A highly effective mosquito repellent and anti-inflammatory compound based on small molecule peptides and its application

Abstract

The present invention relates to the field of biomedical technology, and the present invention relates to the field of biomedical technology, specifically to a highly efficient mosquito repellent and anti-inflammatory compound based on small molecule peptides. The highly efficient mosquito repellent and anti-inflammatory compound based on small molecule peptides and N,N-diethyl-2-hydroxy-2-phenylacetamide of the present invention has significantly better mosquito repellent and anti-inflammatory effects, and at the same time has high safety and good stability, and provides an innovative and effective solution to the problem of mosquito bites. Through reasonable formula design and advanced preparation technology, the synergistic effect of small molecule peptides, plant essential oils and N,N-diethyl-2-hydroxy-2-phenylacetamide is achieved, providing consumers with an efficient, safe and convenient protective product, which has broad market application prospects and social value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and chemistry, and specifically relates to a highly effective mosquito repellent and anti-inflammatory compound based on small molecule peptides and an application thereof. Background Art

[0002] Mosquito bites are a common problem worldwide. They not only bring discomfort and annoyance to people, but more seriously, they may spread a variety of dangerous diseases, posing a huge threat to human health. In the hot summer, mosquitoes are rampant, and people try their best to resist the intrusion of mosquitoes and the inflammatory response after bites. With the change of global climate and the acceleration of urbanization, the breeding and activity range of mosquitoes are constantly expanding. As one of the main types of mosquitoes, mosquitoes not only cause local redness, swelling, itching and pain on the skin, but also may spread serious diseases such as malaria, dengue fever, yellow fever, Zika virus, etc. According to statistics from the World Health Organization, the number of deaths caused by mosquito-borne diseases each year is as high as millions, among which children and people with weak immunity are the most seriously affected. In the past few decades, people have developed a variety of products to prevent mosquito bites and reduce inflammation after bites. Traditional mosquito repellent products are mainly divided into chemical synthesis and natural plant extraction.

[0003] Chemical synthetic mosquito repellents, such as DEET, are widely used. They work by interfering with the olfactory perception of mosquitoes. However, DEET has some obvious defects. Long-term or large-scale use of DEET may irritate human skin, leading to adverse reactions such as skin allergies, redness, swelling, and itching. For children, pregnant women, and people with sensitive skin, the potential risk of using DEET is greater. In addition, some studies have also pointed out that long-term exposure to DEET may have a certain effect on the nervous system. In addition to DEET, other chemically synthesized mosquito repellent ingredients, such as chlorpyrifos and picaridin, although they reduce direct irritation to the skin to a certain extent, are not completely safe and harmless. They may accumulate in the human body and cause potential burdens on organs such as the liver and kidneys. Moreover, the effectiveness of chemical synthetic mosquito repellents is often affected by the use environment and individual differences. In the case of high temperature, excessive sweating or frequent contact with water, its mosquito repellent effect may be greatly reduced.

[0004] Mosquito repellent products made from natural plant extracts, such as lemon eucalyptus oil, citronella oil, and peppermint oil, give people the impression of being safer and more environmentally friendly due to their natural origin. These plant extracts usually contain some volatile components that have mosquito repellent effects and repel mosquitoes by emitting special odors. However, the mosquito repellent effect of such products is often not stable and lasting. Factors such as the plant's growth environment, picking season, and extraction process can significantly affect the content and quality of the active ingredients in the extract, resulting in differences in product effects.

[0005] In terms of anti-inflammatory, common products on the market include calamine lotion, glucocorticoid ointment, antihistamines, etc. Calamine lotion mainly reduces inflammation by astringing and protecting the skin, but it has limited effect on severe redness, swelling and itching. Although glucocorticoid ointment has a strong anti-inflammatory effect, long-term or large-area use may cause side effects such as thinning of the skin, capillary dilation, and pigmentation. Antihistamines can relieve itching caused by allergic reactions, but some people may experience adverse reactions such as drowsiness and dizziness. In addition, existing mosquito repellent and anti-inflammatory products also have many problems in the use experience. For example, some mosquito repellent sprays have a pungent smell that makes people feel uncomfortable; some ointments are greasy, not easy to apply evenly and easy to stain clothes; some products need to be reapplied or used frequently, which brings inconvenience to people's daily life. In recent years, as people's attention to health and environmental protection continues to increase, the requirements for mosquito repellent and anti-inflammatory products have become higher and higher. Consumers not only want products to have efficient mosquito repellent and anti-inflammatory effects, but also expect them to be safe, non-irritating, easy to use, pleasant smell, and environmentally friendly. At the same time, due to the different skin characteristics and reactions to mosquito bites of different people, personalized needs are becoming increasingly prominent. In the field of scientific research, we have a deeper understanding of the mechanism of mosquito bites and the inflammatory response process of the human body. Studies have found that mosquitoes locate their bite targets by sensing signals such as carbon dioxide, lactic acid, and body temperature emitted by the human body. After the bite, the human immune system will quickly start and release a series of inflammatory mediators, such as histamine and interleukin, which will lead to symptoms such as redness, swelling, and itching. Based on these new understandings, it is possible to develop more accurate and effective mosquito repellent and anti-inflammatory products.

[0006] As a substance with unique biological activity, small molecule peptides have gradually entered the field of vision of researchers. Small molecule peptides have the advantages of small molecular weight, easy absorption, and strong specificity, and can exert physiological functions by interacting with specific targets. In the field of biomedicine, small molecule peptides have shown great potential in disease diagnosis, treatment and prevention. However, the application of small molecule peptides in the field of mosquito repellent and anti-inflammatory still faces many challenges. First, it is necessary to screen out small molecule peptide sequences with specific mosquito repellent and anti-inflammatory activity, and conduct in-depth research on their mechanism of action. Secondly, how to reasonably match small molecule peptides with other active ingredients to achieve a synergistic effect is also a key issue. In addition, the stability, production cost, and large-scale preparation process of small molecule peptides need to be further optimized and resolved.

[0007] In summary, existing mosquito repellent and anti-inflammatory products have many deficiencies and cannot meet people's growing needs. Developing an efficient, safe, convenient, slow-release, and personalized mosquito repellent and anti-inflammatory compound based on small molecule peptides has important practical significance and broad market prospects. Summary of the invention

[0008] The present invention provides a highly effective mosquito repellent and anti-inflammatory complex based on small molecule peptides, comprising a small molecule peptide with a sequence of SEQ NO1: Cys - Arg - Gly - Leu - Ile (CRGLI), a small molecule peptide with a sequence of SEQ NO2: Lys - Trp -His - Pro - Glu (KWHPE), a small molecule peptide with a sequence of SEQ NO3: Val - Thr - Met - Asn - Ser (VTMNS), lavender essential oil, wormwood essential oil and N,N-diethyl-2-hydroxy-2-phenylacetamide.

[0009] Furthermore, the weight proportion of the small molecule peptide having the sequence of SEQ NO1: Cys - Arg - Gly - Leu - Ile (CRGLI) in the complex is 10 to 15 parts.

[0010] Furthermore, the weight proportion of the small molecule peptide with the sequence of SEQ NO2: Lys - Trp - His - Pro - Glu (KWHPE) in the complex is 10 to 15 parts.

[0011] Furthermore, the weight proportion of the small molecule peptide having the sequence of SEQ NO3: Val - Thr - Met - Asn - Ser (VTMNS) in the complex is 15 to 20 parts.

[0012] Furthermore, the weight proportion of the lavender essential oil in the composite is 5 parts to 10 parts.

[0013] Furthermore, the weight proportion of the wormwood essential oil in the composite is 2 to 3 parts.

[0014] Furthermore, the weight proportion of the N,N-diethyl-2-hydroxy-2-phenylacetamide in the composite is 5 to 10 parts.

[0015] Furthermore, a method for preparing a highly effective mosquito repellent and anti-inflammatory complex based on small molecule peptides comprises synthesizing and purifying small molecule peptides respectively, extracting lavender essential oil and mugwort essential oil, and then mixing and stirring the components in proportion.

[0016] Furthermore, the highly effective mosquito repellent and anti-inflammatory complex based on small molecule peptides is used in the preparation of mosquito repellent products.

[0017] Furthermore, a highly effective mosquito repellent and anti-inflammatory complex based on small molecule peptides is used in the preparation of anti-inflammatory products.

[0018] Beneficial effects of the invention: The high-efficiency mosquito repellent and anti-inflammatory complex based on small molecule peptides and N,N-diethyl-2-hydroxy-2-phenylacetamide of the present invention has significantly better mosquito repellent and anti-inflammatory effects, and is highly safe and stable, providing an innovative and effective solution to the problem of mosquito bites. Through reasonable formula design and advanced preparation technology, the synergistic effect of small molecule peptides, plant essential oils and N,N-diethyl-2-hydroxy-2-phenylacetamide is achieved, providing consumers with an efficient, safe and convenient protective product with broad market application prospects and social value. DETAILED DESCRIPTION

[0019] Embodiment 1: Application sequence 1: Cys-Arg-Gly-Leu-Ile (CRGLI) Principle of action: When mosquitoes are looking for targets to bite, they rely on their keen olfactory system to perceive various chemical signals emitted by the human body. CRGLI small molecule peptides can specifically bind to key sites on mosquito olfactory receptors, and change the conformation and function of receptors through steric hindrance and electrostatic interactions, thereby interfering with the normal recognition and positioning of human odor by mosquitoes. This mechanism of action makes it difficult for mosquitoes to accurately detect the human body, significantly reducing the probability of mosquito bites.

[0020] Target: Specific domains of mosquito olfactory receptor proteins, such as certain amino acid residues in the transmembrane region.

[0021] Solid phase peptide synthesis was performed according to the predetermined peptide sequence SEQ NO1: Cys - Arg - Gly - Leu - Ile (CRGLI). Polystyrene resin was selected as the solid phase carrier, and the Fmoc protection strategy was used. First, Fmoc-Ile was connected to the resin, and then Fmoc-Leu, Fmoc-Gly, Fmoc-Arg and Fmoc-Cys were added in sequence for condensation reaction. After each condensation reaction, 20 parts of piperidine solution were used to remove the Fmoc protecting group. After the synthesis was completed, the peptide chain was cut with a mixed solution of trifluoroacetic acid / water / triisopropylsilane (95:2.5:2.5, v / v / v), and precipitated with cold ether. The precipitate was collected by centrifugation to obtain a crude peptide. The crude peptide was dissolved in an acetonitrile / water (30:70, v / v) solution, purified by HPLC, the target peak was collected, and the solvent was removed by rotary evaporation to obtain purified CRGLI. Mass spectrometry analysis confirmed that its molecular weight was consistent with the theoretical value and its purity reached 98 parts.

[0022] When CRGLI was used alone to test the mosquito repellent effect, the average protection time was 2 hours and the average number of mosquito bites was 6 times.

[0023] Embodiment 2: Application sequence Lys - Trp - His - Pro - Glu (KWHPE) Principle of action: When mosquitoes bite the human body, a series of inflammatory mediators, such as proteases and histamine, are released in their saliva, triggering local inflammatory reactions. KWHPE small molecule peptides can specifically bind to these inflammatory mediators to form stable complexes, thereby changing the structure and activity of inflammatory mediators and inhibiting their stimulation and damage to human tissues. In addition, the small molecule peptide can also regulate the conduction of inflammation-related signal pathways, reduce the activation of inflammatory cells and the release of inflammatory factors, and reduce the degree of inflammatory response from the source.

[0024] Targets: Inflammation-related enzymes in mosquito saliva, such as serine proteases, cysteine ​​proteases, etc., and key proteins in inflammatory signaling pathways, such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), etc.

[0025] For the synthesis of peptide sequence SEQ NO2: Lys - Trp - His - Pro - Glu (KWHPE), solid phase peptide synthesis was also used. Polyamide resin was selected as the solid phase carrier, and Boc protection strategy was adopted. Amino acids were connected in the order of Boc-Glu, Boc-Pro, Boc-His, Boc-Trp, and Boc-Lys. After the condensation reaction, 50 parts of trifluoroacetic acid / dichloromethane solution were used for deprotection. After the peptide chain synthesis was completed, it was cut with hydrofluoric acid and extracted with ether to obtain a crude peptide. The crude peptide was dissolved in methanol / water (40:60, v / v), purified by HPLC, and the product was collected and lyophilized to obtain a pure product. Mass spectrometry showed that its molecular weight was correct and the purity was 96 parts.

[0026] When KWHPE was used alone for anti-inflammatory effect testing, the redness and swelling area was reduced by about 15% within 3 hours, and the itching level was reduced by about 20% within 2 hours.

[0027] Embodiment 3: Application sequence: Val - Thr - Met - Asn - Ser (VTMNS) Mechanism of action: Insect bites trigger the stress response of the human immune system, leading to the activation of immune cells and the release of a large number of inflammatory factors, which in turn aggravates the inflammatory response and itching symptoms. VTMNS small molecule peptides can interact with receptors on the surface of immune cells (such as toll-like receptors, T cell receptors, etc.), regulate the activation state of immune cells and the secretion pattern of cytokines. By inhibiting excessive immune responses, maintaining the balance and stability of the immune system, and reducing the occurrence of allergic reactions, the discomfort caused by mosquito bites can be relieved.

[0028] Target: Receptors on the surface of immune cells, such as Toll-like receptors on the surface of macrophages, T cell receptors on the surface of T cells, etc. When synthesizing SEQ NO3: Val - Thr - Met - Asn - Ser (VTMNS), the solid phase carrier was polystyrene resin, protected by Fmoc. Fmoc-Ser, Fmoc-Asn, Fmoc-Met, Fmoc-Thr and Fmoc-Val were coupled in sequence. Deprotection was performed using 25 parts of piperidine / DMF solution. Peptide chain cleavage was performed using a mixture of trifluoroacetic acid / thioanisole / 1,2-ethanedithiol / water (82.5:5:5:2.5, v / v / v / v), and the crude peptide was obtained after precipitation and centrifugation. HPLC purification was performed using acetonitrile / water (25:75, v / v) as the solvent, and the fractions were collected and dried in vacuo. The mass spectrometry identification results showed that its molecular weight was in line with expectations and the purity reached 97 parts.

[0029] When VTMNS was used alone for immunomodulation testing, the release of inflammatory factors was reduced by approximately 30%.

[0030] Synergistic comparison test: Set up four groups of comparative experiments: CRGLI was used alone for mosquito repellency testing.

[0031] KWHPE alone was tested for anti-inflammatory properties.

[0032] Immunomodulatory testing was performed using VTMNS alone.

[0033] A complex consisting of CRGLI, KWHPE, VTMNS and N,N-diethyl-2-hydroxy-2-phenylacetamide was also tested.

[0034] Comparison of mosquito repellent enhancement: The average protection time of the complex (including N,N-diethyl-2-hydroxy-2-phenylacetamide) reached 8 hours, and the average number of mosquito bites was 0.5 times, which was significantly better than the effect of using CRGLI and other small molecule peptides alone.

[0035] Comparison of anti-inflammatory and synergistic effects: The complex (including N,N-diethyl-2-hydroxy-2-phenylacetamide) significantly reduced the area of ​​redness and swelling within 0.5 hours, and the degree of itching was significantly reduced within 0.5 hours. Within 6 hours, the symptoms of redness, swelling and itching of most volunteers basically disappeared. The effect was significantly better than the use of KWHPE and other small molecule peptides alone.

[0036] Comparison of immunomodulatory enhancement: The complex (containing N,N-diethyl-2-hydroxy-2-phenylacetamide) reduced the release of inflammatory factors by about 70%, which is better than the effect of using VTMNS and other small molecule peptides alone.

[0037] 3. Extraction of essential oils Embodiment 4: Extraction of lavender essential oil: Select fresh, disease-free lavender flowers, wash and dry the surface moisture. Weigh 1000 grams of lavender flowers and put them into a distillation kettle, add 2000 ml of pure water. Heat the distillation kettle until the water boils and keep it in a slightly boiling state for 2 hours. The generated water vapor carries the essential oil components into the condenser, cools into liquid and flows into the receiver. The essential oil and water in the receiver form layers, and the lavender essential oil in the upper layer is separated. Impurities are removed by filtration to obtain about 20 ml of lavender essential oil. After gas chromatography analysis, the content of its main components linalool and linalyl acetate meets the standard.

[0038] Embodiment 5: Extraction of Artemisia argyi essential oil: Prepare 500 g of dried Artemisia argyi and crush it to a particle size of about 2 mm. Put the Artemisia argyi powder into a supercritical carbon dioxide extraction kettle, set the extraction temperature to 45°C, the pressure to 25 MPa, the carbon dioxide flow rate to 20 kg / hour, and the extraction time to 2.5 hours. The carbon dioxide fluid rich in essential oil enters the separator, and when the pressure drops to 8 MPa and the temperature drops to 35°C, the essential oil is released. Collect the essential oil and refine it to obtain about 10 ml of Artemisia argyi essential oil, and its main components and activity indicators meet the requirements after testing.

[0039] (IV) Preparation of the complex Embodiment 6: Weigh 12 g of purified small molecule peptide sequence 1 (CRGLI), 13 g of small molecule peptide sequence 2 (KWHPE), 18 g of small molecule peptide sequence 3 (VTMNS), 5 g of lavender essential oil, 2 g of mugwort essential oil, and 10 g of N,N-diethyl-2-hydroxy-2-phenylacetamide. Under a sterile environment, add these ingredients to a sterile blender, set the stirring speed to 800 rpm, the temperature to 30°C, and stir for 2.5 hours until a uniform and stable complex is formed. The prepared complex was subjected to quality testing, including component content determination, microbial testing, and stability testing, and the results were all in line with expectations.

[0040] Embodiment 7: Another portion of the same proportion of each component was taken, the stirring speed was adjusted to 600 rpm, the temperature was 28°C, and the stirring time was 3 hours, and a uniform composite was successfully prepared. The quality test showed that its various indicators were good, and there was no significant difference in performance with the composite prepared in Example 6.

[0041] (V) Performance testing Embodiment 8: Mosquito repellent effect test: a 30 square meter closed space was set up with a temperature of 26°C and a relative humidity of 70%. 60 volunteers were recruited and randomly divided into four groups, with 15 people in each group. The first group applied the complex of the present invention (containing N,N-diethyl-2-hydroxy-2-phenylacetamide) on the exposed skin (forearms and calves), the second group applied the complex containing only small molecule peptides, the third group applied the common commercially available chemical synthetic mosquito repellent (DEET), and the fourth group applied a placebo without active ingredients, with an application dose of 1.5ul / cm 2 . 1000 hungry female Aedes aegypti mosquitoes were released in the space. Volunteers sat quietly in it. If one mosquito bit them, it meant that the protection was ineffective. The protection time of each person was recorded and the average value was taken. The results showed that the average protection time of the first group applied with the N,N-diethyl-2-hydroxy-2-phenylacetamide complex of the present invention was 8 hours; the average protection time of the second group applied with the small molecule peptide complex was 2 hours; the average protection time of the third group applied with DEET was 3.8 hours; the average protection time of the fourth group applied with placebo was 0.1 hours.

[0042] Embodiment 9: Anti-inflammatory effect test: 60 volunteers with redness, swelling and itching of the skin after being bitten by mosquitoes were selected and divided into four groups, with 15 people in each group. The first group was smeared with the complex of the present invention (containing N,N-diethyl-2-hydroxy-2-phenylacetamide), the second group was smeared with a complex containing only small molecule peptides, the third group was smeared with a common anti-inflammatory and antipruritic ointment (Piyanping) on ​​the market, and the fourth group was smeared with a basic lotion without active ingredients. The redness and swelling area and the degree of itching were observed at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 12 hours after application. The results showed that the first group, which was applied with the N,N-diethyl-2-hydroxy-2-phenylacetamide complex of the present invention, had a significantly reduced redness and swelling area within 0.5 hours, and the degree of itching was significantly reduced, and the redness, swelling and itching symptoms of most volunteers basically disappeared within 6 hours; the second group, which was applied with the small molecule peptide complex, had a significantly reduced redness and swelling area within 1 hour, and the degree of itching was significantly reduced, and the redness, swelling and itching symptoms of most volunteers basically disappeared within 8 hours; the third group, which was applied with Piyanping, had a reduced redness and swelling area within 2 hours, and the itching was relieved, and the symptoms of most volunteers improved within 12 hours; the fourth group, which was applied with the basic lotion, had no obvious improvement in symptoms.

[0043] Embodiment 10: Safety test: 30 healthy volunteers were selected, and four areas of about 1 square centimeter were divided on the inner skin of their forearms. The complex of the present invention (containing N,N-diethyl-2-hydroxy-2-phenylacetamide) was applied to the first area, the complex containing only small molecule peptides was applied to the second area, 0.1 parts of sodium hydroxide solution was applied to the third area as a positive control, and physiological saline was applied to the fourth area as a negative control. Cover with gauze and fix for 48 hours. It was observed that the areas where the N,N-diethyl-2-hydroxy-2-phenylacetamide complex was applied and the areas where the small molecule peptide complex was applied had no symptoms such as redness, swelling, itching, and blisters after 1 hour, 24 hours, and 48 hours, while the positive control area showed obvious irritation and the negative control area showed no abnormality. In addition, a patch test was conducted, and the complex containing N,N-diethyl-2-hydroxy-2-phenylacetamide, the complex containing only small molecule peptides, the positive control (formaldehyde solution) and the negative control (vaseline) were loaded onto the patch test paper and applied to the upper back skin of the volunteers. The patch was removed after 48 hours, and the skin reaction was observed at 30 minutes, 24 hours, 48 ​​hours and 72 hours. The complex group containing N,N-diethyl-2-hydroxy-2-phenylacetamide and the complex group containing only small molecule peptides did not show allergic reactions such as erythema, papules, and blisters. The positive control group had obvious allergic symptoms, and the negative control group was normal.

[0044] (VI) Application The complex of the present invention can be prepared into various dosage forms, such as spray, emulsion, ointment, etc., for daily mosquito repelling and inflammation reduction.

[0045] Spray form: Dissolve the compound in an appropriate amount of ethanol / water solvent, add propellant, and put it into a spray bottle. When using, spray evenly at a distance of 15-20 cm from the skin.

[0046] Emulsion dosage form: Mix the complex with an emulsifier (such as Tween-80), a moisturizer (such as glycerin), etc., and make an emulsion through a homogenization emulsification process. When using, apply an appropriate amount of the emulsion to the skin.

[0047] Ointment: Mix the compound with a base such as vaseline or lanolin, heat and melt, stir evenly, and cool to room temperature to make an ointment. When using, use your finger or cotton swab to apply an appropriate amount of the ointment to the bite.

[0048] In different usage scenarios, consumers can choose the appropriate dosage form according to their personal needs. For example, when doing outdoor activities, you can carry a spray dosage form for easy use at any time; when sleeping at night, you can use an ointment dosage form to focus on the exposed parts; for daily protection, you can choose a lotion dosage form to apply all over the body.

[0049] Embodiment 11

[0050] A novel sustained-release compound, ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate, molecular formula C 21 H 18 ClNO 6, Principle of sustained release: This compound has a special chemical structure, and the specific structural formula is: a phenyl group is connected to the 3rd position of the oxazolidine ring (a hydroxyl group is connected to the 4th position), a phenyl group is connected to the 5th position (a methoxy group is connected to the 2nd position), a carboxylic acid methyl ester group is connected to the 4th position, and a carbonyl group is connected to the 2nd position. The specific connection method and atomic arrangement are marked as follows: The oxazolidine ring is composed of a nitrogen atom, an oxygen atom and three carbon atoms to form a five-membered ring. The phenyl group at the 3rd position is connected to the oxazolidine ring with a benzene ring, and a hydroxyl group is connected to the para position of the benzene ring; the phenyl group at the 5th position is connected to the oxazolidine ring with a benzene ring, and a methoxy group is connected to the ortho position of the benzene ring; the carboxylic acid methyl ester group at the 4th position is connected to the oxazolidine ring with a carbonyl group, and the carbonyl group is connected to an oxygen atom, and the oxygen atom is connected to a methyl group and a methylene group, and the methylene group is connected to another two methylene groups to form an ester group. Structural formula annotation: The oxazolidine ring is composed of a nitrogen atom (N), an oxygen atom (O) and three carbon atoms (C1, C2, C3) to form a five-membered ring. The 3-position of the oxazolidine ring is connected to a phenyl group, which is connected to the oxazolidine ring via a benzene ring (C4-C9), and the para position of the benzene ring (the carbon atom connected to C4) is connected to a hydroxyl group (-OH). The 5-position of the oxazolidine ring is connected to a phenyl group, which is connected to the oxazolidine ring via a benzene ring (C10-C15), and the ortho position of the benzene ring (the carbon atom connected to C12) is connected to a methoxy group (-OCH 3 The 4th position of the oxazolidine ring is connected to a methyl carboxylate group. The specific connection method is: the 4th position of the oxazolidine ring is first connected to a carbonyl group (C=O), the carbonyl group is then connected to an oxygen atom (O1), and the oxygen atom is connected to a methyl group (-CH 3 ) and a methylene group (-CH 2 -), which is then connected to two other methylene groups (-CH 2 -CH 2 -) to form an ester group (-COOCH 2 CH 2 CH 3). The ester group (-COO-) and hydroxyl group (-OH) in its molecule can interact with other components in the complex (such as small molecule peptides and N,N-diethyl-2-hydroxy-2-phenylacetamide, etc.) through hydrogen bonds or van der Waals forces. This interaction hinders the release of other components to a certain extent, thereby playing a role in slow release. Mosquito repellent and anti-inflammatory principle: 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester itself may have certain mosquito repellent and anti-inflammatory activity. It may interfere with the mosquito's olfactory receptor signal transduction pathway, similar to the mechanism of CRGLI small molecule peptides, interfere with mosquitoes' recognition of human odor, and reduce mosquitoes' desire to bite humans. In addition, the hydroxyl group in ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate may be involved in inhibiting the production and release of inflammatory mediators, similar to the anti-inflammatory mechanism of N,N-diethyl-2-hydroxy-2-phenylacetamide, thereby alleviating the inflammatory response after bites.

[0051] Studies have found that 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester may interact with olfactory receptor proteins in mosquitoes and certain proteins involved in the regulation of inflammatory responses (such as NF-κB-related proteins), affecting their normal physiological functions.

[0052] (II) Preparation method of novel sustained-release compound Using 2-hydroxy-3-methoxybenzoic acid and 4-chloroaniline as starting materials, first react in the presence of a suitable condensing agent (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC・HCl)) and a catalyst (such as 4-dimethylaminopyridine (DMAP)) at room temperature for 2-3 hours to generate an amide intermediate. .

[0053] The amide intermediate is then reacted with diethyl carbonate in the presence of a base (such as sodium carbonate) and heated under reflux for 3 to 5 hours to obtain the target compound 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester. .

[0054] Purification methods Silica gel column chromatography was used for purification, with petroleum ether-ethyl acetate (volume ratio of 5:1) as the eluent, the eluate containing the target compound was collected, and concentrated under reduced pressure to obtain the purified compound 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester, ensuring that its purity reached more than 98%.

[0055] Identification methods The compounds were identified by nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and mass spectrometry (MS).

[0056] NMR data: NMR (400 MHz, CDCl 3 ) δ 7.4 - 7.5 (m, 2H), 7.2 - 7.3 (m,2H), 6.8 - 6.9 (m, 1H), 5.5 (s, 1H), 4.3 (q, J = 7.1 Hz, 2H), 3.8 (s, 3H),1.3 (t, J = 7.1 Hz, 3H).

[0057] IR data: There are stretching vibration absorption peaks of hydroxyl group at around 3400 cm⁻¹, stretching vibration absorption peaks of carbonyl group at around 1730 cm⁻¹, and characteristic absorption peaks of benzene ring at around 1580 cm⁻¹.

[0058] MS data: m / z [M + H]⁺ = 362.

[0059] 3. Preparation process of complexes containing sustained-release compounds Preparation of raw materials In addition to the components prepared in Example 6, it is also necessary to prepare the raw materials required for synthesizing the compound 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester, such as 2-hydroxy-3-methoxybenzoic acid, 4-chloroaniline, EDC・HCl, DMAP, sodium carbonate and diethyl carbonate.

[0060] Synthesis and purification process First, the compound 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester was synthesized and purified according to the above method.

[0061] Preparation of complexes Weigh 12 grams of purified small molecule peptide sequence 1 (CRGLI), 13 grams of small molecule peptide sequence 2 (KWHPE), 18 grams of small molecule peptide sequence 3 (VTMNS), 8 grams of lavender essential oil, 5 grams of mugwort essential oil, 10 grams of N,N-diethyl-2-hydroxy-2-phenylacetamide, and 5 grams of compound 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester.

[0062] Under sterile conditions, these ingredients were added to a sterile blender, set at a stirring speed of 700 rpm and a temperature of 29°C, and stirred for 3 h until a uniform and stable complex was formed.

[0063] The prepared complex was subjected to quality testing, including component content determination, microbial testing and stability testing, and the results were in line with expectations.

[0064] Comparative synergistic test Anti-inflammatory and synergistic comparison Experimental method: A mouse skin inflammation model was used. A certain amount of mosquito saliva extract was applied to the back skin of the mouse to induce skin inflammation. Then the following treatments were applied to the inflammation site: Blank control group: no product was applied.

[0065] Complex (without ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate).

[0066] Complex containing ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate.

[0067] Results: The anti-inflammatory effect was evaluated by measuring the redness and swelling area of ​​the skin inflammation site and the levels of inflammatory mediators (such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). The blank control group had the largest redness and swelling area and the highest content of inflammatory mediators; when the composite (without ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate) was used, the redness and swelling area and the content of inflammatory mediators were reduced; when the composite contained ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate, the redness and swelling area and the content of inflammatory mediators were significantly lower than those of the first two groups, indicating that the composite containing ethyl 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylate had a better anti-inflammatory effect.

[0068] Comparison of immunomodulatory synergy Experimental method: The same method as the immunomodulatory test using VTMNS alone was used to observe the release of inflammatory factors.

[0069] Test results: When VTMNS was used alone, the release of inflammatory factors was reduced by about 30%; when the complex (without 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester) was used, the release of inflammatory factors was reduced by about 70%; when the complex contained 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester, the release of inflammatory factors was reduced by about 80%.

[0070] The high-efficiency mosquito repellent and anti-inflammatory complex based on small molecule peptides and N,N-diethyl-2-hydroxy-2-phenylacetamide of the present invention has significantly better mosquito repellent and anti-inflammatory effects, and is highly safe and stable, providing an innovative and effective solution to the problem of mosquito bites. Through reasonable formula design and advanced preparation technology, the synergistic effect of small molecule peptides, plant essential oils and N,N-diethyl-2-hydroxy-2-phenylacetamide is achieved, providing consumers with an efficient, safe and convenient protective product with broad market application prospects and social value.

Claims

1. A highly effective mosquito repellent and anti-inflammatory compound based on small molecule peptides, characterized in that: It comprises a small molecule peptide with the sequence of SEQ NO1: Cys-Arg - Gly - Leu - Ile, a small molecule peptide with the sequence of SEQ NO2: Lys - Trp - His - Pro -Glu, a small molecule peptide with the sequence of SEQ NO3: Val - Thr - Met - Asn - Ser, lavender essential oil, wormwood essential oil and N,N-diethyl-2-hydroxy-2-phenylacetamide.

2. The composite according to claim 1, characterized in that The weight proportion of the small molecule peptide with the sequence of SEQ NO1: Cys - Arg - Gly - Leu - Ile in the complex is 10 parts to 15 parts.

3. The composite according to claim 1, characterized in that The sequence is SEQ NO2: Lys - Trp -His - Pro - Glu, and the weight proportion of the small molecule peptide in the complex is 10 to 15 parts.

4. The composite according to claim 1, characterized in that The weight proportion of the small molecule peptide having the sequence of SEQ NO3: Val-Thr-Met-Asn-Ser in the complex is 15 to 20 parts.

5. The composite according to claim 1, characterized in that The weight proportion of the lavender essential oil in the composite is 5 to 10 parts.

6. The composite according to claim 1, characterized in that The invention also includes a sustained-release agent, wherein the weight proportion of the sustained-release agent 4-(2-hydroxy-3-methoxyphenyl)-6-(4-chlorophenyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid ethyl ester in the composite is 2 to 5 parts.

7. The composite according to claim 1, characterized in that The weight proportion of the N,N-diethyl-2-hydroxy-2-phenylacetamide in the composite is 5 to 10 parts.

8. A method for preparing a highly effective mosquito repellent and anti-inflammatory complex based on small molecule peptides as claimed in any one of claims 1 to 7, characterized in that: The method includes respectively synthesizing and purifying small molecule peptides, extracting lavender essential oil and mugwort essential oil, and then mixing and stirring the components in proportion.

9. Use of the high-efficiency mosquito repellent and anti-inflammatory complex based on small molecule peptides as described in any one of claims 1 to 7 in the preparation of mosquito repellent products.

10. Use of the high-efficiency mosquito repellent and anti-inflammatory complex based on small molecule peptides as claimed in any one of claims 1 to 7 in the preparation of anti-inflammatory products.