then dry it, stain it with 0.1% crystal violet for 20 minutes, wash it once with PBS, and dry it;

[0074] Result statistics: The number of cells in the field of view of the Transwell chamber was recorded under a microscope, and the relative invasion ratio of the cells was counted. The specific results are shown in Tables 4-5.

[0075] Table 4. Effects of drugs on invasion of human prostate cancer cells LNCAP ( n=3).

[0076]

[0077] Note: *P<0.05, **P<0.01, all compared with the blank control group.

[0078] As shown in Table 4, compared with the blank control group (DMSO solution), the number of human prostate cancer cell LNCAP in the Transwell chamber after being treated with beauvericin or indole compound A1 was significantly lower than that in the blank control group, indicating that beauvericin or indole compound A1 can effectively inhibit the invasion of human prostate cancer cell LNCAP, and the result was statistically significant (P<0.05). The number of human prostate cancer cell LNCAP in the Transwell chamber after being treated with beauvericin + indole compound A1 (mass ratio of 1:0.3) was significantly lower than the number of cells treated with beauvericin or indole compound A1, indicating that the combination of beauvericin + indole compound A1 can significantly reduce the invasion of human prostate cancer cell LNCAP, and the result was statistically significant (P<0.01).

[0079] Table 5. Effects of drugs on migration of human prostate cancer cells PC-3 ( n=3).

[0080]

[0081]

[0082] Note: *P<0.05, **P<0.01, all compared with the blank control group.

[0083] As shown in Table 5, compared with the blank control group (DMSO solution), the number of human prostate cancer cells PC-3 in the Transwell chamber after being treated with beauvericin or indole compound A1 was significantly lower than that in the blank control group, indicating that beauvericin or indole compound A1 can effectively inhibit the invasion of human prostate cancer cells PC-3, and the results were statistically significant (P<0.05). The number of human prostate cancer cells PC-3 in the Transwell chamber after being treated with beauvericin + indole compound A1 (mass ratio of 1:0.3) was significantly lower than the number of cells treated with beauvericin or indole compound A1, indicating that the combination of beauvericin + indole compound A1 can significantly reduce the invasion of human prostate cancer cells PC-3, and the results were statistically significant (P<0.01).

[0084] It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above embodiments are described. If there is no contradiction, technicians in this field can combine and combine the different embodiments and features of different embodiments described in this specification.

[0085] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A beauvericin composition for inhibiting the proliferation of human prostate cancer cells, characterized in that: The composition comprises beauvericin and an indole compound, wherein the mass ratio of beauvericin to the indole compound is 1:(0.2-0.5); the structure of the indole compound is as follows: 。 2. The beauvericin composition for inhibiting the proliferation of human prostate cancer cells according to claim 1, characterized in that: The preparation method of the indole compound comprises the following steps: Heptanoic acid is chlorinated and then reacted with an indole compound Reaction to obtain indole compounds.

3. The beauvericin composition for inhibiting the proliferation of human prostate cancer cells according to claim 2, characterized in that: The reagent for the chlorination is SOCl2; The mass ratio of the heptanoic acid to the indole compound is 2.1-2.5:

1.

4. The beauvericin composition for inhibiting the proliferation of human prostate cancer cells according to claim 2, characterized in that: The temperature of the chlorination is 80-100°C and the time is 12-20h; The reaction temperature is 110-130° C. and the reaction time is 12-18 hours.

5. The beauvericin composition for inhibiting the proliferation of human prostate cancer cells according to claim 3, characterized in that: The mass ratio of the heptanoic acid to SOCl2 is 1:1.2-1.

5.

6. Use of the beauvericin composition for inhibiting the proliferation of human prostate cancer cells according to any one of claims 1 to 5 in the preparation of a drug for inhibiting the proliferation of human prostate cancer cells.

7. The use according to claim 6, characterized in that: The human prostate cancer cells include human prostate cancer cells LNCAP or human prostate cancer cells PC-3.

8. The use according to claim 6, characterized in that: The medicine comprises an effective amount of a beauvericin composition for inhibiting the proliferation of human prostate cancer cells and a pharmaceutically acceptable carrier.

9. The use according to claim 6, characterized in that: The drug is administered orally or by injection.

10. The use according to claim 6, characterized in that: The dosage form of the medicine is suspension, granule, capsule, powder, tablet, solution, pill or injection.

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