Application of total coumarin of kadsura longepedunculata in preparation of colon cancer oxaliplatin drug-resistant sensitizer

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of total coumarin of Italian Korean Root to preparation of an oxaliplatin drug-resistant sensitizer for colon cancer. According to the application disclosed by the invention, the total coumarin of the dalbergia odorifera is combined with the oxaliplatin, so that the sensitivity of drug-resistant cells HCT116 / L of human colon cancer to the oxaliplatin can be increased, the drug-resistant cells can be sensitive to the oxaliplatin, cell proliferation can be inhibited, and the total coumarin of the dalbergia odorifera has a certain inhibition effect on the drug-resistant cells HCT116 / L of the human colon cancer OXA. A drug-resistant strain colon cancer nude mouse subcutaneous transplantation tumor model is constructed, TCPT can effectively inhibit the growth of tumor blocks and promote the sensitivity of the oxaliplatin-resistant colon cancer to oxaliplatin, so that a related theoretical basis and an experimental basis are provided for preparing a colon cancer oxaliplatin-resistant sensitizer and preparing a pharmaceutical composition for treating the oxaliplatin-resistant colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of total coumarins from Pileostegia tomentella in the preparation of a sensitizer for oxaliplatin resistance in colon cancer. Background Art

[0002] Colon cancer is a malignant tumor evolved from colonic epithelial tissues and is one of the most common malignant tumors globally. With the improvement of people's living standards and the change of dietary structure, the incidence rate of colorectal cancer (CRC) has been increasing year by year, and the onset age is also getting younger.

[0003] Currently, the common treatment methods for colon cancer patients include surgery, radiotherapy, chemotherapy, immunotherapy, etc. Surgical resection combined with standard adjuvant chemotherapy strategies, such as the combined chemotherapy regimen of 5-fluorouracil, calcium folinate, and oxaliplatin (OXA), can effectively improve the disease-free survival period and overall survival period of patients. High recurrence rate and multi-drug resistance are the key challenges in current anti-CRC. During the long-term chemotherapy process of patients, the sensitivity of cancer cells to chemotherapy drugs gradually weakens or even develops drug resistance. Oxaliplatin (OXA) is currently a first-line chemotherapy drug used for the treatment of colon cancer. However, the primary and secondary drug resistance of cancer cells to OXA greatly limits its clinical efficacy. How to improve the chemotherapy efficacy has become an urgent clinical problem to be solved in CRC treatment. Therefore, finding effective drugs to enhance the sensitivity of tumor cells to oxaliplatin is of great significance for clinical tumor treatment.

[0004] Ethnic medicines and their active ingredients have unique effects in overcoming or reversing tumor drug resistance and improving the quality of life of patients due to their characteristics of multi-target, multi-stage, low toxicity, and few side effects, and have great superiority as tumor chemotherapy sensitizers. Total coumarins from Pileostegia tomentella (TCPT) are the effective part of the traditional Yao medicine "Pileostegia tomentella" in Guangxi. Summary of the Invention

[0005] Aiming at the above deficiencies, the present invention provides the application of total coumarins from Pileostegia tomentella in the preparation of a sensitizer for oxaliplatin resistance in colon cancer. The combination of total coumarins from Pileostegia tomentella and oxaliplatin can promote the sensitivity of drug-resistant cells HCT116 / L to oxaliplatin, inhibit cell activity, and inhibit the growth of tumors in mice. The specific technical solutions are as follows:

[0006] The application of total coumarins from Pileostegia tomentella in the preparation of a sensitizer for oxaliplatin resistance in colon cancer.

[0007] On the other hand, the present invention also provides the application of total coumarins from Pileostegia tomentella in the preparation of a drug for treating oxaliplatin-resistant colon cancer.

[0008] On the other hand, the present invention also provides a pharmaceutical composition for treating oxaliplatin-resistant colon cancer, and the pharmaceutical composition comprises total coumarins of Gymnema sylvestre and oxaliplatin.

[0009] Preferably, in the above-mentioned pharmaceutical composition, the mass ratio of oxaliplatin to total coumarins of Gymnema sylvestre is 1:50.

[0010] Preferably, in the above-mentioned pharmaceutical composition, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0011] Preferably, in the above-mentioned pharmaceutical composition, the excipient comprises one or several of a diluent, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a flavoring agent or a transdermal absorption enhancer.

[0012] Preferably, in the above-mentioned pharmaceutical composition, the dosage form of the pharmaceutical composition is an injection preparation.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention adopts the combination of total coumarins of Gymnema sylvestre and oxaliplatin to increase the sensitivity of human colon cancer drug-resistant cells HCT116 / L to oxaliplatin, can make the drug-resistant cells sensitive to oxaliplatin, inhibit cell proliferation, and total coumarins of Gymnema sylvestre has a certain inhibitory effect on human intestinal cancer OXA-resistant cells HCT116 / L. On the basis of constructing a subcutaneous xenograft tumor model of drug-resistant colon cancer nude mice, TCPT can effectively inhibit the growth of tumor masses, promote the sensitivity of oxaliplatin-resistant CRC tumors to oxaliplatin, which provides a relevant theoretical basis and experimental basis for the preparation of an oxaliplatin sensitizer for colon cancer and the preparation of a pharmaceutical composition for treating oxaliplatin-resistant colon cancer. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings according to these drawings without creative efforts.

[0016] Figure 1 It is a graph showing the inhibitory rate of TCPT on the proliferation of HCT116 / L cells detected by the CCK-8 method in Example 1 of the present invention;

[0017] Figure 2 It is the inhibitory rate of the combined action of TCPT and OXA on the proliferation of HCT116 / L cells detected by the CCK-8 method in Example 1 of the present invention;

[0018] Figure 3The figures are the photos and weight comparison of the HCT116 / L colon cancer subcutaneous transplanted tumor in nude mice in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Unless otherwise defined, all technical terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

[0020] Reagents and instruments: Cell Counting Kit-8, KN658, Dojin, Japan; biological safety cabinet (ESCO AC2-4S1, Singapore); CO2 cell incubator (MCO-18AIC, Panasonic, Japan); multifunctional detector (Synergy H1, Biotek, USA).

[0021] Samples and cells: TCPT was prepared in our laboratory; human colon cancer OXA-resistant cells HCT116 / L were purchased from Shanghai Jinxuan Biotechnology Service Center (STR identification passed).

[0022] Nude mice: SPF nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (license number SCXK(Su)2018-0008). This animal experiment was approved by the Experimental Animal Ethics Committee of Guangxi Academy of Traditional Chinese Medicine (no. 2023032704).

[0023] Preparation of TCPT: 250g of Cibotium sylvestris was ground into a coarse powder. The extract was then extracted five times, 2 hours each time, by reflux with 8-fold 95% ethanol. The extracts were filtered and combined, the solvent recovered, and concentrated to yield 46g of an extract. The extract was suspended in water and extracted sequentially with petroleum ether (60-90°C) and ethyl acetate. The ethyl acetate-extracted fraction was separated by silica gel chromatography using a gradient elution ratio of ethyl acetate-methanol (100:0-90:0-80:20-50:50). The fractions were collected and combined by thin-layer chromatography to yield 6.28g of total coumarins.

[0024] Example 1 Inhibitory test of total coumarins from Xanthophyllum sylvestris on human colon cancer OXA-resistant cells HCT116 / L

[0025] Test method:

[0026] (1) Cell culture:

[0027] Human colon cancer OXA-resistant HCT116 / L cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (37°C, 5% CO2). When the cell fusion rate reached 80-90%, they were digested with 0.25% trypsin for subsequent grouping experiments.

[0028] (2) CCK-8 assay to detect cell proliferation

[0029] HCT116 / L cells in the logarithmic growth phase were digested and counted, and the cells were counted as 5×10 4 Cells were seeded into 96-well plates at 100 μg / well and allowed to adhere for 24 h. A gradient of TCPT concentrations (50, 100, 150, and 200 μg / mL) was added, with triplicate wells set up for each concentration. Drug-free RPMI-1640 medium was used as a blank control. After 48 h, 10 μL of CCK-8 was added to each well and incubated for another 2 h. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The cell proliferation inhibition rate was calculated and a growth curve was plotted.

[0030] Inhibition rate (%) = 1-(OD value of experimental group / OD value of control group) × 100%.

[0031] Following the CCK-8 assay protocol described above, HCT116 / L cells were treated with TCPT (100 and 150 μg / mL) combined with OXA (1, 2, and 5 μg / mL) for 24 and 48 hours. The synergistic effect of the drugs was evaluated by calculating the contract inhibition ratio (Q) using the Kim Jong-kyun method. Q = E(a+b) / (Ea+Eb - Ea×Eb), where E(a+b) represents the combined inhibition ratio of drug A and drug B, and Ea and Eb represent the inhibition ratios of drug A and drug B, respectively. A Q value greater than 1.15 indicates synergy, 0.85-1.15 indicates additive effect, and <0.85 indicates antagonism.

[0032] Data are expressed as mean ± standard deviation ( x ±s). SPSS 22.0 software was used for statistical analysis. The t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. The LSD-t-test was used for overall variance (p>0.05). When the normality condition was not met, the data were appropriately transformed.

[0033] Test results:

[0034] CCK-8 test results are shown in Figure 1 The results showed that the proliferation inhibition rate of OXA-resistant colon cancer HCT116 / L cells increased with the increase of TCPT concentration. The IC50 value of TCPT in HCT116 / L cells after 48 hours was 180.01 μg / mL.

[0035] The results of the TCPT combined with OXA test on the drug resistance sensitivity of HCT116 / L are shown inFigure 2 and Table 1. The results showed that the best inhibitory effect on HCT116 / L cells was achieved by the combined action of 100 μg / mL TCPT and 2 μg / mL OXA for 48 h. The Q value was 1.2, which was greater than 1.15, indicating a synergistic effect. This demonstrated that the combined use of TCPT and oxaliplatin could effectively inhibit the proliferation of drug-resistant HCT116 / L cells.

[0036] Table 1 Q values of the combined use of TCPT and OXA

[0037]

[0038] Example 2 Inhibitory test of total coumarins from Melodinus suaveolens Champ. ex Benth. on the growth of subcutaneous xenografts of drug-resistant colon cancer in nude mice

[0039] Test method:

[0040] HCT116 / L cells in the logarithmic growth phase were adjusted to a concentration of 1×10 7 cells / mL with serum-free medium and inoculated subcutaneously at 0.2 mL / mouse on the outer side of the right back of nude mice. When the maximum diameter of the tumor reached (6±0.5) mm, the mice were randomly divided into 3 groups, with 4 mice in each group: blank control group, high-dose TCPT group (200 mg / Kg), and low-dose TCPT group (100 mg / Kg). They were intraperitoneally injected once a day. The blank control group was given the corresponding volume of normal saline. The administration was continued for 20 days, and the general condition of the nude mice and the tumor formation were observed daily. The above animals were approved by the Ethics Committee of Guangxi University of Chinese Medicine (Ethical number: 2023032710).

[0041] Data were expressed as mean ± standard deviation and statistically analyzed using SPSS 22.0 software. The t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the overall variance p﹥0.05, the LSD-t test was used. When the normality condition was not met, the data were appropriately transformed.

[0042] Test results:

[0043] The test results are shown in Figure 3 . Compared with the blank control group, the tumor weights of the tumor-bearing mice in the high- and low-dose TCPT groups were significantly reduced (P<0.01). The results indicated that TCPT could effectively inhibit the growth of subcutaneous xenografts of drug-resistant colon cancer in nude mice.

[0044] The foregoing description of specific exemplary embodiments of the invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that, according to the above teaching, many modifications and variations are possible. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical application, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Application of total coumarins from Melodinus chunii Hand.-Mazz. in preparing an oxaliplatin resistance sensitizer for colon cancer.

2. Application of total coumarins from Melodinus chunii Hand.-Mazz. in preparing a drug for treating oxaliplatin-resistant colon cancer.

3. A pharmaceutical composition for treating oxaliplatin-resistant colon cancer, characterized in that, The pharmaceutical composition comprises total coumarins from Melodinus chunii Hand.-Mazz. and oxaliplatin.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of oxaliplatin to total coumarins from Melodinus chunii Hand.-Mazz. is 1:

50.

5. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, wherein The excipient comprises one or several of diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents or transdermal absorption promoters.

7. The pharmaceutical composition according to claim 3, wherein The dosage form of the pharmaceutical composition is an injection preparation.

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