Use of patchouli oil or patchouli alcohol in the preparation of a trpv channel modulator

Abstract

The application discloses a use of patchouli oil or patchouli alcohol in preparation of a TRP channel modulator. The patchouli oil or patchouli alcohol has a function of regulating a TRP channel, and particularly has a synergistic effect with a TRPM2 inhibitor in anti-inflammatory effect, and is a promising TRPM2 inhibitor. In addition, the patchouli alcohol or patchouli oil has a function of improving colitis-related injury, and can inhibit expression of IL-1beta, TNF-alpha, IL-6, TRPM2, TRPM6 and TRPM7 genes.

Description

Technical Field

[0001] This invention relates to the fields of medicine and cosmetics. Specifically, this invention relates to the use of patchouli oil or patchouli alcohol or compositions comprising therein in the preparation of TRP channel modulators, and their use in the preparation of medicaments for treating, preventing and / or improving diseases or symptoms associated with TRP channel abnormalities. Background Technology

[0002] The TRP family is an important membrane channel protein that functions as a molecular receptor. It can be activated by various physical and chemical stimuli, thereby transmitting the sensation of pain / itch. TRP channels play a crucial role in both health and disease. Currently, drug development targeting TRP channels has expanded beyond early pain relief to include new clinical indications such as inflammation and pain. Activation of TRP channels can mediate the release of neuropeptides, such as CGRP and SP, thereby triggering an inflammatory response. Furthermore, TRP channels are expressed in skin keratinocytes, intestinal epithelial cells, and various immune cells, regulating phagocytic activity and cytokine secretion, and participating in various pathophysiological processes related to the immune microenvironment, skin homeostasis, itching and pain, and barrier integrity. Therefore, TRP channels are closely related to inflammatory bowel disease (IBD), visceral hypersensitivity (VHS), intestinal fibrosis and gut microbiota, rosacea, atopic dermatitis, psoriatic sensitive skin, and other conditions. TRP channels are a class of ion channel proteins, including multiple subtypes such as TRPC, TRPV, TRPM, TRPA, and TRPP. TRPCs are involved in the regulation of calcium ion influx, TRPVs are thermosensing channels, TRPMs regulate calcium concentration and temperature perception, TRPAs are chemoreceptors, and TRPPs are associated with polycystic kidney disease. They play important roles in cell physiology, sensory transmission, and pain regulation. A deeper understanding of the types and functions of TRP channels will help to further understand their roles in physiological and pathological processes. Current TRP channel inhibitors face challenges including specificity and selectivity issues, lack of high efficacy and reversibility, insufficient pharmacokinetic and pharmacochemical studies, unclear mechanisms of action, and limitations in drug delivery and feasibility. Despite these challenges, research and development of TRP channels remain crucial. With continuous research and technological advancements, it is believed that more specific, efficient, and controllable TRP channel inhibitors can be developed, accelerating their application in disease treatment.

[0003] Inflammation bowel disease (IBD) is a chronic intestinal disease affecting the immune system. It is divided into Crohn's disease and ulcerative colitis. Both are inflammatory diseases caused by nonspecific infections leading to intestinal mucosal lesions and related tissue destruction. IBD is often accompanied by skin problems or diseases, which is related to the interaction between the skin and gut (gut-skin axis). Therefore, treating the gut can have beneficial effects on the skin. The gut and skin are closely related, influencing each other through the gut-skin axis, which is regulated by diet, microbial metabolites, neuroendocrine pathways, and the central nervous system. For example, gluten in the diet can affect skin health, causing food allergies, celiac disease, and psoriasis. Conversely, skin exposure to UVB can indirectly increase serum vitamin D levels and gut microbiota diversity. Disorders of the intestinal mucosal immune function directly affect the skin, making psoriasis more likely. Furthermore, pain caused by pathological changes in internal organs may project to specific skin areas. In summary, skin and intestinal diseases are closely linked in two ways: first, intestinal dysfunction and even structural damage can be caused by skin conditions; second, both the gut and skin can be affected by the same processes. When the gut and skin are compromised, nerve sensitivity and inflammation are often important manifestations.

[0004] Patchouli is the dried aerial part of *Pogostemon cablin* (Blanco) Benth., a plant in the Lamiaceae family. Patchouli oil (PO) is the effective anti-inflammatory component of patchouli, mainly containing patchouli alcohol, patchouli ketone, and patchoulene, with patchouli alcohol being the most abundant (the 2020 edition of the Chinese Pharmacopoeia stipulates that it should contain no less than 26% patchouli alcohol). Initially, patchouli was known as a spice and was widely loved for its unique aroma. It was first recorded as a medicinal herb in the *Mingyi Bielu* (Records of Famous Physicians) from the Northern and Southern Dynasties period, stating that it is "slightly warm, treats edema caused by wind and dampness, removes foul odors, and stops cholera and abdominal pain." Subsequently, various ancient medical texts, such as *Bencao Jing Jizhu* (Collected Annotations on the Materia Medica), *Xinxiu Bencao* (Newly Revised Materia Medica), *Bencao Shiyi* (Supplement to the Materia Medica), and *Jiayou Bencao* (Jiayou Materia Medica), all adopted this record. In his "Illustrated Materia Medica" from the Song Dynasty, Su Songzhi considered agastache to be an essential medicine for treating vomiting and nausea. Summary of the Invention

[0005] The inventors discovered through research that patchouli oil and patchouli alcohol have the effect of regulating TRP channels, especially exhibiting a synergistic effect with TRPM2 inhibitors in anti-inflammatory and other functions, making them promising TRPM inhibitors. Furthermore, patchouli alcohol and patchouli oil can improve colitis-related damage and inhibit the expression of IL-1β, TNF-α, IL-6, TRPM2, TRPM6, and TRPM7 genes. Based on this, the technical solution of this invention is as follows: A first aspect of the invention provides the use of patchouli oil or patchouli alcohol in the preparation of pharmaceuticals, reagents, or cosmetics having one or more of the following effects: a) TRPM channel inhibitors; b) Inhibit calcium ion influx into macrophages; c) Inhibit the upregulation of TRPM2, TRPM6 and / or TRPM7 gene expression; d) Inhibit the release of IL-1β, TNF-α, IL-6 and / or upregulate the expression of IL-17A; e) Inhibit the downregulation of IκBα expression; f) Treating, preventing, and / or improving symptoms of inflammation-related bowel disease or itching, pain, or sensitivity of the skin; preferably, the inflammation-related bowel disease or itching, pain, or sensitivity of the skin is associated with an abnormality of the TRPM channel, particularly the TRPM2, TRPM6, or TRPM7 channel.

[0006] Preferably, for the above-mentioned use a), the TRPM channel is TRPM2, 6 or 7, especially TRPM2.

[0007] Preferably, in use b) above, the calcium ion influx is induced by lipopolysaccharide or caused by disease.

[0008] Preferably, for the above-mentioned use (c), the upregulation of TRPM2, TRPM6 and / or TRPM7 gene expression is induced by lipopolysaccharide or caused by disease.

[0009] Preferably, for the above-mentioned use d), it specifically inhibits the release or upregulation of IL-1β, TNF-α, IL-6 and / or IL-17A in macrophages; preferably, the release or upregulation of IL-1β, TNF-α, IL-6 and / or IL-17A is induced by lipopolysaccharide or caused by disease.

[0010] Preferably, for the above-mentioned use (e), it specifically inhibits the downregulation of IκBα expression in macrophages; preferably, the downregulation of IκBα expression is induced by lipopolysaccharide or caused by disease.

[0011] Preferably, in the above uses (b)-e), the disease is selected from colitis, and is one or more diseases with symptoms of itching, pain or sensitivity of the skin, preferably colitis.

[0012] Preferably, according to the above-mentioned uses, patchouli oil can be applied in the form of patchouli oil gel, wherein the raw materials of the gel include the following components by weight: 5 parts patchouli oil, 3-5 parts polyethylene glycol glyceryl laurate (GELUCIRE 50 / 13), 0.5-2 parts vitamin E polyethylene glycol succinate (VE-TPGS), and 0.05-0.2 parts SiO2; The gel preparation method is as follows: Heat and melt the prescribed amounts of polyethylene glycol glyceride and vitamin E polyethylene glycol succinate, add the prescribed amount of patchouli oil, and stir until uniform. After stirring until uniform, add the prescribed amount of SiO2 and continue stirring until uniform, resulting in a brownish-yellow liquid. After cooling, patchouli oil gel is obtained.

[0013] A second aspect of the invention provides a composition comprising: (1) patchouli oil or patchouli alcohol; (2) a TRPM2 channel inhibitor; and (3) optional pharmaceutically acceptable excipients, or a carrier or excipient for a cosmetic.

[0014] In some embodiments, the TRPM2 channel inhibitor is one or more selected from ML-SI3, JNJ-28583113, and rosiglitazone maleate, preferably ML-SI3.

[0015] According to the above composition, in some embodiments, the mass ratio of patchouli oil or patchouli alcohol to TRPM2 channel inhibitor is 1:0.01 to 99.99, preferably 1:0.5 to 20, more preferably 1:1 to 5, for example 1:2 to 3 or 1:2.5.

[0016] A third aspect of the invention provides the use of the above-described composition in the preparation of a pharmaceutical, reagent, or cosmetic having one or more of the following effects: a) TRPM channel inhibitors; b) Inhibit calcium ion influx into macrophages; c) Inhibit the upregulation of TRPM2, TRPM6 and / or TRPM7 gene expression; d) Inhibit the release of IL-1β, TNF-α, IL-6 and / or upregulate the expression of IL-17A; e) Inhibit the downregulation of IκBα expression; f) Treating, preventing, and / or improving symptoms of inflammation-related bowel disease or itching, pain, or sensitivity of the skin; preferably, the inflammation-related bowel disease or itching, pain, or sensitivity of the skin is associated with an abnormality of the TRPM channel, particularly the TRPM2, TRPM6, or TRPM7 channel.

[0017] Preferably, for the above-mentioned use a), the TRPM channel is TRPM2, 6 or 7, especially TRPM2.

[0018] Preferably, in use b) above, the calcium ion influx is induced by lipopolysaccharide or caused by disease.

[0019] Preferably, for the above-mentioned use (c), the upregulation of TRPM2, TRPM6 and / or TRPM7 gene expression is induced by lipopolysaccharide or caused by disease.

[0020] Preferably, for the above-mentioned use d), it specifically inhibits the release or upregulation of IL-1β, TNF-α, IL-6 and / or IL-17A in macrophages; preferably, the release or upregulation of IL-1β, TNF-α, IL-6 and / or IL-17A is induced by lipopolysaccharide or caused by disease.

[0021] Preferably, for the above-mentioned use (e), it specifically inhibits the downregulation of IκBα expression in macrophages; preferably, the downregulation of IκBα expression is induced by lipopolysaccharide or caused by disease.

[0022] Preferably, in the above uses (b)-e), the disease is selected from colitis, and is one or more diseases with symptoms of itching, pain or sensitivity of the skin, preferably colitis.

[0023] A fourth aspect of the present invention provides the use of patchouli oil or patchouli alcohol in improving colitis-related damage and / or inhibiting the expression of IL-1β, TNF-α, IL-6, TRPM2, TRPM6 or TRPM7 genes in a zebrafish model of enteritis. Preferably, the zebrafish model of enteritis is constructed by induction with trinitrobenzenesulfonic acid. Attached Figure Description

[0024] Figure 1 The results of the effects of patchouli oil, patchouli alcohol, TRPM2 channel inhibitor, TRPM8 channel inhibitor, and combination of drugs on LPS-induced calcium ion influx in macrophages are shown in Example 1.

[0025] Figure 2 The anti-inflammatory effects of patchouli oil, patchouli alcohol, TRPM2 channel inhibitors, TRPM8 channel inhibitors, and the combination of drugs on inflammatory macrophages were demonstrated in Example 1 by RT-PCR detection.

[0026] Figure 3 The anti-inflammatory effects of patchouli oil, patchouli alcohol, TRPM2 channel inhibitors, TRPM8 channel inhibitors, and the combination of drugs on inflammatory macrophages were demonstrated by ELISA in Example 1.

[0027] Figure 4 Typical intestinal area diagrams of TNBS-induced zebrafish colitis model induced by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 3 are shown.

[0028] Figure 5 The results of the statistical analysis of intestinal area in a TNBS-induced zebrafish colitis model using patchouli oil, patchouli alcohol, and patchouli oil gel in Example 3 are shown.

[0029] Figure 6 Typical images of intestinal macrophages in a TNBS-induced zebrafish colitis model are shown in Example 4, using patchouli oil, patchouli alcohol, and patchouli oil gel.

[0030] Figure 7 The results of the statistical analysis of the number of intestinal macrophages in a TNBS-induced zebrafish colitis model using patchouli oil, patchouli alcohol, and patchouli oil gel in Example 4 are shown.

[0031] Figure 8 The diagram shows a typical number of goblet cells in a TNBS-induced zebrafish colitis model induced by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 5.

[0032] Figure 9 The results of the statistical analysis of the number of intestinal goblet cells in a TNBS-induced zebrafish colitis model using patchouli oil, patchouli alcohol, and patchouli oil gel in Example 5 are shown.

[0033] Figure 10 Typical intestinal pathological sections in a TNBS-induced zebrafish colitis model are shown in Example 6, using patchouli oil, patchouli alcohol, and patchouli oil gel.

[0034] Figure 11 The statistical results of the relative expression levels of IL-1β mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown.

[0035] Figure 12 The statistical results of the relative expression levels of TNF-α mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown.

[0036] Figure 13 The statistical results of the relative expression levels of IL-6 mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown.

[0037] Figure 14The statistical results of the relative expression levels of TRPM2 mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown.

[0038] Figure 15 The statistical results of the relative expression levels of TRPM6 mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown.

[0039] Figure 16 The statistical results of the relative expression levels of TRPM7 mRNA in a TNBS-induced zebrafish colitis model by patchouli oil, patchouli alcohol, and patchouli oil gel in Example 7 are shown. Detailed Implementation

[0040] To better illustrate the present invention, the following detailed description, in conjunction with embodiments, will provide a clear and complete picture, but these should not be construed as limiting the scope of protection of the present invention. Based on the embodiments of the present invention, those skilled in the art can make various improvements and adjustments to the implementation schemes within the spirit and substance of the present invention.

[0041] Example In the following examples, the materials / reagents and instruments / equipment used are listed in Tables 1-2 below.

[0042] Table 1. Materials and Reagents

[0043] Among them, ML-SI3 is a TRPM2 inhibitor, CAS number: 891016-02-7, and its structure is as follows: .

[0044] AMG-333 is a TRPM8 antagonist, CAS number: 1416799-28-4, its structure is as follows: .

[0045] The preparation method of patchouli oil gel is as follows: Melt 4 parts of polyethylene glycol glyceryl laurate (GELUCIRE 50 / 13) and 1 part of vitamin E polyethylene glycol succinate (VE-TPGS) by heating. Add 5 parts of patchouli oil and stir evenly with a stirring rod. After even mixing, add 0.1 parts of SiO2 and continue stirring until uniform, controlling the speed to avoid foaming. After even mixing, the mixture is a brownish-yellow liquid, which is then placed in a refrigerator to cool. After cooling, the preparation is a brownish-yellow, semi-transparent gel-like solid that retains its solid form at room temperature.

[0046] Macrophages (PBMC, RAW264.7) were provided by the Department of Biopharmaceuticals, School of Pharmacy, Fudan University.

[0047] Table 2. Instruments and Equipment

[0048] In the following examples, all data were statistically analyzed using Microsoft Excel and plotted using GraphPad 5.0 and SPSS 20.0. P<0.05, P<0.01, A p-value < 0.001 is considered statistically significant.

[0049] Example 1: Effects of patchouli oil and patchouli alcohol on TRP ion channels in inflammatory macrophages 1. Calcium ion detection Macrophages (PBMC, RAW264.7) were harvested, and the cell concentration was adjusted to 5 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates. After the cells adhered fully, the supernatant culture medium was removed. A negative control group (containing only culture medium) and a model group (containing culture medium containing 100 ng / mL lipopolysaccharide (LPS)) were set up. The experimental groups, in addition to the same amount of LPS as the model group, also contained patchouli oil (4 μg / mL), patchouli alcohol (4 μg / mL), ML-SI3 (10 μM), AMG-333 (1 μM), patchouli oil (4 μg / mL) + ML-SI3 (10 μM), patchouli alcohol (4 μg / mL) + ML-SI3 (10 μM), patchouli oil (4 μg / mL) + AMG-333 (1 μM), and patchouli alcohol (4 μg / mL) + AMG-333 (1 μM). After 24 h of incubation, cells were collected, and 200 μL of sample lysis buffer was added to each well for thorough lysis. Centrifuge at 12000g for 5 minutes at 4℃ and collect the supernatant. Prepare the standard solution and construct the standard curve according to the instructions of the calcium content colorimetric reagent kit (Beyotime, S1063S). Detect the calcium ion content in the sample. The detection results are shown in [Figure number missing]. Figure 1 .

[0050] In addition, the synergistic index (CI) was used to evaluate the synergistic relationship between drugs. In the experiment, the concentrations of patchouli oil (PO), patchouli alcohol (PA), ML-SI3, and AMG-333 were kept consistent in both the single-dose and combination groups: patchouli oil (4 μg / mL), patchouli alcohol (4 μg / mL), ML-SI3 (10 μM), and AMG-333 (1 μM). Therefore, the CI value was calculated based on the different effects produced by consistent dosage. The formula for calculating the synergistic index (CI) is as follows: CI=(DA +D B ) / D A+B Among them, D A D represents the efficacy index of drug A; B D represents the efficacy index of drug B. A+B The efficacy index of the combination of drugs A and B.

[0051] CI>1 indicates an antagonistic effect, CI=1 indicates an additive effect, and CI<1 indicates a synergistic effect.

[0052] The calculated synergistic index (CI) of patchouli oil or patchouli alcohol and ML-SI3 or AMG-333 is shown in Table 3.

[0053] Table 3. Synergistic Index (CI) of Patchouli Oil or Patchouli Alcohol with ML-SI3 or AMG-333

[0054] like Figure 1 As shown, in PBMC and RAW264.7 cells, the TRPM2 inhibitor ML-SI3 inhibited LPS-induced calcium ion influx compared to the TRPM8 inhibitor AMG-333, indicating that TRPM2 participates in LPS-induced calcium ion influx, and its inhibitor can suppress LPS-induced calcium ion influx. Patchouli oil and patchouli alcohol also inhibited LPS-induced calcium ion influx, and the inhibitory effect was better than that of ML-SI3, suggesting that patchouli oil and patchouli alcohol have excellent TRPM2 inhibitory effects. Furthermore, the inhibitory effect of patchouli oil and patchouli alcohol combined with ML-SI3 on ​​calcium ion influx was better than that of patchouli oil or patchouli alcohol alone. P<0.01, and P<0.001). Further analysis of Table 3 shows that patchouli oil or patchouli alcohol can produce a synergistic effect with the TRPM2 inhibitor ML-SI3, while the TRPM8 inhibitor AMG-333 cannot produce a synergistic effect with patchouli oil or patchouli alcohol.

[0055] 2. RT-PCR Healthy cells (PBMC, RAW264.7) were seeded into 12-well plates at a density of 5 × 10⁶ cells per well. 4 100 cells, 1 mL cell suspension. After cell adhesion, the culture medium was changed. Following section "1. Calcium Ion Detection," control, model, and experimental groups were set up with the same drug concentration. Total RNA was extracted from the cells after drug treatment. (Following SuperScript...) TMThe IVOne-Step RT-PCR Reverse Transcription Kit instructions describe the reverse transcription of RNA into cDNA. PCR primers were designed and synthesized by Sangon Biotech Co., Ltd., using primers retrieved from the GeneBank database, with GAPDH as an internal control. Results are shown below. Figure 2 .

[0056]

[0057] In addition, the synergistic index CI value was calculated using the same method as in section “1. Calcium Ion Detection”, and the calculation results are shown in Tables 4 and 5.

[0058] Table 4. Synergistic Index (CI) of Patchouli Oil or Patchouli Alcohol and ML-SI3

[0059] Table 5. Synergistic Index (CI) of Patchouli Oil or Patchouli Alcohol and AMG-3333

[0060] like Figure 2 As shown in the PCR results, the TRPM8 inhibitor AMG-333 could not inhibit LPS-induced IκBα mRNA expression in macrophages. However, compared with AMG-333, the TRPM2 inhibitor ML-SI3, when acted upon simultaneously with patchouli oil or patchouli alcohol, was more effective in increasing IκBα mRNA expression than either patchouli oil or patchouli alcohol alone. P<0.05, (P<0.01). Combined with the data in Tables 4 and 5, it is shown that patchouli oil or patchouli alcohol can have a synergistic effect with the TRPM2 inhibitor ML-SI3, while the TRPM8 inhibitor AMG-333 did not show a significant synergistic effect with patchouli oil or patchouli alcohol.

[0061] Compared with the control group, LPS increased the expression of IL-17A mRNA in macrophages PBMC and RAW264.7 in the model group; the TRPM8 inhibitor AMG-333 could not inhibit LPS-induced IL-17A mRNA expression in macrophages PBMC and RAW264.7, while ML-SI3 inhibited LPS-induced IL-17A mRNA expression in macrophages compared with AMG-333. Moreover, the effect of ML-SI3 when acting simultaneously with patchouli oil or patchouli alcohol was better than the effect of patchouli oil or patchouli alcohol acting alone. P<0.05, (P<0.01). Further analysis of Table 4 indicates that patchouli oil and patchouli alcohol can have a synergistic effect with the TRPM2 inhibitor ML-SI3.

[0062] 3. ELISA detection of cytokines Macrophages (PBMC, RAW264.7) were harvested, and the cell concentration was adjusted to 5 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates. After the cells adhered fully, the supernatant was removed. Following section "1. Calcium Ion Detection," control, model, and experimental groups were set up with the same drug concentration. After 24 h of incubation, the cell culture supernatant was collected and immediately analyzed. The OD value was the difference between the value measured at 450 nm and the value measured at 570 nm. The results are shown below. Figure 3 .

[0063] In addition, the synergistic index CI value was calculated using the same method as in “1. Calcium ion detection”, and the results are shown in Tables 6 and 7.

[0064] Table 6. Synergistic Index (CI) of Patchouli Oil or Patchouli Alcohol and ML-SI3

[0065] Table 7. Synergistic Index (CI) of Patchouli Oil or Patchouli Alcohol and AMG-333

[0066] like Figure 3 As shown in the ELISA results, compared with the control group, LPS in the model group significantly increased the release of TNF-α and IL-6 from macrophages. ML-SI3, patchouli oil, and patchouli alcohol could inhibit the LPS-induced release of TNF-α and IL-6 from macrophages, and the inhibitory effect of patchouli oil or patchouli alcohol when acting simultaneously with ML-SI3 was better than the effect of patchouli alcohol or patchouli oil alone. P<0.01). Further analysis of Tables 6 and 7 indicates that patchouli oil or patchouli alcohol has a synergistic effect with the TRPM2 inhibitor ML-SI3.

[0067] The TRPM8 inhibitor AMG-333 itself had almost the same effect as the model, and its combination with patchouli oil also had basically the same effect as patchouli oil or patchouli alcohol alone, with no obvious synergistic effect observed.

[0068] Example 2: Determination of Maximum Detectable Concentration (MTC) in a Zebrafish Model of Colitis 3 dpf transgenic neutrophil-positive green fluorescent MPX strain zebrafish (raised by Huante Biotechnology, with husbandry management meeting the requirements of international AAALAC certification (certification number: 001458)) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Drugs were administered in water-soluble form to the patchouli alcohol group, patchouli oil group, and patchouli oil gel group, with drug concentrations shown in Table 8. A normal control group (no drug treatment) and a model control group (administered TNBS in water-soluble form) were also set up. The experimental groups were also administered TNBS in water-soluble form to establish a zebrafish intestinal mucosal injury model. The volume of each well was 3 mL. After treatment at 28℃ for 2 days, the MTC of the drugs in the model zebrafish was measured. The results are shown in Table 8, indicating that under the experimental conditions, the maximum detectable concentration (MTC) of patchouli alcohol, patchouli oil, and patchouli oil gel in the colitis model zebrafish were 2.50, 10.0, and 20.0 μg / mL, respectively.

[0069] Table 8. Results of the experiment to determine the maximum detectable concentration of samples (n = 30)

[0070] Example 3: Evaluation of the efficacy of intestinal mucosal damage repair 3 dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected and placed in 6-well plates. Each well contained 30 zebrafish. The experimental groups were treated with patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL), respectively. The positive control group was treated with 500 µg / mL mesalazine. A normal control group (no drug treatment) and a model control group (treated with TNBS) were also set up. All experimental groups were also treated with TNBS to establish a zebrafish intestinal mucosal injury model. The volume of each well was 3 mL. After treatment at 28 ℃ for 2 days, 10 zebrafish were randomly selected from each group, photographed under a fluorescence microscope, and the images were saved. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the intestinal area of ​​the zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the sample in repairing zebrafish intestinal mucosal damage.

[0071] The results are as follows Figure 4 , 5 As shown, compared with the normal control group, the model control group (TNBS) significantly increased the intestinal mucosal damage area. Compared with the model control group, treatment with patchouli alcohol (1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (10, 20 μg / mL) all significantly reduced the intestinal area of ​​the intestinal mucosal damage model, demonstrating their efficacy in repairing intestinal mucosal damage.

[0072] Example 4: Evaluation of efficacy in reducing intestinal inflammation 3 dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected and placed in 6-well plates. Each well contained 30 zebrafish. The experimental groups were treated with patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL), respectively. The positive control group was treated with 500 µg / mL mesalazine. A normal control group (no drug treatment) and a model control group (treated with TNBS) were also set up. All experimental groups were also treated with TNBS to establish a zebrafish intestinal mucosal injury model. The volume of each well was 3 mL. After treatment at 28 ℃ for 2 days, 10 zebrafish were randomly selected from each group, photographed under a fluorescence microscope, and the images were saved. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the number of macrophages in the zebrafish intestine was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the sample in promoting the reduction of macrophage inflammation in the zebrafish intestine.

[0073] The results are as follows Figure 6 , 7 As shown, compared with the normal control group, the model control group (TNBS) significantly increased the number of intestinal macrophages. Compared with the model control group, patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL) all significantly reduced the number of macrophages in the intestinal mucosal injury model, indicating that they have the effect of promoting macrophage inflammation resolution.

[0074] Example 5: Evaluation of the regulatory efficacy of intestinal goblet cells Wild-type AB strain zebrafish with a 3-day pf growth rate were randomly selected and placed in 6-well plates. Each well contained 30 zebrafish. The experimental groups were treated with patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL), respectively. The positive control group was treated with 500 µg / mL mesalazine. A normal control group and a model control group (treated with TNBS) were also set up. All experimental groups were also treated with TNBS to establish a zebrafish intestinal mucosal injury model. The volume of each well was 3 mL. After treatment at 28 ℃ for 2 days, the zebrafish were stained with Alcian blue. Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope. The images were saved, and data were collected using NIS-Elements D 3.20 advanced image processing software. The number of goblet cells in the zebrafish intestine was analyzed, and the statistical analysis results of this index were used to evaluate the regulatory effect of the sample on the goblet cells in the zebrafish intestine.

[0075] like Figure 8 , 9 As shown, compared with the normal control group, the model control group (TNBS) significantly reduced the number of intestinal goblet cells. Compared with the model control group, treatment with patchouli alcohol (1.25 μg / mL) and patchouli oil gel (5 μg / mL) significantly upregulated the number of intestinal goblet cells in the intestinal mucosal injury model, showing that they have significant goblet cell regulatory effects, and the patchouli oil group showed a trend of goblet cell regulatory effects.

[0076] Example 6: Histopathological Evaluation of Intestinal Tissue Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates. Each well contained 30 zebrafish, treated with water-soluble drugs. The experimental groups were treated with patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL), respectively. The positive control group was treated with 500 µg / mL mesalazine. A normal control group and a model control group were also set up. Except for the normal control group, all experimental groups were treated with water-soluble TNBS to establish a zebrafish intestinal mucosal injury model. The volume of each well was 3 mL. After treatment at 28 ℃ for 2 days, the zebrafish were fixed, dehydrated, embedded, sectioned, and stained with H&E for histopathological analysis.

[0077] Intestinal pathology section results as follows Figure 10 In the normal control group, zebrafish exhibited numerous and prominent intestinal folds, with intestinal epithelial cells tightly connected to cilia and mucosa. In the model control group (TNBS), the number of intestinal folds was reduced, the intestinal lumen was significantly enlarged, and the mucosal tissue was damaged. The zebrafish in the patchouli alcohol, patchouli oil, and patchouli oil gel groups showed significantly restored intestinal lumen expansion compared to the model control group, and the morphology of intestinal tissue cells was similar to that of the normal control group. This indicates that patchouli alcohol, patchouli oil, and patchouli oil gel all have significant ameliorative effects on the pathological structure of the intestinal mucosa in zebrafish with intestinal mucosal damage.

[0078] Example 7: Effects on genes related to a colitis model Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Drugs were administered in water-soluble form to each well. Experimental groups received patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5, 10 μg / mL), and patchouli oil gel (5, 10, 20 μg / mL), respectively. The positive control group received 500 µg / mL mesalazine. A normal control group and a model control group (administered TNBS in water-soluble form) were also included. All other experimental groups received TNBS in water-soluble form to establish a zebrafish intestinal mucosal injury model. Each well had a volume of 3 mL, and three parallel experiments were conducted. Except for the normal control group, [the following steps were performed]. After treatment at 28 ℃ for 2 days, total RNA was extracted from each group of zebrafish using a rapid RNA extraction kit. The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. Total RNA from 2.00 μg zebrafish samples was collected, and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit. The expression of β-actin, IL-1β, TNF-α, IL-6, TRPM2, TRPM6, and TRPM7 genes was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative RNA expression levels of IL-1β, TNF-α, IL-6, TRPM2, TRPM6, and TRPM7 genes were calculated. Primer sequences are shown in the table below.

[0079]

[0080] like Figure 11 As shown, the IL-1β gene was significantly upregulated in the model control group (TNBS) compared to the normal control group. In contrast, patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5 μg / mL), and patchouli oil gel (10 μg / mL) significantly downregulated the IL-1β gene compared to the model control group.

[0081] like Figure 12 As shown, compared with the normal control group, the model control group significantly upregulated the TNF-α gene, while compared with the model control group, patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5 μg / mL) and patchouli oil gel (20 μg / mL) significantly downregulated the TNF-α gene.

[0082] like Figure 13 As shown, compared with the normal control group, the model control group significantly upregulated the IL-6 gene, while compared with the model control group, patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5 μg / mL) and patchouli oil gel (5 μg / mL) significantly downregulated the IL-6 gene.

[0083] like Figure 14 As shown, compared with the normal control group, the TRPM2 gene was significantly upregulated in the model control group, while compared with the model control group, patchouli alcohol (0.625, 1.25, 2.5 μg / mL), patchouli oil (2.5, 5 μg / mL) and patchouli oil gel (20 μg / mL) significantly downregulated the TRPM2 gene.

[0084] like Figure 15 As shown, compared with the normal control group, the TRPM6 gene was significantly upregulated in the model control group, while compared with the model control group, patchouli alcohol (0.625, 1.25 μg / mL), patchouli oil (2.5 μg / mL) and patchouli oil gel (5 μg / mL) significantly downregulated the TRPM6 gene.

[0085] like Figure 16 As shown, compared with the normal control group, the TRPM7 gene was significantly upregulated in the model control group, while compared with the model control group, patchouli alcohol (1.25, 2.5 μg / mL), patchouli oil (5, 10 μg / mL) and patchouli oil gel (5, 10, 20 μg / mL) significantly downregulated the TRPM7 gene.

[0086] In summary, patchouli alcohol, patchouli oil, and patchouli oil gel can all downregulate the genes of IL-1β, TNF-α, IL-6, TRPM2, TRPM6, and TRPM7.

Claims

1. A composition comprising the following components: (1) patchouli oil or patchouli alcohol; (2) TRPM2 channel inhibitors; and (3) optional pharmaceutically acceptable excipients, or cosmetic excipients. The TRPM2 channel inhibitor is ML-SI3, and The mass ratio of patchouli oil or patchouli alcohol to TRPM2 channel inhibitor is 1:0.5~20.

2. The composition according to claim 1, characterized in that, The mass ratio of patchouli oil or patchouli alcohol to TRPM2 channel inhibitor is 1:1~5.

3. The composition according to claim 1, characterized in that, The mass ratio of patchouli oil or patchouli alcohol to TRPM2 channel inhibitor is 1:2~3.

4. The composition according to claim 1, characterized in that, The mass ratio of patchouli oil or patchouli alcohol to TRPM2 channel inhibitors is 1:2.

5.

5. The composition according to claim 1, characterized in that, Patchouli oil is applied in the form of patchouli oil gel, the raw materials of which are the following components by weight: 5 parts patchouli oil, 3-5 parts polyethylene glycol glyceride laurate, 0.5-2 parts vitamin E polyethylene glycol succinate, and 0.05-0.2 parts SiO2. The gel preparation method is as follows: Heat and melt the prescribed amounts of polyethylene glycol glyceride and vitamin E polyethylene glycol succinate, add the prescribed amount of patchouli oil, and stir until uniform. After stirring until uniform, add the prescribed amount of SiO2 and continue stirring until uniform, resulting in a brownish-yellow liquid. After cooling, patchouli oil gel is obtained.

6. Use of the composition according to any one of claims 1-5 in the preparation of a medicament for treating colitis.

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