Pharmaceutical composition for treating tumors and inflammations and application thereof

Abstract

The invention discloses a medicine composition for treating tumors and inflammation and application thereof. The medicine composition has a synergistic effect when used in a combined mode, and the anti-tumor effect, the anti-inflammation effect and the like can be enhanced. According to the application, a cell proliferation activity detection method and a real-time fluorescent quantitative detection method are used for evaluating the combined use of the target compound naringin dihydrochalcone and the antitumor drug cis-platinum, and the result shows that compared with the single use of one of the drugs, the combined use has better mouse monocyte leukemia cell proliferation resistance; and the expression of related inflammatory factors in an LPS-induced cell inflammation model can be obviously inhibited. The invention provides a group of pharmaceutical compositions which have better anti-tumor and anti-inflammatory effects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a pharmaceutical composition for treating tumors and inflammation and application thereof. Background Art

[0002] A tumor is a new organism formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation. It is generally believed that tumor cells are monoclonal, that is, all tumor cells in a tumor are descendants of a mutated cell. At present, tumors are generally divided into two categories: benign and malignant. Benign tumors are generally called "tumors", malignant tumors from epithelial tissues are called "cancers", and those from mesenchymal tissues are called "sarcomas". Some malignant tumors can also be called "tumors" or "diseases", such as malignant lymphoma, seminoma, leukemia, Hodgkin's disease, etc. All malignant tumors are usually called cancer or tumors. Cancer is a major cause of death worldwide. The most common cancers are breast cancer, lung cancer, colon cancer, rectal cancer and prostate cancer. On February 2, 2024, the latest data released by the World Health Organization's International Agency for Research on Cancer (IARC) showed that the number of new cancer cases worldwide reached 20 million, and the number of deaths reached 9.7 million; it is estimated that about 1 / 5 of the people will develop cancer in their lifetime, and about 1 / 9 of men and 1 / 12 of women will die from cancer.

[0003] Inflammation is a common and frequently occurring disease that threatens human health. There are many causes of inflammation, including bacteria, viruses, rickettsia, mycoplasma, fungi, etc. Inflammation caused by biological pathogens is also called infection. The human body has a complex structure, and different parts of the body will experience varying degrees of inflammation. For example, gastroenteritis, hepatitis, appendicitis, pancreatitis, pharyngitis, prostatitis, vaginitis, periarthritis of the shoulder, otitis media, etc. are representative. Ordinary inflammation can be cured by antibacterial and anti-inflammatory treatments, while some uncontrollable inflammations that are difficult to control by various methods are closely related to the occurrence and development of tumors. "Cell" once published a major review of tumor research, summarizing the hot topics in oncology in the past 10 years. Among them, inflammation is listed as the main feature that promotes cancer, and anti-inflammatory drugs may be used to treat tumors.

[0004] Naringin dihydrochalcone is a flavonoid derivative obtained by hydrogenating naringin extracted from natural citrus plants. Naringin dihydrochalcone has strong antioxidant properties and can also reduce blood sugar and blood lipids, inhibit fatty liver, regulate immunity and other important physiological activities. Research experiments have shown that naringin dihydrochalcone can significantly remove stable free radicals and reactive oxygen species (ROS), and can reduce blood sugar levels and blood cholesterol levels in animals. These studies are of great significance in medicine. In addition, naringin dihydrochalcone also has a similar effect to prebiotics, which can affect the symbiotic intestinal group in the body, regulate the body's immunity, and reduce intestinal diseases. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a pharmaceutical composition for treating tumors and inflammation, wherein: the pharmaceutical composition consists of naringin dihydrochalcone and cisplatin, and the administration concentrations of naringin dihydrochalcone and cisplatin are 150 μM and 1 μg / mL, respectively.

[0007] As another aspect of the present invention, the present invention provides the use of the pharmaceutical composition in preparing drugs for treating tumors and inflammatory diseases.

[0008] The purpose of the present invention is mainly implemented through the following solutions:

[0009] The in vitro anti-tumor proliferation activity evaluation method of drugs is as follows: under sterile conditions, mouse mononuclear macrophage leukemia cells RAW264.7 were resuspended at 3×10 4 / mL, and the cells were seeded into 96-well plates, and 150μM naringin dihydrochalcone solution, 1μg / mL cisplatin solution and the combination of the two drugs were added respectively. Cell activity was detected 24 hours later, and the inhibition rate of drugs on RAW264.7 cells was calculated.

[0010] The in vitro anti-inflammatory activity evaluation method of the drug is as follows: under sterile conditions, mouse mononuclear macrophage leukemia cells RAW264.7 were resuspended at 3×10 4 / mL, and the cells were seeded into 96-well plates. 150 μM naringin dihydrochalcone solution, 1 μg / mL cisplatin solution and the combination of the two drugs were added 1 hour later, and LPS (1 μg / mL) was given to continue stimulation for 4 hours. The cell lysate was collected, and total RNA was extracted. The expression of IL-6 and IL-1β was detected using a kit.

[0011] Beneficial effects of the present invention: The present invention combines naringin dihydrochalcone with cisplatin, and detects the effects of the combined application of the two drugs on the proliferation activity of RAW264.7 and inflammatory factors after LPS stimulation in vitro. The results show that the combined use of naringin dihydrochalcone and cisplatin has a synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Compared with the control group, naringin dihydrochalcone and cisplatin alone or in combination had a significant effect on the proliferation activity of RAW264.7 cells in vitro, ***P<0.01. ### P<0.001, indicating a significant statistical difference.

[0013] Figure 2 a Detection of IL-1β expression level in LPS-induced RAW264.7 cell inflammation model in vitro by using naringin dihydrochalcone and cisplatin alone or in combination, compared with the Control group, *P<0.05.

[0014] Figure 2 b Detection of IL-1β expression level in LPS-induced RAW264.7 cell inflammation model in vitro by using naringin dihydrochalcone and cisplatin alone or in combination, compared with the Control group, *P<0.05. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with specific embodiments, according to common technical knowledge and conventional means in the art. The following embodiments are only some preferred embodiments of the present invention and should not be regarded as limiting the present invention. For those of ordinary skill in the art, several improvements can be made without departing from the scope of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

[0016] Example 1

[0017] Study on the inhibitory effect of naringin dihydrochalcone and cisplatin on tumor cell proliferation

[0018] 1. Cell Culture

[0019] Mouse macrophage leukemia cells RAW 264.7 were cultured in DMEM high glucose medium (Gibco, Catalog No.: 11995065) supplemented with 10% (V / V) FBS (Gibco, Catalog No.: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, Catalog No.: 15140122). The culture conditions were 37°C, 5% CO2, and the cells were passaged when they reached 80% confluence.

[0020] 2. Cell Proliferation Activity Detection

[0021] Mouse monocytic leukemia cells RAW 264.7 were selected and cultured at 2×10 3 / mL, 100μL / well were inoculated in a 96-well plate. When the cell coverage rate was about 80%, naringin dihydrochalcone (NDC, 150μM) and cisplatin (Cis, 1μg / mL) were added alone or in combination, with 5 replicates in each group, and a blank control group was set up. After continuous culture for 24h, 10μL CCK8 solution was added to each well, and the cells were incubated in a 37℃, 5% CO2 incubator for 2h. The absorbance OD value was measured at a wavelength of 450nm with an enzyme reader and the cell survival rate was calculated.

[0022] The results are as follows Figure 1 As shown, both naringin dihydrochalcone and cisplatin alone can significantly inhibit the cell proliferation ability of mouse monocytic leukemia cells RAW264.7, while the combined administration has a stronger inhibitory effect than the single naringin dihydrochalcone or cisplatin group, indicating that the combination of the two has a synergistic effect.

[0023] Example 2

[0024] Study on the combined anti-inflammatory activity of naringin dihydrochalcone and cisplatin.

[0025] RAW264.7 cells were seeded in 12-well plates at a density of 2×105 / mL, 1 mL / well. When the cell coverage rate was about 80%, naringin dihydrochalcone (NDC, 150 μM) and cisplatin (Cis, 1 μg / mL) were added alone or in combination. One hour later, LPS was added at a final concentration of 1 μg / mL. LPS stimulation continued for 4 hours.

[0026] (1) RNA extraction and qPCR determination

[0027] After completely discarding the culture medium of the cultured cells, 0.5 mL of RA2 in the RNA extraction kit was added to each well for lysis; the cell lysate was collected and Total RNA was extracted according to the kit instructions (Vazyme, catalog number: RC112), and its concentration was measured using an ultra-micro UV-visible spectrophotometer;

[0028] (2) Reverse transcription reaction

[0029] A 20 μL reverse transcription system was established, including 4 μL 5× Buffer, X μL Total RNA (500 ng), and DEPC H2O (16X) μL placed in 8 tubes, marked with serial numbers, and placed in a PCR instrument and set to 50°C, 15 min; 85°C, 5 s; 16°C∝ for reverse transcription;

[0030] (3) Fluorescence quantification

[0031] Add 80 μL DEPC H2O to the transcribed cDNA to make 100 μL, centrifuge and mix, and set aside at 20°C; multiply the number of samples by (SYBR 10 μL + DEPC H2O 10 μL + primer 0.5 μL), take 18 μL / well to a 96-well plate, add 2 μL cDNA to form a 20 μL system, and centrifuge and mix at 1200 rpm for 1 min; put the sample plate into the CFX Connect Real Time System (real-time fluorescence quantitative PCR instrument) for detection, and perform detection according to the program of (95°C, 2 min; 95°C, 20 s; 57°C, 20 s; 72°C, 20 s) for a total of 39 cycles, 95°C, 1 min; 55°C, 30 s; 95°C, 30 s;

[0032] (4) Analysis

[0033] The 2ΔΔCt method was used to analyze the experimental results. The calculation formula is as follows: ΔCt target gene = Ct target gene Ct reference gene, ΔΔCt target gene = ΔCt experimental group target gene ΔCt control group target gene. 2ΔΔCt represents the experimental group

[0034] The expression fold of the target gene relative to the control group.

[0035] The primers are shown in Table 1 below.

[0036]

[0037] The results are as follows Figure 2 As shown, when naringin dihydrochalcone and cisplatin were used alone, they could reduce the expression of inflammatory factors IL-6 and IL-1β in the LPS-stimulated RAW264.7 cell inflammation model, but there was no statistical difference. After combined use, the expression of inflammatory factors was significantly reduced compared with the model group, and it was statistically significant, indicating that the combined use of the two has a synergistic effect on the inhibition of inflammation.

Claims

1. A pharmaceutical composition for treating tumors and inflammation, characterized in that: The pharmaceutical composition comprises naringin dihydrochalcone and cisplatin, and the administration concentrations of the naringin dihydrochalcone and cisplatin are 150 μM and 1 μg / mL respectively.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of drugs for treating tumors and inflammation.

3. The use according to claim 3, characterized in that: The administration method is intragastric administration.

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