Application of origanum oil in preparation of medicine for treating colorectal cancer

Abstract

The invention provides application of origanum oil in preparation of a medicine for treating colorectal cancer, and relates to the technical field of medicines. The invention discovers that the origanum oil has the effect of resisting colorectal cancer for the first time, in-vivo and in-vitro pharmacodynamic tests verify that the invention provides a new application of the origanum oil in preparation of the medicine for treating colorectal cancer. We verify that the origanum oil can relieve weight loss of mice caused by AOM / DSS and reduce death rate, significantly inhibit growth of colorectal cancer tumor cells and induce death of the colorectal cancer tumor cells, and further research finds that the origanum oil can induce cell ferroptosis to resist colorectal cancer by regulating and controlling a cAMP / AMPK signal channel through OR51E2. The invention provides a research basis for taking the origanum oil as a new medicine for treating the colorectal cancer, and provides a new thought for treating the colorectal cancer.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of oregano oil in preparing a drug for treating colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the malignant tumors that seriously threatens human health. The incidence and mortality are both high, and the disease burden is heavy. The incidence of CRC is tending to be younger, so early screening, early diagnosis, and early treatment of the disease are particularly important. With the continuous deepening of research on the pathogenesis of malignant tumors, a variety of "killing and antagonistic" intervention methods such as radiotherapy, chemotherapy, surgery, targeted therapy, and immunotherapy have been established, which have the advantages of shrinking tumors and prolonging survival, but at the same time also lead to a series of adverse reactions. Traditional Chinese medicine adheres to the holistic concept, emphasizes syndrome differentiation and treatment, and is based on thousands of years of clinical practice. The prevention and treatment of malignant tumors has the characteristics of multiple components, multiple targets, and multiple mechanisms. A large number of studies have shown that traditional Chinese medicine has unique advantages in treating CRC in improving tumor response rate, improving quality of life, reducing adverse reactions related to radiotherapy and chemotherapy, promoting postoperative gastrointestinal function recovery, and prolonging survival.

[0003] Oregano, also known as Manpoxiang, Tushou, and Dihuoxiang, is a perennial aromatic plant of the genus Origanum in the family Lamiaceae. The whole herb of oregano can be used as medicine, which has the effects of dispelling exterior pathogens, regulating qi, clearing away heat, and removing dampness. It is often used to prevent influenza, treat jaundice, heatstroke, diarrhea, vomiting, qi obstruction and food stagnation, abdominal distension due to food accumulation in children, and skin itching. Oregano oil, also known as oregano volatile oil or oregano essential oil, is a light yellow oily liquid with an aromatic smell extracted from oregano, accounting for 1% to 10% of oregano. Oregano oil mainly contains aromatic phenols, mucilage, terpenes, resins, tannic acid and bitter substances, etc. It has multiple pharmacological activities such as antioxidant, antibacterial, immune regulation and anti-inflammatory, and is widely used in animal production, food analysis, medicine and health, and daily necessities. Studies have shown that oregano oil has a certain inhibitory effect on liver cancer HepG2 and lung cancer A549 cells. Oregano oil has certain application prospects in anti-cancer. However, there are currently no reports on the application of oregano oil in colorectal cancer. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies in the prior art, the present invention provides the use of oregano oil in preparing a drug for treating colorectal cancer.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides the use of oregano oil in the preparation of a drug for treating colorectal cancer.

[0009] In a second aspect, the present invention provides the use of oregano oil in the preparation of a drug for inhibiting colorectal cancer cell HCT116.

[0010] In a third aspect, the present invention provides the use of oregano oil in the preparation of a drug for increasing the expression level of cAMP and AMKP.

[0011] In a fourth aspect, the present invention provides the use of oregano oil in the preparation of a drug for inhibiting the expression level of OR51E2 gene.

[0012] In a fifth aspect, the present invention provides the use of oregano oil in the preparation of a drug for increasing the expression level of PRKAA1 and / or PRKAA2 and / or CAMKK2 genes.

[0013] In a sixth aspect, the present invention provides the use of oregano oil in the preparation of a drug for inhibiting the expression level of GSS and / or GPX4 and / or SLC7A11 genes.

[0014] In the above application, oregano oil and olive oil are evenly mixed in a mass ratio of 1:9-19 for use.

[0015] In the above application, oregano oil is a commercially available single essential oil with a purity of more than 99%, or oregano oil with a purity of more than 99% obtained by extracting oregano using supercritical carbon dioxide extraction.

[0016] (III) Beneficial effects

[0017] The present invention first discovered that oregano oil has an anti-colorectal cancer effect, and verified it through in vitro and in vivo efficacy tests, and proposed a new application of oregano oil in the preparation of drugs for the treatment of colorectal cancer. We verified that oregano oil can alleviate the weight loss of mice caused by AOM / DSS and reduce mortality, significantly inhibit the growth of colorectal cancer tumor cells, and induce the death of colorectal cancer tumor cells. We further found that oregano oil can significantly increase the expression levels of cAMP and AMPK in the serum of AOM / DSS-induced mice, significantly inhibit the expression of OR51E2, GSS, GPX4, and SLC7A11 gene mRNA in colorectal tissue, and increase the expression of PRKAA1, PRKAA2, and CAMKK2 gene mRNA. Oregano oil intervention can regulate the expression of cAMP / AMPK signaling pathways and ferroptosis-related proteins, indicating that oregano oil can regulate the cAMP / AMPK signaling pathway through OR51E2 to induce cell ferroptosis and resist colorectal cancer. This study provides data support for oregano oil as a new drug for the treatment of colorectal cancer and provides new ideas for the treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of in vitro and in vivo efficacy study of oregano oil against colorectal cancer; A: changes in body weight of mice in each group during the experiment, compared with AOM / DSS group, *P<0.05, **P<0.01; B: mortality of mice in each group during the experiment; C: colon length of mice in each group, compared with AOM / DSS group, *P<0.05, **P<0.01; D: H&E staining results of colon tissue of mice in each group; E: proliferation rate of colorectal cancer cell HCT116 under different doses of oregano oil treatment, compared with blank control group, *P<0.05, **P<0.01; F: bright field observation of the effect of oregano oil on the morphology of colorectal cancer cells under inverted microscope; G: results of cell cloning experiment, compared with blank control group, *P<0.05, **P<0.01.

[0019] Figure 2 Oregano oil can mediate OR51E2 to regulate cAMP / AMPK signaling pathway to induce cell ferroptosis and resist colorectal cancer; A: ELISA kit was used to detect the changes in the expression levels of cAMP and AMPK in the serum of each group of mice induced by AOM / DSS. Compared with the AOM / DSS group, *P<0.05, **P<0.01; B: RT-qPCR was used to detect the mRNA levels of OR51E2, PRKAA1, PRKAA2, and CAMKK2 genes in the colorectal tissues of each group of mice induced by AOM / DSS. =The level was observed in the control group, *P<0.05, **P<0.01 compared with the AOM / DSS group; C: RT-qPCR was used to detect the mRNA levels of GSS, GPX4 and SLC7A11 genes in the colorectal tissues of each group of mice induced by AOM / DSS. Compared with the AOM / DSS group, *P<0.05, **P<0.01; D: IHC was used to detect the expression levels of p-AMPK, CAMKK2, GSS, GPX4 and SLC7A11 proteins in the colorectal tissues of each group of mice induced by AOM / DSS. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. It is worth noting that the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1

[0022] 1. Methods and Materials

[0023] 1.1 Chemicals and Reagents

[0024] Oregano oil with a purity of 99% was provided by Ji'an Red Apple Natural Spice Oil Co., Ltd., dissolved in olive oil and stored at 4°C. Azoxymethane (AOM), dextran sulfate sodium (DSS), and 5-Fu were provided by Shanghai Sigma High-Tech Co., Ltd. MTT kit, paraformaldehyde fixative, RNA extraction kit, reverse transcription kit, primers, primary antibody, and secondary antibody were all provided by Wuhan Saiweier Biotechnology Co., Ltd.

[0025] 1.2 Construction of AOM / DSS mice and drug treatment methods

[0026] After the purchased C57BL / 6 animals were adaptively fed for 1 week, the model was constructed. Among them, 10 healthy mice were used as the blank control group (NC group) and were gavaged with normal saline. A colorectal cancer model associated with colorectal inflammation was constructed with 40 mice, which were randomly divided into a model group (AOM / DSS), a low-dose oregano oil group (OV 50 mg / kg), a high-dose oregano oil group (OV 100 mg / kg), and a positive group (5-Fu20 mg / kg), with 10 mice in each group. Except for the blank control group, mice in other groups were intraperitoneally injected with 12.5 mg / kg of AOM. After 1 week of feeding, except for the blank control group, the other groups drank 2.5% DSS sterile water for 7 days, and then drank normal water for 14 days. This was a DSS cycle. Three cycles were repeated to construct a mouse colorectal cancer model associated with colorectal inflammation. During the three DSS cycles, mice in the blank control group were gavaged with an equal amount of normal saline every day, and mice in the other groups were gavaged with corresponding doses of oregano oil or intraperitoneally injected with 5-Fu.

[0027] 1.3 Cell culture

[0028] Colorectal cancer cell HCT116 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). The cells were cultured in DMEM complete culture medium supplemented with 10% fetal bovine serum, 100 mg / mL streptomycin, and 100 mg / mL penicillin. Both cells were kept at 37°C and 5% CO. 2 The cells were incubated in a cell culture incubator. The cell status was photographed under an inverted microscope using bright field.

[0029] 1.4 Statistical analysis

[0030] All experiments included in this study were repeated ≥ 3 times, and the experimental data were expressed as "mean ± standard deviation" (mean ± SD). IBM SPSS Statistics 19 software was used for statistical analysis, and one-way ANONA was used for inter-group difference analysis. P < 0.05 indicated statistical difference, and P < 0.01 indicated significant statistical difference.

[0031] 2. Detection indicators

[0032] 2.1 H&E staining

[0033] The colorectum of the mouse was cut longitudinally and rolled into a "Swiss roll", fixed with 4% paraformaldehyde, and embedded in paraffin at 58°C. After the paraffin solidified, the fixed wax block was placed in a rotary microtome and cut into 5-μm thick tissue sections, which were fixed on positively charged slides, stained with hematoxylin and eosin using standard procedures, and then observed under an optical microscope for histopathological analysis.

[0034] 2.2IHC technology

[0035] After dewaxing with xylene, ethanol, and 70% ethanol, the tissue sections were incubated with citrate antigen retrieval buffer and treated with 3% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. After blocking with 3% bovine serum albumin (BSA) solution, the sections were incubated with primary antibodies against p-AMPK (1:100), CAMKK2 (1:100), GSS (1:100), GPX4 (1:100), and SLC7A11 (1:100) at 4°C. The next day, the sections were incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies at room temperature for 1 h, developed with chromogenic 3,3'-diaminobenzidine (DAB) solution, and then counterstained with hematoxylin for nuclei and observed under an optical microscope.

[0036] 2.3ELISA kit

[0037] Collect blood from the inner canthus of the mouse eye, let it stand at low temperature for 30 minutes, put it in a centrifuge at 4°C, centrifuge it at 4000×g for 15 minutes, and obtain the supernatant. Take 10-20 mg of colorectal tissue, add normal saline according to the ratio of colorectal tissue: normal saline (1:9), lyse the colorectal tissue on ice with an electric homogenizer, centrifuge it at 4°C, 4000×g for 15 minutes, and obtain the supernatant. According to the instructions of the ELISA kit, incubate the antibody in a 96-well plate, add the sample and standard for processing, draw a standard curve, and calculate the content of cAMP and AMPK in the sample based on it.

[0038] 2.4 RT-qPCR

[0039] After extracting total RNA according to the kit requirements, immediately measure the RNA concentration of the sample in the NanoDrop nucleic acid protein concentration meter. Referring to the kit instructions, incubate at 42°C for 30 minutes (reverse transcription), incubate at 80°C for 10 minutes (reverse transcriptase inactivation), and incubate at 4°C for 5 minutes (termination reaction) to obtain the first-strand cDNA. Combined with the PCR kit instructions, pre-denaturation was performed at 95°C for 2 minutes, and amplification was performed at 95°C for 15 seconds and 60°C for 30 seconds for 40 cycles. β-actin was used as the internal reference gene, according to formula 2 (－△△Ct) Calculate the relative expression of mRNA.

[0040] Table 1 Mouse RT-qPCR primer sequences

[0041]

[0042]

[0043] 2.5 MTT experiment

[0044] The cells were inoculated in a 96-well plate, and a control group, a 5-Fu group (20 μg / mL), and groups treated with different doses of oregano oil (0-2000 μg / mL) were set up, with 6 replicate wells in each group. At the same time, a cell-free well was set up as a blank control to eliminate the interference caused by background factors of the culture plate. The drug was administered 24 hours after inoculation, and MTT solution was added to the culture medium of each group 24 hours after administration, and the plates were cultured in an incubator for 4 hours. Then, 150 μL of DMSO solution was added, and the plates were shaken at a low speed on a shaker for 10 minutes away from light. The plates were placed on a preheated microplate reader to detect the absorbance at 450 nm, and the IC value of oregano oil against colorectal cancer cells HCT116 was calculated. 50 to determine subsequent dosing.

[0045] 2.6 Cell cloning

[0046] The cells were cultured at 1×10 3 The cells were inoculated with a density of 100 μg / mL in 6-well plates, and then treated with 12.5 μg / mL, 25 μg / mL, 50 μg / mL of oregano oil and 20 μg / mL of 5-Fu prepared with DMSO, respectively. The drug-containing culture medium was replaced every 3 days, and the drug was administered continuously for 14 days. Finally, 1 mL of 4% paraformaldehyde was added to the cells to fix the cells for 15 minutes, and then crystal violet was added for staining for 15 minutes, and the cells were washed 3 times with PBS. The cell colony formation was photographed under white light and the statistics were recorded.

[0047] 3. Results

[0048] 3.1 In vivo efficacy study of oregano oil against colorectal cancer

[0049] In an in vivo study of the AOM / DSS-induced mouse colorectal cancer model, oregano oil was found to alleviate the weight loss of mice caused by AOM / DSS ( Figure 1 A) and reduced the mortality rate of mice ( Figure 1 B). After the experiment, the mice's colon and rectum were removed to observe the formation of intestinal tumors and measure the length of the intestinal tract. It was found that oregano oil could significantly reduce the formation of tumors in the intestine and inhibit the shortening of the length of the colon and rectum ( Figure 1 C). H&E pathological staining results clearly showed that obvious crypt foci and inflammatory infiltration lesions were formed in the intestine of AOM / DSS mice, and the administration of oregano oil could significantly alleviate the above symptoms ( Figure 1 D).

[0050] 3.2 In vitro efficacy study of oregano oil against colorectal cancer

[0051] In vitro experimental studies on HCT116 cells found that oregano oil can inhibit the growth of colorectal cancer cells in a dose-dependent manner. The IC 50 57.41 μg / mL ( Figure 1 E), so the subsequent drug administration concentrations in the cell experiment were set at 12.5μg / mL, 25μg / mL, and 50μg / mL. The effect of oregano oil on the morphology of colorectal cancer cells was observed under an inverted microscope. It was found that after the administration of oregano oil, colorectal cancer cells began to shrink and become round, and cell death / apoptosis occurred ( Figure 1 F). Cell cloning experiments found that oregano oil treatment can significantly inhibit cell cloning ( Figure 1 G) In summary, oregano oil can produce anti-colorectal cancer effects.

[0052] 3.3 Study on the mechanism of oregano oil against colorectal cancer

[0053] The ELISA kit was used to detect the serum of each group of mice induced by AOM / DSS, and it was found that oregano oil could increase the levels of serum cAMP and AMPK, suggesting that the anti-colorectal cancer effect of oregano oil may be related to the cAMP / AMPK signaling pathway ( Figure 2 A). Further, the above speculation was verified by RT-qPCR and IHC, and it was found that oregano oil can reduce the abundance of OR51E2 gene mRNA, increase the abundance of PRKAA1, PRKAA2, and CAMKK2 gene mRNA, and reduce the abundance of GSS, GPX4, and SLC7A11 gene mRNA ( Figure 2 B, C). At the same time, IHC was used to detect the expression levels of related proteins, and the results were consistent with the RT-qPCR results ( Figure 2 D), indicating that oregano oil can mediate OR51E2 to regulate the cAMP / AMPK signaling pathway to induce cell ferroptosis and fight colorectal cancer.

[0054] 4. Conclusion

[0055] The present invention discovers for the first time that oregano oil has an anti-colorectal cancer effect, and verifies it through in vivo and in vitro efficacy tests. The experimental results show that oregano oil can alleviate the weight loss of mice caused by AOM / DSS, reduce mortality, significantly inhibit the growth of colorectal cancer tumor cells, and induce the death of colorectal cancer tumor cells. Further studies show that oregano oil can regulate the cAMP / AMPK signaling pathway through OR51E2 to induce cell ferroptosis to resist colorectal cancer. The above results show that oregano oil can be used as a new drug for the treatment of colorectal cancer, providing a new idea for the treatment of colorectal cancer.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of oregano oil in the preparation of drugs for the treatment of colorectal cancer.

2. Application of oregano oil in the preparation of drugs for inhibiting colorectal cancer cells HCT116.

3. Application of oregano oil in the preparation of drugs for increasing the expression levels of cAMP and AMKP.

4. Application of oregano oil in the preparation of drugs for inhibiting OR51E2 gene expression level.

5. Use of oregano oil in the preparation of a drug for increasing the expression level of PRKAA1 and / or PRKAA2 and / or CAMKK2 genes.

6. Use of oregano oil in the preparation of drugs for inhibiting the expression level of GSS and / or GPX4 and / or SLC7A11 genes.

7. The use according to any one of claims 1 to 6, characterized in that: Oregano oil and olive oil are mixed evenly in a mass ratio of 1:9-19.

8. The use according to any one of claims 1 to 6, characterized in that: Oregano oil is a single essential oil with a purity of more than 99% available on the market, or oregano oil with a purity of more than 99% obtained by extracting oregano using supercritical carbon dioxide extraction.

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