A polypeptide of asaia spp. for reducing toxicity of arecoline and application thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a polypeptide of aspongopus capable of reducing toxicity of arecoline and application thereof. The polypeptide of aspongopus is obtained by defatting, enzymolysis and concentration treatment of aspongopus as raw material. The polypeptide of aspongopus prepared in the present application can relieve the decrease of GSH and SOD and the increase of LDH, ROS, TNF-alpha and IL-1beta caused by arecoline, and reduce the cell necrosis rate. By improving the immune and antioxidant capacity, the polypeptide of aspongopus can strengthen the regeneration program response of cells and tissues, reduce the inflammatory response, and maintain the normal and healthy level of cells and tissues, and has a certain effect of relieving the toxicity of arecoline.

Description

Technical fields:

[0001] This invention belongs to the field of biotechnology, specifically relating to a nine-fragrant insect polypeptide that can reduce arecoline toxicity and its application. Background technology:

[0002] Areca nut is a commonly used traditional Chinese medicine with high medicinal and economic value. Its main component is arecoline, which modern toxicological studies have reported to have oral submucosal fibrosis toxicity, reproductive toxicity, hepatotoxicity, nephrotoxicity, immunosuppressive toxicity, and neurotoxicity. Long-term chewing of areca nut products can lead to slow healing of lesions and the formation of a local inflammatory microenvironment. Furthermore, arecoline toxicity may weaken tissue regeneration processes, triggering a vicious cycle.

[0003] Previous studies have shown that total saponins from Panax notoginseng can alleviate the cytotoxicity of arecoline by inhibiting the activation of the ERK / JNK / p38MAPK pathway in oral mucosal fibroblasts; curcumin can inhibit cell proliferation and migration and enhance apoptosis in arecoline-treated normal oral mucosal fibroblasts; melatonin can reduce arecoline-induced DNA damage. However, current research on the cytotoxicity of arecoline in China is insufficient, the mechanism of arecoline's cytotoxicity remains unclear, and there are few studies on peptide components that can effectively alleviate arecoline cytotoxicity. Therefore, it is necessary to strengthen the development of products that alleviate arecoline cytotoxicity.

[0004] Nine-scented worm (Aspongopus chinensis Dallas) is the dried body of the stink bug (Aspongopus chinensis Dallas). It has a salty and warm nature, and enters the liver, spleen, and kidney meridians. In the 2015 edition of the Chinese Pharmacopoeia, nine-scented worm is listed as a medicinal material and processed product, possessing the effects of regulating qi and relieving pain, warming the middle jiao and tonifying yang. It is commonly used for symptoms such as stomach cold and distending pain, liver and stomach qi pain, kidney deficiency and impotence, and lower back and knee pain. In Traditional Chinese Medicine, nine-scented worm is frequently used to treat impotence, stomach ailments, and liver cancer. As a traditional Chinese medicinal and edible animal resource, nine-scented worm is a high-quality protein source. Existing research is mainly based on nine-scented worm hemolymph, nine-scented worm oil, or nine-scented worm water extract. Studies have shown that the protein content of nine-scented worm is second only to fat; however, current research on nine-scented worm protein and peptides is limited. Therefore, to expand the application range of nine-scented worm, it is important to research and develop new bioactive peptides. Summary of the Invention:

[0005] The purpose of this invention is to provide a nine-fragrant insect polypeptide that can reduce arecoline toxicity, its preparation method and application, wherein the nine-fragrant insect polypeptide has arecoline toxicity mitigation activity and can be used in the research and development of health food and other fields.

[0006] To address the aforementioned problems, one of the technical solutions provided by this invention is a method for preparing stink bug polypeptides. This method uses stink bugs as raw material and obtains the polypeptides through defatting, enzymatic hydrolysis, and concentration. Specifically, it includes the following steps:

[0007] 1) After crushing the stink bugs, degrease them for 6-8 hours, then dry and grind them into powder;

[0008] 2) After being ground into powder, the stink bugs undergo a second degreasing treatment for 4-6 hours, are dried, pulverized, and passed through a 40-80 mesh sieve;

[0009] 3) Take the sieved stink bug powder, add water at a solid-liquid ratio of 1:20-30 and cook for 2-6 hours to obtain the cooking liquid;

[0010] 4) After steaming, enzymatic hydrolysis is performed using a combination of neutral protease and alkaline protease. The pH is adjusted to 7-11, the hydrolysis temperature is controlled at 45℃-65℃, and the hydrolysis time is 4h-8h. After the hydrolysis is completed, the enzyme is inactivated, cooled, centrifuged, and the supernatant is concentrated to 1 / 4 to 3 / 4 of the original volume to obtain the nine-fragrant insect polypeptide that can alleviate arecoline cytotoxicity.

[0011] Furthermore, in step 1), the defatting is performed using Soxhlet extraction;

[0012] Furthermore, the filter paper package containing the stink bug is placed into the extraction tube, and 1.5-2 times the amount of petroleum ether is injected. The extractor is connected and the cooling water is turned on. Extraction is carried out in a water bath at 40-50℃ for 6-8 hours. After extraction, the filter paper package is removed and dried.

[0013] Further, in step 2), the degreasing is carried out using Soxhlet extraction. The filter paper package containing the stink bug is placed into the extraction tube, and 1.5-2 times the amount of petroleum ether is injected. The extractor is connected and the cooling water is turned on. Extraction is carried out in a water bath at 40-50℃ for 4-6 hours. After extraction, the filter paper package is removed and dried.

[0014] Furthermore, in step 3), the steaming time is 5 hours;

[0015] Further, in step 4), 0.25%-1.25% (w / v) of neutral protease and 0.75%-3.75% (w / v) of alkaline protease are added according to the volume of the cooking liquid.

[0016] Further, in step 4), the enzymatic hydrolysis conditions are as follows: neutral protease and alkaline protease are added at pH 10 and hydrolysis temperature of 55℃. 0.25%-1.25% (w / v) of neutral protease and 0.75%-3.75% (w / v) of alkaline protease are added according to the volume of the cooking liquid. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100℃ for 5-10 min.

[0017] Further, in step 4), the supernatant is concentrated and then freeze-dried to obtain solid *Stachys chinensis* polypeptide.

[0018] The second technical solution provided by this invention is a nine-fragrant insect polypeptide prepared from the first technical solution;

[0019] The third technical solution provided by the present invention is the application of the nine-fragrant insect polypeptide described in technical solution two, especially its application in reducing or alleviating arecoline toxicity; or its application in the preparation of food, health products or drugs that reduce or alleviate arecoline toxicity.

[0020] Furthermore, the application involves preparing the aforementioned nine-fragrant insect polypeptide into mouthwash and other products for use by betel nut users to treat, reduce, or alleviate oral damage.

[0021] Beneficial effects:

[0022] (1) The present invention provides a method for extracting stink bug polypeptides. According to the stink bug polypeptide preparation method provided by the present invention, after enzymatic hydrolysis of stink bug, small molecule peptides are the main component, with a relative molecular weight of less than 1000 Da accounting for 88.1%. Furthermore, stink bug polypeptides are animal peptides with rich amino acid composition, which are easier to absorb. They have the effects of improving immune function, anti-oxidation, and anti-aging. They also have nutritional and functional properties, which can enhance the body's resistance and delay aging damage.

[0023] (2) The nine-fragrant insect polypeptide prepared in this invention can alleviate the decrease of GSH and SOD and the increase of LDH, ROS, TNF-α and IL-1β caused by arecoline and reduce the cell necrosis rate. By improving immunity and antioxidant capacity, strengthening the regeneration process of cells and tissues, and reducing inflammatory response, it can maintain the normal health level of cells and tissues and has a certain arecoline toxicity relief effect.

[0024] (3) The nine-fragrant insect polypeptide prepared by the present invention can alleviate the oral cavity damage in mice caused by arecoline, and can exert the nutritional and health care effects of nine-fragrant insect. It is expected to be a new resource health food with broad application prospects. Attached image description:

[0025] Figure 1 Flowchart for the preparation of nine-fragrant insect polypeptide;

[0026] Figure 2 This is a diagram showing the relative molecular mass distribution of polypeptides from the stink bug (Nepeta cataria).

[0027] Figure 3 The results of the in vitro antioxidant assay of *Corydalis yanhusuo* polypeptide;

[0028] Figure 4 The effect of *Corydalis yanhusuo* polypeptide on cellular GSH;

[0029] Figure 5 The effect of *Corydalis yanhusuo* polypeptide on cellular SOD;

[0030] Figure 6 The effect of *Corydalis yanhusuo* polypeptide on cellular LDH;

[0031] Figure 7 The effect of *Nine-fragrant insect polypeptide* on reactive oxygen species in cells;

[0032] Figure 8 The effect of *Nine-fragrant insect polypeptide* on cell necrosis rate;

[0033] Figure 9 The effect of *Corydalis yanhusuo* polypeptide on GSH in mouse skin;

[0034] Figure 10 The effect of *Corydalis yanhusuo* polypeptide on SOD in mouse skin;

[0035] Figure 11 The effect of *Nine-fragrant insect polypeptide* on LDH in mouse skin;

[0036] Figure 12 The effect of *Corydalis yanhusuo* polypeptide on ROS in mouse skin;

[0037] Figure 13 The effect of *Corydalis yanhusuo* polypeptide on TNF-α in mouse skin;

[0038] Figure 14 The effect of *Corydalis yanhusuo* polypeptide on IL-1β in mouse skin.

[0039] Note: In the above figures, the model group showed significant differences compared to the blank group* (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001); and the drug group showed significant differences compared to the model group # (p < 0.05), ## (p < 0.01), ### (p < 0.001), #### (p < 0.0001). Detailed implementation method:

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0041] The present invention will be further explained and illustrated below through specific embodiments.

[0042] Example 1: A method for preparing a polypeptide from the stink bug.

[0043] Preparation process as follows Figure 1As shown, after washing and drying, the stink bugs were initially pulverized until the insect bodies were broken. Following pulverization, a first degreasing treatment was performed using Soxhlet extraction. The filter paper packet containing the stink bugs was placed into the extraction tube using long tweezers, and 1.67 times the volume of petroleum ether (one siphon volume) was injected to completely submerge the sample packet. All parts of the extractor were connected, and the cooling water flow was turned on. Extraction was carried out in a constant-temperature water bath at 48℃, ensuring the condensed petroleum ether dripped in a continuous stream (140 drops / min). The extraction time was 8 hours. After extraction, the filter paper packet was removed using long tweezers, and the petroleum ether was allowed to evaporate in a ventilated area.

[0044] After the first defatting, the sample package was dried, and the stink bugs were removed and pulverized until no large pieces of insect remained. The package was repackaged and subjected to a second defatting process, again using Soxhlet extraction. The filter paper package containing the stink bugs was placed into the extraction tube using long tweezers, and 1.67 times the volume of petroleum ether (one siphon volume) was injected to completely submerge the sample package. All parts of the extractor were connected, and the cooling water flow was turned on. Extraction was performed in a constant-temperature water bath at 46°C, ensuring the condensed petroleum ether dripped in a continuous stream (100 drops / min). The extraction time was 6 hours. After extraction, the filter paper package was removed using long tweezers, and the sample package was dried at 60°C for 3 hours. After drying, the stink bugs were removed, powdered, and passed through a 40-mesh sieve. The resulting powder was stored at 4°C.

[0045] The protein content of the degreased *Corydalis yanhusuo* powder was determined by the Dumas nitrogen determination method, and the protein content of the powder was 71.26%, indicating that the sample after degreasing was mostly protein.

[0046] Enzymatic hydrolysis: Distilled water was added to defatted powder at a solid-liquid ratio of 1:25, and the mixture was cooked for 5 hours to obtain a cooking liquid. After cooking, the pH was adjusted to 10. At the same time, neutral protease and alkaline protease were added at an enzymatic hydrolysis temperature of 55℃. The amount of neutral protease added was 1% (w / v) of the cooking liquid, and the amount of alkaline protease added was 3% (w / v) of the cooking liquid. Enzymatic hydrolysis was carried out for 6 hours. After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. The supernatant was centrifuged and the peptide yield was determined. The concentrate was then concentrated to half of the original volume to obtain a concentrated solution of *Corydalis yanhusuo* peptides. Solid *Corydalis yanhusuo* peptides were obtained by freeze drying.

[0047] It should be noted that the method for determining the polypeptide yield in this invention involves using trichloroacetic acid precipitation of large protein molecules combined with the biuret method to determine the polypeptide concentration and calculate the polypeptide yield. In this example, the polypeptide yield of *Corydalis yanhusuo* is 35.74%.

[0048] Example 2: Determination of the molecular weight and amino acid composition of the nine-fragrant insect polypeptide.

[0049] The molecular weight of the *Nine-Fragrance Insect* polypeptide sample prepared in Example 1 was determined using the method described in GB / T22729-2008, namely, high-performance gel filtration chromatography (HPLC). 20 mg of the lyophilized *Nine-Fragrance Insect* polypeptide was weighed into a 10 mL volumetric flask, diluted to volume with a mobile phase (acetonitrile:water:trifluoroacetic acid = 45:55:0.1), dissolved by shaking and sonication, filtered through a 0.22 μm nylon filter membrane, and then analyzed by the HPLC. Figure 2 It can be seen that 88.1% of the peptides in *Corydalis yanhusuo* have a relative molecular mass of <1000 Da, and are mainly small molecule peptides.

[0050] The amino acid composition of *Corydalis yanhusuo* polypeptide was determined according to the determination method in GB 5009.235-2016 National Food Safety Standard - Determination of Amino Acids in Food. The results are as follows.

[0051] Table 1. Amino acid composition of nine-fragrant insect polypeptides

[0052]

[0053] Example 3: In vitro antioxidant test of stink bug polypeptide

[0054] The lyophilized *Corydalis yanhusuo* polypeptide prepared in Example 1 was subjected to DPPH free radical scavenging test, ABTS free radical scavenging test and total reducing power determination, while glutathione of the same concentration was used as a positive control.

[0055] (1) Determination of DPPH free radical scavenging rate: Different samples were diluted to solutions with mass concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg / mL, respectively. 2 mL of each concentration solution was placed in a test tube, and 2 mL of 0.2 mmol / L DPPH solution was added. The mixture was shaken thoroughly, centrifuged at 4000 rpm for 6 min, and allowed to stand for 30 min. The absorbance was measured at 517 nm using anhydrous ethanol as a reference. The scavenging capacity was calculated according to the following formula.

[0056] DPPH free radical scavenging capacity = [1-(A s -A c ) / A0]×100%

[0057] Where As is the absorbance of the experimental group, Ac is the absorbance of the control group with anhydrous ethanol instead of DPPH solution, and A0 is the absorbance of the blank group with anhydrous ethanol instead of sample solution.

[0058] DPPH is a stable nitrogen-centered chromogenic free radical that produces a characteristic absorption peak at 517 nm. Antioxidants in the sample can reduce or even eliminate the absorbance of DPPH·. Results are shown below. Figure 3 The DPPH free radical scavenging rate of the nine-fragrant insect polypeptide increased with increasing concentration, indicating that the nine-fragrant insect polypeptide has a certain antioxidant capacity.

[0059] (2) ABTS free radical scavenging rate determination: Different samples were diluted to solutions with mass concentrations of 0.2, 0.4, 0.6, 0.8 and 1 mg / mL, respectively. 0.02 mL of each diluted sample solution was taken and 3.80 mL of the diluted ABTS mixed solution was added to each solution, for a total of 4.00 mL. The solutions were mixed and reacted for 6 min. The absorbance was measured at 734 nm with deionized water as a blank control. The scavenging rate was calculated according to the following formula.

[0060] ABTS free radical scavenging rate (%) = (A0 – A i )×100 / A0

[0061] Where A0 is the absorbance of deionized water + ABTS, A i The absorbance is the sample solution + ABTS.

[0062] ABTS reacts with a suitable oxidant to generate a blue-green ABTS cation radical (ABTS·+), which produces a characteristic absorption peak at 734 nm. The formation of ABTS·+ is inhibited in the presence of antioxidants, resulting in a decrease in absorbance. See the results below. Figure 3 The ABTS scavenging rate of the nine-fragrant insect polypeptide increases with increasing concentration, reaching up to 90%, and it has good antioxidant properties.

[0063] (3) Dilute the samples to solutions with mass concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg / mL, respectively. Take 2.5 mL of each solution in a test tube, and add 2.5 mL each of phosphate buffer (pH 6.6) and 5% potassium ferricyanide solution. Shake well and incubate in a water bath at 50°C for 20 min. Then add 2.5 mL of 10% trichloroacetic acid solution, shake thoroughly, and centrifuge at 3000 rpm for 10 min. Take 2.5 mL of the supernatant, add an equal volume of distilled water and 0.1% FeCl3 solution, shake well, and let stand for 10 min. Measure the absorbance at 700 nm. The reducing power is calculated using the following formula.

[0064] Total reducing capacity = A s -A0

[0065] Where A S A0 represents the absorbance of the experimental group, while A0 represents the absorbance of the blank group using anhydrous ethanol instead of the sample.

[0066] The sample eliminates free radicals by donating electrons through its own reduction process; the stronger the reducing power, the stronger the antioxidant activity. Results are shown below. Figure 3 The total reducing power of the nine-fragrant insect polypeptide increases with increasing concentration, indicating that the nine-fragrant insect polypeptide has a certain reducing power and can play an antioxidant role.

[0067] Example 4: Culture, Model Establishment and Sample Intervention of Human Gastric Mucosal Epithelial Cells

[0068] Step 1: Cell culture, human gastric mucosal epithelial cells were cultured at a concentration of 2×10⁻⁶. 5 The cells were seeded at a density of 1 / mL in 25T cell culture flasks, and an appropriate amount of DMEM medium containing 10% fetal bovine serum was added. The flasks were then incubated at 37-31°C, 53% CO2, and 95% relative humidity for 48 hours, resulting in a final cell density of 80%.

[0069] Step 2: Cell plating. Cultured human gastric mucosal epithelial cells are digested with trypsin for 2 minutes to prepare a cell suspension. Then, the suspension is prepared by adding 1×10⁻⁶ cells / mL of trypsin. 4 The inoculum was uniformly seeded at a density of 1 / mL into 6-well plates and incubated in an incubator at 37-31℃, 531% CO2, and 95% relative humidity for 24 hours.

[0070] Step 3: Change the cell medium, discard the old medium, add an appropriate amount of DMEM medium containing 10% fetal bovine serum to each well, and incubate the 6-well plate in an incubator at 37-31°C, 53% CO2, and 95% relative humidity for 24 hours.

[0071] Step 4: Divide the cultured cells into a blank group, a model group, and a sample group;

[0072] Model establishment: Discard the old culture medium for the model group and sample group, and add DMEM culture medium containing 90 μg / mL of arecoline and 10% fetal bovine serum to each well (discard the old culture medium for the blank group, and add DMEM culture medium containing 10% fetal bovine serum to each well). Place the 6-well plate in an incubator at 37-31℃, 531% CO2, and 95% relative humidity for 24 hours.

[0073] Step 5: Nine-fragrant insect polypeptide intervention

[0074] The old culture medium was discarded from each group. The blank group and the model group were added to DMEM culture medium containing 10% fetal bovine serum. The sample groups were treated as follows:

[0075] Sample group 1: DMEM medium containing 250 μg / mL of *Nine-fragrant insect polypeptide* and 10% fetal bovine serum was added to each well (the *Nine-fragrant insect polypeptide* was prepared as solid *Nine-fragrant insect polypeptide* in Example 1).

[0076] Sample group 2: DMEM medium containing 500 μg / mL of worm peptide and 10% fetal bovine serum was added to each well;

[0077] Sample group 3: DMEM medium containing 10% fetal bovine serum and a final concentration of 1000 μg / mL of phlegm-containing polypeptide was added to each well;

[0078] All well plates were incubated in an incubator at 3731℃, 531% CO2, and 95% relative humidity for 24 hours.

[0079] Step 6: Cell Preparation

[0080] Human gastric mucosal epithelial cells from the blank group, model group, and sample group were digested and treated with pancreatic enzyme digestion for 2 minutes. The cells were washed twice and prepared into a cell suspension for later use.

[0081] Step 7: Indicator Measurement

[0082] The indicators were measured according to the detailed instructions for staining the ROS detection kit, SOD detection kit, GSH-PX detection kit, LDH detection kit, and apoptosis and necrosis detection kit. The results are as follows:

[0083] GSH results are as follows Figure 4 As shown in the figure, GSH helps maintain normal cellular immune system function and has antioxidant and detoxifying effects. Arecoline leads to GSH depletion in cells, resulting in a decrease in GSH levels. In this example, the GSH level in the control group was 41.71 / activity unit, while the GSH level in the model group was 32.74 / activity unit, a significant decrease compared to the control group. After drug intervention at 250, 500, and 1000 μg / mL, the GSH levels were 40.47, 41.96, and 45.86 / activity units, respectively, showing a concentration-dependent effect. This indicates that the *Nine-Fragrance Insect Peptide* can improve the GSH activity reduction caused by arecoline, and the high-dose *Nine-Fragrance Insect Peptide* showed the best effect.

[0084] SOD results are as follows Figure 5 As shown in the figure, SOD, as an intracellular antioxidant enzyme, plays an important role in oxidative homeostasis. Arecoline reduces intracellular SOD levels, inhibits cell growth, and interferes with cell signal transduction. The SOD level in the control group was 58.09 U / mgprot, while the SOD level in the model group was 44.65 U / mgprot, a significant decrease compared to the control group. After drug intervention at 250, 500, and 1000 μg / mL, the SOD levels were 54.62, 57.17, and 61.75 U / mgprot, respectively, showing a concentration-dependent effect. This indicates that *Corydalis jiunifolia* polypeptide can improve the SOD activity reduction caused by arecoline, with high-dose *Corydalis jiunifolia* polypeptide showing the best effect.

[0085] LDH results are as follows Figure 6As shown, when cells are damaged, LDH is released from within the cells. Arecoline-induced cell damage promotes LDH release, causing it to rise. The LDH level in the control group was 0.34 / activity unit, while the LDH level in the model group was 0.41 / activity unit, a significant increase compared to the control group. After drug intervention at 250, 500, and 1000 μg / mL, the LDH levels were 0.4, 0.39, and 0.37 / activity unit, respectively, with the 1000 μg / mL sample group showing a significant decrease. This indicates that high-dose *Corydalis yanhusuo* polypeptide can reduce the increase in LDH levels caused by arecoline.

[0086] ROS results are as follows Figure 7 As shown. When cells are in a normal state, intracellular antioxidants can neutralize reactive oxygen species (ROS). The decrease in antioxidant activity caused by arecoline reduces the neutralization of ROS, triggering oxidative stress and ultimately leading to genotoxicity or gene mutations. The average fluorescence intensity in the arecoline control group was 30.33, while the average fluorescence intensity in the model group was 231.67, significantly higher than the control group. After drug intervention at 250, 500, and 1000 μg / mL, the average fluorescence intensities were 187.33, 139.33, and 96.00, respectively, significantly reduced. Nine-fragrant insect polypeptide can inhibit the production of ROS.

[0087] Necrosis rate results as follows Figure 8 As shown in the figure, the necrosis rate in the control group was 18.47%, while the necrosis rate in the model group was 32.37%, significantly higher than that in the control group. After drug intervention at concentrations of 250, 500, and 1000 μg / mL, the necrosis rates were 24.57%, 23%, and 21.7%, respectively, all significantly reduced. The results indicate that *Corydalis jiunifolia* polypeptide can reduce arecoline-induced cell death, with the highest dose showing the best effect.

[0088] Example 5: Protection of arecoline-induced oral cavity damage in mice by nine-fragrant insect polypeptide.

[0089] Step 1: Animal grouping and administration

[0090] Seven-week-old male specific pathogen-free (SPF) BALB / c mice were acclimatized for seven days and then randomly divided into three groups (n=10): normal group (NC); model group (MC); and high-dose group of *Corydalis yanhusuo* polypeptide (HG, 1000 μg / mL, *Corydalis yanhusuo* polypeptide prepared in Example 1).

[0091] In the normal control group, drinking water was applied evenly to the upper and lower jaws of mice using cotton swabs. In the other groups, arecoline solution (200 mg / mL) was applied evenly to the upper and lower jaws of mice using cotton swabs once daily. Simultaneously, the mice's mouths were opened with hemostatic forceps, and their jaws were stimulated with arecoline solution using a small brush for 2 minutes, once daily. From day 14, distilled water was used to apply to the upper and lower jaws of mice in the normal and model groups, while the high-dose group of *Chenopodium clavatum* polypeptide was treated with 1000 μg / mL *Chenopodium clavatum* polypeptide solution once daily. The experiment continued until day 28. After the experiment, skin tissue from the upper and lower jaws of the mice was collected for later use.

[0092] Step 2: Homogenize the skin tissue in an ice bath using 0.9% NaCl solution (1:9, w / v). Centrifuge the homogenate at 4000 rpm for 20 min. Determine the levels of GSH, SOD, LDH, ROS, TNF-α, and IL-1β in the mouse skin homogenate according to the kit instructions.

[0093] Skin homogenate GSH results as follows Figure 9 As shown, the GSH level in the blank group was 79.75 U / mg, while the GSH level in the model group was 50.93 U / mg, a significant decrease. After intervention with *Corydalis yanhusuo* polypeptide, the GSH level increased to 65.58 U / mg. This indicates that *Corydalis yanhusuo* intervention can improve the GSH decrease caused by arecoline.

[0094] Skin homogenate SOD results as follows Figure 10 As shown in the figure, the SOD level in the blank group was 198.39 U / mg, while the SOD level in the model group was 140.29 U / mg, a significant decrease. After intervention with *Corydalis yanhusuo* polypeptide, the SOD level increased to 165.24 U / mg. This indicates that *Corydalis yanhusuo* intervention can increase SOD levels and enhance the antioxidant capacity of mice.

[0095] Skin homogenate LDH results as follows Figure 11 As shown in the figure, the LDH level in the control group was 10.88 U / mg, while the LDH level in the model group was 14.54 U / mg, which was significantly higher than that in the control group. After intervention with *Corydalis yanhusuo* polypeptide, the LDH level decreased to 11.15 U / mg. This indicates that *Corydalis yanhusuo* intervention can reduce LDH levels and alleviate arecoline-induced skin damage to the upper and lower jaws of mice.

[0096] ROS results of skin homogenate are as follows Figure 12 As shown in the figure, the ROS level in the control group was 1, while the LDH level in the model group was 1.64, which was significantly higher than that in the control group. After intervention with *Corydalis yanhusuo* peptides, the ROS level decreased to 1.3. This indicates that *Corydalis yanhusuo* intervention can reduce reactive oxygen species levels and alleviate arecoline-induced oxidative damage to the skin of the upper and lower jaws of mice.

[0097] Skin homogenate TNF-α results as follows Figure 13As shown, TNF-α increases under many pathological conditions, and arecoline-induced skin damage in mice promotes TNF-α production. The TNF-α level in the control group was 297.36 ng / L, while the TNF-α level in the model group was 498.98 ng / L, significantly higher than the control group. After intervention with *Corydalis yanhusuo* polypeptide, TNF-α decreased to 406.04 ng / L. This indicates that *Corydalis yanhusuo* intervention can reduce TNF-α levels and alleviate arecoline-induced inflammatory responses in the upper and lower jaw skin of mice.

[0098] Results of skin homogenate IL-1β Figure 14 As shown, the combined effect of arecoline and physical friction induces a stress response that promotes IL-1β release. The IL-1β level in the control group was 106.45 ng / L, while the IL-1β level in the model group was 394.82 ng / L, significantly higher than the control group. After intervention with *Corydalis yanhusuo* polypeptide, IL-1β decreased to 183.16 ng / L. This indicates that *Corydalis yanhusuo* intervention can reduce IL-1β levels and alleviate arecoline-induced inflammatory responses in the upper and lower jaw skin of mice.

[0099] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0100] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An application of a nine-fragrant insect polypeptide, characterized in that, It is used in the preparation of topical medications that reduce or alleviate oral skin damage or inflammation caused by arecoline; The preparation method of the nine-fragrant insect polypeptide specifically includes the following steps: 1) After crushing the stink bugs, degrease them, dry them, and then grind them into powder; 2) The powdered stink bugs undergo a second degreasing process and are then dried; 3) Take the powder of nine-fragrant insects, add water and steam to obtain the steaming liquid; 4) After steaming and boiling, enzymatic hydrolysis is performed using a combination of neutral protease and alkaline protease. After the enzymatic hydrolysis is completed, the enzyme is inactivated, cooled, centrifuged, and the supernatant is collected and concentrated to obtain the nine-fragrant insect polypeptide. The enzymatic hydrolysis conditions are as follows: a combination of neutral protease and alkaline protease is used for enzymatic hydrolysis, the pH is adjusted to 7-11, the hydrolysis temperature is controlled at 45℃-65℃ and the hydrolysis time is 4 h-8 h. After the hydrolysis is completed, the enzyme is inactivated, and after cooling, the supernatant is collected by centrifugation and concentrated to 1 / 4 to 3 / 4 of the original volume to obtain the nine-fragrant insect polypeptide.

2. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, Specifically, the steps include the following: 1) After crushing the stink bugs, degrease them for 6-8 hours, then dry and grind them into powder; 2) After being ground into powder, the stink bugs undergo a second degreasing treatment for 4-6 hours, are dried, pulverized, and passed through a 40-80 mesh sieve; 3) Take the sieved stink bug powder, add water at a solid-liquid ratio of 1:20-30 and cook for 2-6 hours to obtain the cooking liquid; 4) After steaming, enzymatic hydrolysis is performed using a combination of neutral protease and alkaline protease. The pH is adjusted to 7-11, the hydrolysis temperature is controlled at 45℃-65℃, and the hydrolysis time is 4 h-8 h. After the hydrolysis is completed, the enzyme is inactivated, cooled, centrifuged, and the supernatant is concentrated to 1 / 4 to 3 / 4 of the original volume to obtain the nine-fragrant insect polypeptide.

3. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, In step 1), the degreasing is performed using Soxhlet extraction. The filter paper package containing the stink bug is placed into the extraction tube, and 1.5-2 times the amount of petroleum ether is injected. The extractor is connected and the cooling water is turned on. Extraction is carried out in a water bath at 40-50℃ for 6-8 hours. After extraction, the filter paper package is removed and dried.

4. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, In step 2), the defatting is performed using Soxhlet extraction. The filter paper package containing the stink bug is placed into the extraction tube, and 1.5-2 times the amount of petroleum ether is injected. The extractor is connected and the cooling water is turned on. Extraction is carried out in a water bath at 40-50℃ for 4-6 hours. After extraction, the filter paper package is removed and dried.

5. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, In step 4), add 0.25%-1.25% of neutral protease and 0.75%-3.75% of alkaline protease according to the volume of the cooking liquid.

6. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, In step 4), the enzymatic hydrolysis conditions are as follows: neutral protease and alkaline protease are added at pH 10 and hydrolysis temperature of 55℃. 0.25%-1.25% of neutral protease and 0.75%-3.75% of alkaline protease are added according to the volume of the cooking liquid. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100℃ for 5-10 minutes.

7. The application of the nine-fragrant insect polypeptide as described in claim 1, characterized in that, In step 4), the supernatant is concentrated and then freeze-dried to obtain solid stink bug polypeptide.

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