Application of patchouli oil in medicine

Abstract

The invention relates to application of patchouli oil in medicines. According to the application of the patchouli oil in preparation of the medicine for treating the colorectal cancer, the patchouli oil and olive oil are used after being uniformly mixed according to the mass ratio of 1: 9-1: 19. The patchouli oil can relieve weight loss, caused by azomethane oxide / dextran sodium sulfate, of mice and reduce the death rate, proliferation of colorectal cancer tumor cells can be remarkably inhibited, and death of the colorectal cancer tumor cells is induced.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of patchouli oil in medicines. Background Art

[0002] Patchouli is the dried aerial part of the plant Pogostemon cablin (Blanco) Benth. of the Lamiaceae family. It is pungent in taste and slightly warm in nature. It enters the spleen, stomach, and lung meridians. It has the effects of aromatic turbidity removal, nausea and vomiting, and heat relief. It has unique advantages in the treatment of gastrointestinal diseases. Patchouli oil is a fat-soluble substance extracted from patchouli. The active ingredients are patchouli alcohol, patchouli ketone, patchoulene, etc. It has multiple pharmacological activities such as antioxidant, antibacterial, immune regulation and anti-inflammatory. It is widely used in animal production, food analysis, medicine and health, and daily necessities. Studies have shown that patchouli oil has a certain inhibitory effect on malignant tumor cells such as liver cancer and lung cancer. Patchouli oil has certain application prospects in anti-cancer.

[0003] 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 latest malignant tumor registration data show that the incidence of CRC in my country is second only to lung cancer, and the mortality rate ranks fourth, with higher male than female, and higher urban than rural characteristics. Studies have shown that the incidence of CRC is tending to be younger, so early screening, early diagnosis, and early treatment of CRC 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 reducing tumor size and prolonging survival, but at the same time lead to a series of adverse reactions. Studies have shown that the active ingredients of patchouli can be used to protect the intestinal mucosal barrier, repair damaged intestinal segments, promote gastrointestinal motility and secretion, regulate intestinal microbiota, relieve gastrointestinal discomfort, regulate immune response and inflammatory factor levels, and have antibacterial effects, and have the potential to be used in the prevention and treatment of colorectal cancer. Summary of the invention

[0004] The purpose of the present invention is to provide an application of patchouli oil in medicine to solve the technical problem of large side effects when colorectal cancer is treated by radiotherapy, chemotherapy, surgery, targeted therapy and immunotherapy.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The invention discloses an application of patchouli oil in medicine, and an application of the patchouli oil in preparing a medicine for treating colorectal cancer. The patchouli oil and olive oil are mixed evenly in a mass ratio of 1:9 to 1:19 and then used.

[0007] Furthermore, the patchouli oil is used in the preparation of a drug for inhibiting colorectal cancer cells HCT116.

[0008] Furthermore, the patchouli oil is used in the preparation of drugs for increasing the expression levels of cAMP and AMKP.

[0009] Furthermore, the patchouli oil is used in the preparation of a drug for increasing the expression level of the OR13G1 gene.

[0010] Furthermore, the patchouli oil is used in the preparation of a drug for increasing the expression level of PRKAA1 and / or PRKAA2 and / or CAMKK2 genes.

[0011] Furthermore, the patchouli oil is used in the preparation of a drug for inhibiting the expression level of GSS and / or GPX4 and / or SLC7A11 genes.

[0012] Furthermore, the purity of the patchouli oil is above 99%.

[0013] Beneficial effects of the present invention:

[0014] Patchouli oil can alleviate the weight loss and mortality of mice induced by azoxymethane (AOM) / dextran sulfate sodium (DSS), and can significantly inhibit the proliferation of colorectal cancer cells and induce the death of colorectal cancer cells.

[0015] Patchouli oil can significantly increase the expression levels of cAMP and AMPK in the serum of AOM / DSS mice, significantly inhibit the expression of GSS, GPX4, and SLC7A11 gene mRNA in colorectal tissue, and increase the expression of OR13G1, PRKAA1, PRKAA2, and CAMKK2 gene mRNA. Patchouli oil intervention can regulate the expression of cAMP / AMPK signaling pathway and ferroptosis-related proteins, indicating that patchouli oil can regulate the cAMP / AMPK signaling pathway through OR13G1 to induce cell ferroptosis and resist colorectal cancer. The present invention provides data support for patchouli oil as a new drug for the treatment of colorectal cancer, and provides a new idea for the treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1is a graph showing the anti-colorectal cancer effect of patchouli oil in Example 1, wherein A is a graph showing the weight change of mice during the experiment, B is a graph showing the mortality rate of mice during the experiment, C is a graph showing the actual length of the mouse colon under different treatments, D is a graph showing the length of the mouse colon under different treatments, E is a graph showing the results of H&E staining of the mouse colon tissue under different treatments, F is a graph showing the proliferation rate of colorectal cancer cells HCT116 under treatment with different doses of patchouli oil, G is a graph showing the effect of patchouli oil on the morphology of colorectal cancer cells observed under an inverted microscope, and H is a graph showing the results of a cell cloning experiment, *P<0.05, **P<0.01;

[0017] Figure 2 Figure 1 is a study on the mechanism of patchouli oil against colorectal cancer in Example 1, wherein A is a graph showing the changes in cAMP and AMPK expression levels in mouse serum induced by AOM / DSS, B is a graph showing the mRNA levels of OR13G1, PRKAA1, PRKAA2, and CAMKK2 genes in colorectal tissue of mice induced by AOM / DSS, C is a graph showing the mRNA levels of GSS, GPX4, and SLC7A11 genes in colorectal tissue of mice induced by AOM / DSS, and D is a graph showing the expression of p-AMPK, CAMKK2, GSS, GPX4, and SLC7A11 proteins in colorectal tissue of mice induced by AOM / DSS, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0019] Patchouli 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, sodium dextran sulfate, 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. Colorectal cancer cells HCT116 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0020] AOM / DSS mouse construction method and drug treatment method: After purchasing C57BL / 6 animals and feeding them adaptively for 1 week, the model was constructed. Among them, 10 healthy mice were used as the blank control group (NC) and were gavaged with normal saline. 40 mice were used to construct a colorectal cancer model associated with colorectal inflammation, and were randomly divided into a model group (AOM / DSS), a low-dose patchouli oil group (PC 50mg / kg), a high-dose patchouli oil group (PC 100mg / kg), and a positive group (5-Fu 20mg / kg), with 10 mice in each group. Except for the blank control group, the mice in the other groups were intraperitoneally injected with 12.5mg / kg of AOM. After 1 week of feeding, except for the blank control group, the mice in the other groups drank 2.5% DSS sterilized water for 7 days, and then drank normal water for 14 days continuously. This was a DSS cycle, and 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 normal saline every day, and mice in the other groups were gavaged with corresponding doses of patchouli oil or intraperitoneally injected with 5-Fu. Mice in the high-dose patchouli oil group and the low-dose patchouli oil group were gavaged with patchouli oil mixed with olive oil at a mass ratio of 1:9 and 1:19, respectively.

[0021] Colorectal cancer cell HCT116 culture: Colorectal cancer cell HCT116 was cultured in DMEM complete culture medium supplemented with 10% fetal bovine serum, 100 mg / mL streptomycin and 100 mg / mL penicillin. Colorectal cancer cell HCT116 was incubated in a cell culture incubator at 37°C and 5% CO2. The cell state was photographed in bright field under an inverted microscope.

[0022] H&E staining: The colorectum of the mouse was cut longitudinally and rolled into a "Swiss roll", fixed with 4% paraformaldehyde fixative, 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.

[0023] IHC technique: 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 (volume ratio 1:100), CAMKK2 (volume ratio 1:100), GSS (volume ratio 1:100), GPX4 (volume ratio 1:100), and SLC7A11 (volume ratio 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, counterstained with hematoxylin for nuclei, and observed under an optical microscope.

[0024] ELISA kit: 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 colorectal tissue: normal saline (volume ratio 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.

[0025] RT-qPCR: After extracting total RNA according to the requirements of the kit, 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-chain cDNA. Combined with the PCR kit instructions, pre-denaturation was performed at 95°C for 2 minutes, and amplification was performed for 40 cycles at 95°C for 15 seconds and 60°C for 30 seconds. Use β-actin as the internal reference gene, and according to the CT value obtained from the RT-qPCR experiment, according to formula 2 (－△△Ct) Calculate the relative expression of mRNA.

[0026] Table 1 Mouse RT-qPCR primer sequences

[0027]

[0028] Example 1

[0029] In vivo efficacy of patchouli oil against colorectal cancer

[0030] from Figure 1As shown in A and 1B, patchouli oil can alleviate the weight loss of mice caused by AOM / DSS and reduce the mortality of mice. After the experiment, the colorectum of the mice was removed to observe the formation of intestinal tumors and measure the length of the intestinal tube. Figure 1 As shown in C and 1D, patchouli oil can significantly reduce the formation of tumors in the intestine and inhibit the shortening of colorectal length. Figure 1 E It can be seen that obvious crypt foci and inflammatory infiltration lesions are formed in the intestine of AOM / DSS mice, and the above symptoms can be significantly alleviated after administration of patchouli oil.

[0031] Example 2

[0032] In vitro efficacy of patchouli oil against colorectal cancer

[0033] from Figure 1 As can be seen from Figure F, patchouli oil can inhibit the growth of colorectal cancer cells in a dose-dependent manner. The IC 50 The concentration of 90.98 μg / mL was used for the subsequent cell experiments, so the concentrations were set at 25 μg / mL, 50 μg / mL, and 100 μg / mL. Figure 1 As can be seen in G, after the administration of patchouli oil, the colorectal cancer cells began to shrink and become round, and cell morphology changed. Figure 1 As can be seen from H, patchouli oil can significantly inhibit cell cloning after treatment. In summary, patchouli oil can produce anti-colorectal cancer effects.

[0034] Example 3

[0035] Study on the mechanism of patchouli oil against colorectal cancer

[0036] The serum of each group of mice induced by AOM / DSS was detected by ELISA kit. Figure 2 A shows that patchouli oil can increase serum cAMP and AMPK levels, indicating that the anti-colorectal cancer effect of patchouli oil may be related to the cAMP / AMPK signaling pathway. Figure 2 As can be seen from B and 2C, patchouli oil can increase the mRNA abundance of OR13G1 gene, increase the mRNA abundance of PRKAA1, PRKAA2, and CAMKK2 genes, and reduce the mRNA abundance of GSS, GPX4, and SLC7A11 genes. Figure 2 D It can be seen that the results are consistent with the RT-qPCR results, indicating that patchouli oil can mediate OR13G1 to regulate the cAMP / AMPK signaling pathway to induce cell ferroptosis and fight colorectal cancer.

Claims

1. An application of patchouli oil in medicine, characterized in that: The patchouli oil is used in the preparation of a drug for treating colorectal cancer, wherein the patchouli oil and olive oil are evenly mixed in a mass ratio of 1:9-1:19 and then used.

2. The use of patchouli oil in medicine according to claim 1, characterized in that: The patchouli oil is used in the preparation of a drug for inhibiting colorectal cancer cells HCT116.

3. The use of patchouli oil in medicine according to claim 1, characterized in that: The patchouli oil is used in preparing a drug for increasing the expression levels of cAMP and AMKP.

4. The use of patchouli oil in medicine according to claim 1, characterized in that: The patchouli oil is used in the preparation of a drug for improving the expression level of the OR13G1 gene.

5. The use of patchouli oil in medicine according to claim 1, characterized in that: The patchouli oil is used in the preparation of a drug for increasing the expression level of PRKAA1 and / or PRKAA2 and / or CAMKK2 genes.

6. The use of patchouli oil in medicine according to claim 1, characterized in that: The patchouli oil is used in the preparation of a drug for inhibiting the expression level of GSS and / or GPX4 and / or SLC7A11 genes.

7. Use of patchouli oil in medicine according to any one of claims 1 to 6, characterized in that: The purity of the patchouli oil is above 99%.

Images