Application of homocapsaicin II in preparation of antitumor drugs

Abstract

The invention discloses an application of homocapsaicin II in preparation of an anti-tumor drug, the homocapsaicin II is used in the anti-tumor drug independently or in combination with 5-fluorouracil or in combination with oxaliplatin, and when the homocapsaicin II is used in preparation of an anti-colorectal cancer drug, the drug concentration is 20-200 [mu] M. It is found that high capsaicin II can inhibit growth of intestinal cancer cells in vivo and in vitro, and nude mice have good tolerance to the natural compound. When the compound is used together with a first-line chemotherapeutic drug 5-fluorouracil, the drug concentration of the 5-fluorouracil can be remarkably reduced while the same treatment effect is achieved, a synergistic cancer inhibition effect is achieved, and a new research and development approach is found for anti-colorectal cancer drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an application of high capsaicin II in the preparation of anti-tumor drugs. Background Art

[0002] At present, the incidence and mortality of tumors are high, and chemotherapy is one of the main treatment methods, but the efficacy is not satisfactory. Natural products have always been the main source of chemotherapy drugs, including anticancer drugs. With the rise of targeted therapy at the end of the last century, the research and development of natural products for anticancer effects basically stopped. Because targeted therapy is generally prone to drug resistance, has a short effective period, and has unsatisfactory overall efficacy, natural products have returned to the center of anticancer chemotherapy drug research and development at the beginning of this century. From 2000 to 2010, more than a dozen tumor chemotherapy drugs approved by the FDA were all natural products or their derivatives.

[0003] Currently, chemotherapy is still one of the main treatment options for tumors, but due to the low specificity and strong toxicity of current first-line chemotherapy drugs, the side effects on patients are very strong. Therefore, finding a chemotherapy drug with strong specificity and low side effects is still an urgent need for current tumor treatment.

[0004] Capsaicin is mainly extracted from peppers. Capsaicin is a type of capsaicin, and capsaicin II is a type of capsaicin. At present, there are no relevant reports on the inhibitory effect of capsaicin II on tumors. Summary of the invention

[0005] The purpose of the present invention is to provide the application of capsaicin II in the preparation of anti-tumor drugs. Capsaicin II can inhibit the growth of colorectal cancer cells in vivo and in vitro, and nude mice have good tolerance to this natural compound. When used in combination with the first-line chemotherapy drug 5-fluorouracil, the drug concentration of 5-fluorouracil can be significantly reduced while achieving the same therapeutic effect, and it has a synergistic anti-cancer effect, thus finding a new research and development approach for anti-colorectal cancer drugs.

[0006] To achieve the above-mentioned purpose of the invention, the present invention provides the use of capsaicin II in the preparation of anti-tumor drugs, wherein the capsaicin II is used in the anti-tumor drugs alone or in combination with 5-fluorouracil.

[0007] Furthermore, the tumor is colorectal cancer.

[0008] Furthermore, when capsaicin II is used to prepare drugs against colorectal cancer, the drug concentration is 20-200 μM.

[0009] Preferably, when capsaicin II is used to prepare anti-colorectal cancer drugs, the drug concentration is 50-150 μM.

[0010] Furthermore, when capsaicin II and 5-fluorouracil are used in combination to prepare anti-colorectal cancer drugs, the concentrations of capsaicin II and 5-fluorouracil are the same, both of which are 20-200 μM.

[0011] Furthermore, when capsaicin II and oxaliplatin are used in combination to prepare anti-colorectal cancer drugs, the concentration of capsaicin II is 20-200 μM, and the concentration of oxaliplatin is 4-128 μM.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The present invention found that capsaicin II can inhibit the growth of colorectal cancer cells in vitro and in vivo, and nude mice have good tolerance to this natural compound. The prior art has not reported that the natural product capsaicin II has a significant effect in anti-tumor drugs. Therefore, this natural product may be developed into a new anti-cancer drug, such as for the treatment of colorectal cancer.

[0014] (2) The drug used in the prior art to treat colorectal cancer is 5-fluorouracil. The present invention compares the anti-tumor effects of capsaicin II and 5-fluorouracil on colorectal cancer and finds that capsaicin II has better anti-tumor effects and less side effects on nude mice than 5-fluorouracil. This indicates that compared with the current conventional colorectal cancer drug 5-fluorouracil, capsaicin II has better anti-cancer effects and less toxicity, and can be used as an anti-colorectal cancer drug to replace 5-fluorouracil.

[0015] (3) Capsaicin II is a natural product with good biocompatibility and good activity in inhibiting colorectal cancer. When used in combination with first-line chemotherapy drugs 5-fluorouracil or oxaliplatin, it can significantly reduce the drug concentration of 5-fluorouracil and oxaliplatin while achieving the same therapeutic effect. It has a synergistic anti-cancer effect and finds a new research and development path for anti-colorectal cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The molecular formula of capsaicin II (A) and the HPLC detection chart (B);

[0017] Figure 2 Figure 2 is the nuclear magnetic resonance (NMR) test result of capsaicin II; (A) is 1 H-NMR, (B) 1 C-NMR;

[0018] Figure 3 The mass spectrometry results of capsaicin II are shown in Figure 1. (A) The mass-to-nuclear ratio of the mass spectrometry result is 8.27×10 4 , (B) is the mass spectrometry result with a mass-to-nuclear ratio of 4.28×10 4 ;

[0019] Figure 4 Schematic diagram of the effect of different concentrations of high capsaicin II on the activity of colorectal cancer cells HCT116 and colorectal cancer cells SW480 in vitro;

[0020] Figure 5 Schematic diagram of the effect of different concentrations of high capsaicin II on the activity of immortalized intestinal cells NCM460 in vitro;

[0021] Figure 6 Schematic diagram of the correlation between the use of capsaicin II and 5-fluorouracil, (A) is the fitting curve between the drug concentration and the cell activity; (B) is a schematic diagram of the correlation between the combined drug index and the cell activity inhibition rate;

[0022] Figure 7 The diagram is a correlation diagram between the use of capsaicin II and oxaliplatin, (A) is a fitting curve between the drug concentration and cell activity; (B) is a correlation diagram between the combined drug index and the cell activity inhibition rate;

[0023] Figure 8 Schematic diagram of the effect of capsaicin II and 5-fluorouracil on the tumor size of intestinal cancer mice in the mouse model; (A) is a schematic diagram of macroscopic measurement, and (B) is a statistical diagram of mouse tumor size;

[0024] Fig. 9 Schematic diagram of the effect of capsaicin II and 5-fluorouracil on mouse body weight in the mouse model. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments.

[0026] Unless otherwise specified, the raw materials, reagents, etc. used in the following examples were obtained from commercial sources.

[0027] Example 1

[0028] In vitro method to verify whether capsaicin II has the effect of inhibiting the activity of colorectal cancer cells

[0029] 1. Experimental samples:

[0030] (1) High in Capsaicin II

[0031] Capsaicin II was purchased from Sichuan Dester Biological Company with CAS number: 71240-51-2 and molecular formula: C 19 H 29 NO3, molecular weight: 319.44. Structural formula: Figure 1 As shown in A, the purification method is HPLC, and the purity is 99.64% ( Figure 1 B). The compounds were identified using NPM and Mass. The identification results are shown in Figure 2 and Figure 3 .

[0032] (2) Cells

[0033] HCT116 and SW480 human colorectal cancer cell lines, NCM460 human immortalized small intestinal epithelial cells, both cells were purchased from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences in Shanghai, and cultured in a constant temperature closed incubator at 37°C and 5% CO2 using DMEM medium containing 10% PBS, 100U / m penicillin, and 100mg / L streptomycin. Cells in the logarithmic growth phase were used for experiments.

[0034] (3) Reagents and test kits

[0035] 5-Fluorouracil and oxaliplatin were purchased from Merck & Co Inc, USA, with CAS numbers of 51-21-8 and 61825-94-3, respectively. MTT kit and Tunel cell apoptosis test kit were purchased from Bio-Tech Biotech Co., Ltd., with product numbers of C0009S and C1086, respectively.

[0036] 2. Operation process:

[0037] Take HCT116, SW480 and NCM460 cells in the logarithmic growth phase and adjust the cell density to 8x10 3 / well, inoculated in 96-well culture plates, and after adherence, high capsaicin II with final concentrations of 0, 20, 50, 100, and 200 μM was added to the cell culture medium solution. After treatment for 24 and 48 hours, the changes in cell morphology were observed under an inverted microscope. The MTT method was used to detect the effects of the test samples on tumor cell viability.

[0038] The experimental results showed that 24 hours after administration, compared with the control group, the culture medium solution with a final concentration of 20, 50, 100, and 200 μM capsaicin II significantly inhibited the activity of HCT116 and SW480 cells (P < 0.01) ( Figure 4 ), but had no significant inhibitory effect on immortalized human intestinal epithelial cells NCM460 ( Figure 5 ). The effect of capsaicin II on the intestinal cancer cell lines HCT116 and SW480 was dose-dependent. The results suggest that capsaicin II in the present invention has the effect of inhibiting the activity of colorectal cancer cells, but has no significant toxicity to immortalized normal intestinal cells within a drug concentration of 200 μM.

[0039] Example 2: Verification of the synergistic effect of capsaicin II and 5-fluorouracil

[0040] Operation process:

[0041] Take the colon cancer cells HCT116 in the logarithmic growth phase and adjust the cell density to 8x10 3 / well, inoculated in 96-well culture plates, incubated for 24 hours, discarded the supernatant, and added high capsaicin II with a final concentration of 0, 20, 50, 100, and 200 μM or 5-fluorouracil with a final concentration of 0, 20, 50, 100, and 200 μM in the cell culture medium solution in the single drug group, and added high capsaicin II and 5-fluorouracil at the same concentration in the combined drug group (high capsaicin II + 5-fluorouracil: 0+0, 20+20, 50+50, 100+100, 200+200 μM). After 48 hours, the changes in cell morphology were observed under an inverted microscope. The MTT method was used to detect the effect of the test samples on the viability of tumor cells, and Compusyn was used to analyze the combined drug index of high capsaicin II and 5-fluorouracil.

[0042] result:

[0043] like Figure 6 As shown, Figure 6 AThe horizontal axis is the concentration, and the vertical axis is the inhibition rate. The three curves represent the activity inhibition rate of capsaicin II, 5-fluorouracil, and combined use of drugs on tumor cells as the concentration changes. The larger the vertical axis value, the stronger the inhibition rate. Figure 6 The results showed that both capsaicin II and 5-fluorouracil had significant inhibitory effects on colorectal cancer cells in the concentration range of 0-200 μM, and compared with 5-fluorouracil, capsaicin II and combined drugs showed more significant inhibitory effects under the same concentration conditions.

[0044] Figure 6 B is a schematic diagram of the combined drug index, the horizontal axis is the cell activity inhibition rate, and the vertical axis is the combined drug index. A combined drug index less than 1 represents a combined effect of the two drugs, that is, the combined effect of the two drugs is better than the sum of the effects of the two drugs at the same concentration. Figure 6 The combined drug results of B showed that capsaicin II and 5-fluorouracil showed a certain combined inhibitory effect on colorectal cancer cells.

[0045] Example 3: Verification of the synergistic effect of capsaicin II and oxaliplatin

[0046] Operation process:

[0047] Take the colon cancer cells HCT116 in the logarithmic growth phase and adjust the cell density to 8x10 3 / well, inoculated in 96-well culture plates, incubated for 24 hours, discarded the supernatant, added high capsaicin II with a final concentration of 0, 20, 50, 100, 200 μM or oxaliplatin with a final concentration of 0, 4, 8, 16, 32 μM in the cell culture medium solution for the single drug group, and added gradient concentrations of high capsaicin II and oxaliplatin (high capsaicin II + oxaliplatin: 0+0, 20+4, 50+8, 100+16, 200+32 μM) to the combined drug group. After 48 hours, the changes in cell morphology were observed under an inverted microscope. The MTT method was used to detect the effect of the test samples on tumor cell viability, and Compusyn was used to analyze the combined drug index of high capsaicin II and oxaliplatin.

[0048] result:

[0049] like Figure 7 As shown, Figure 7 A The horizontal axis is the concentration, and the vertical axis is the inhibition rate. The two curves represent the activity inhibition rate of capsaicin II and oxaliplatin on tumor cells with the change of concentration. The triangle represents the activity inhibition rate of tumor cells under the condition of combined drug use. The larger the vertical axis value, the stronger the inhibition rate. Fig. 9 The results showed that both capsaicin II and oxaliplatin had significant inhibitory effects on colorectal cancer cells in the concentration range of 0-200 μM, and compared with oxaliplatin, capsaicin II and combined drugs showed synergistic inhibitory effects under the same concentration conditions.

[0050] Figure 7 B is a schematic diagram of the combined drug index, the horizontal axis is the cell activity inhibition rate, and the vertical axis is the combined drug index. A combined drug index less than 1 represents a combined effect of the two drugs, that is, the combined effect of the two drugs is better than the sum of the effects of the two drugs at the same concentration. Figure 7 The combined drug results of B showed that capsaicin II and oxaliplatin showed a good combined drug effect on colorectal cancer cells, and as the concentrations of the two drugs increased, the combined drug effect became better.

[0051] Example 4: Animal experiments verify the inhibitory effect of capsaicin II on colorectal cancer cell growth

[0052] Operation process:

[0053] Take HCT116 cells in the logarithmic growth phase and adjust the cell density to 1×10 7100ul was taken to inject mice to build a tumor model. After the model was built, the control group, high capsaicin II treatment group, and 5-fluorouracil treatment group were injected with equal volumes of normal saline, 30mg / kg / d high capsaicin II solution, and 20mg / kg / d 5-fluorouracil solution every day. The tumor size and weight of the mice were measured every three days. The mice were killed after 16 days, and the tumor tissue blocks were obtained by dissection to measure the size of the tissue blocks.

[0054] result:

[0055] like Figure 8 A and Figure 8 B shows that the tumor size of mice in the high capsaicin II treatment group grew slowly during the 16-day experiment, while in the control group, the tumor cells grew rapidly, indicating that high capsaicin II 25mg / kg / d injection treatment of colorectal cancer model mice had a significant inhibitory effect on the growth of colorectal cancer tumor cells after 16 days. The 5-fluorouracil injection group mice also showed a slow growth of tumor cells compared to the control group mice, but compared to the high capsaicin II treatment group mice, the colorectal cancer cells in the 5-fluorouracil treatment group mice still showed a faster growth rate, indicating that high capsaicin II showed a better inhibitory effect on colorectal cancer cell growth than 5-fluorouracil.

[0056] Example 5: Toxicity of Capsaicin II to Mice

[0057] The mouse intestinal cancer model is an in situ tumor grown by subcutaneous injection of intestinal cancer cells, which has little effect on the growth of mice. Therefore, without treatment, the intestinal cancer model mice can grow normally during the experimental period. If the drug treatment significantly inhibits the growth of mice and causes weight loss, it means that the drug is more toxic to the mice; otherwise, it means that the toxicity is relatively small.

[0058] Operation process:

[0059] Take HCT116 cells in the logarithmic growth phase and adjust the cell density to 1×10 7 100ul was taken to inject mice to build a tumor model. After the model was built, the control group, high capsaicin II treatment group, and 5-fluorouracil treatment group were injected with equal volumes of normal saline, 25mg / kg / d high capsaicin II solution, and 25mg / kg / d 5-fluorouracil solution every day. The body weight of the mice was measured every three days. The mice were killed after 16 days and their body weight was measured.

[0060] result:

[0061] like Fig. 9As shown, during the 16-day experiment, the body weight of mice injected with high capsaicin II did not show significant differences compared with the control group mice (colon cancer model mice, not treated with drugs), indicating that the toxic effect of high capsaicin II on mice was not significant under the injection dose of 25 mg / kg / d. The body weight of 5-fluorouracil mice showed a significant downward trend compared with the control group and the high capsaicin II treatment group, indicating that 5-fluorouracil has strong toxic side effects on mice. This suggests that high capsaicin II has the potential to be developed as a drug for the treatment of colorectal cancer, or as an auxiliary drug for the treatment of colorectal cancer.

[0062] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0063] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. The application of high capsaicin II in the preparation of anti-tumor drugs, characterized in that: The capsaicin II is used in the anti-tumor drug alone or in combination with 5-fluorouracil or in combination with oxaliplatin.

2. The use of high capsaicin II according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is colorectal cancer.

3. The use of high capsaicin II according to claim 2 in the preparation of anti-tumor drugs, characterized in that: When high capsaicin II is used to prepare drugs for treating colorectal cancer, the drug concentration is 20-200 μM.

4. The use of high capsaicin II according to claim 3 in the preparation of anti-tumor drugs, characterized in that: When capsaicin II is used to prepare anti-colorectal cancer drugs, the drug concentration is 50-150 μM.

5. The use of high capsaicin II according to claim 2 in the preparation of anti-tumor drugs, characterized in that: When capsaicin II and 5-fluorouracil are used in combination to prepare anti-colorectal cancer drugs, the concentrations of capsaicin II and 5-fluorouracil are the same, both of which are 20-200 μM.

6. The use of high capsaicin II according to claim 2 in the preparation of anti-tumor drugs, characterized in that: When capsaicin II and oxaliplatin are used in combination to prepare anti-colorectal cancer drugs, the concentration of capsaicin II is 20-200 μM, and the concentration of oxaliplatin is 4-128 μM.

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