Preparation method and application of periplaneta americana water extract

Abstract

This invention provides a method for preparing and applying an aqueous extract of American cockroaches, belonging to the technical field of preparation and application of aqueous extracts of natural insects. The main method involves freeze-drying and pulverizing American cockroaches under two months old, followed by water extraction, enzymatic hydrolysis with trypsin, and defatting to prepare the extract. The American cockroach extract prepared by this invention has moisturizing, anti-inflammatory, antioxidant, and anti-allergic effects, and is odorless and has a fine texture, making it suitable for use in the cosmetics field.

Description

Technical Field

[0001] This invention belongs to the field of preparation and application technology of water extracts of natural insects, specifically relating to a method for preparing and applying water extracts of American cockroaches. Background Technology

[0002] Increased environmental pollution leads to excessive exposure of the body to pollutants such as ultraviolet radiation, inducing the production of excessive free radicals, such as superoxide anions and hydroxyl radicals. Poor lifestyle habits and work stress also increase free radicals in the modern human body. As people age, free radicals gradually accumulate. Excessive fat and free radicals can lead to cross-linking and polymerization of biomolecules and the accumulation of lipofuscin in organs and tissues. This damages and reduces organ and tissue cells, ultimately leading to aging. With improved living standards and increased awareness of skincare, the concept of healthy skin is gradually taking shape. Therefore, the application of general soothing, anti-allergic, and repairing ingredients in cosmetics is limited. As living conditions gradually improve, people's performance requirements for cosmetic products are gradually increasing. The emergence of natural herbal and insect anti-allergic active ingredients fills this gap and is favored by the market and consumers.

[0003] The American cockroach, scientifically known as *Periplaneta americana*, is an insect widely distributed globally, particularly common in tropical and subtropical regions. They are relatively large, with adults typically measuring 27 to 32 millimeters in length. They have reddish-brown bodies and well-developed wings. It is an important animal-based traditional Chinese medicine, rich in amino acids, polysaccharides, polypeptides, and fats. Water extracts of the American cockroach can increase collagen synthesis, promote wound healing, improve skin elasticity and luster, and inhibit inflammatory responses, showing good therapeutic effects on burns, ulcers, and other skin injuries. Simultaneously, it can scavenge free radicals, protect cells from oxidative damage, and delay cell aging; its water extract can also be used as a raw material in cosmetic formulations.

[0004] Existing methods for preparing American cockroach extract mainly employ the following methods: Water extraction: American cockroaches are mixed with water and soaked, then refluxed to extract the water extract. Alcohol precipitation: The obtained American cockroach water extract is precipitated with alcohol, concentrated, and the oil is removed to obtain a solution. Filtration and concentration: The solution is filtered and concentrated to obtain the American cockroach extract. These existing methods for preparing American cockroach extract suffer from problems such as low utilization rate, unsatisfactory yield, and complex composition.

[0005] The active ingredients extracted using current technologies are not fully understood, and their molecular mechanisms of action require further investigation. Current technologies use ethanol as a solvent for extraction, yielding mostly alcohol-soluble substances, primarily medicinal nucleic acids such as pyrimidines, uracil, hypoxanthine, thymine, and inosine, as well as alkaloids. Their efficacy is concentrated in anti-liver cancer, liver function protection, and treatment of ulcerative colitis. The functional effects of American cockroach extracts in current technologies are more focused on tissue repair, anti-tumor activity, and liver function protection against liver cancer; reports on its skincare effects are limited, and those that are extracted are often combined with other components, resulting in varying effects. There is a market need to research and develop an American cockroach extract suitable for skincare applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying an aqueous extract of American cockroach.

[0007] The water extract of cockroaches prepared by this invention has significant soothing, anti-allergic and repairing effects, and helps to resist the adverse effects of the external environment on the skin.

[0008] The technical solution of the present invention is as follows:

[0009] A method for preparing an aqueous extract of the American cockroach includes the following steps:

[0010] (1) Mix the crushed American cockroaches with deionized water at a mass ratio of 1:(5-20) and extract at 40-60℃ for more than 60 minutes under stirring conditions; then adjust the temperature to 20-50℃ and the pH to 6.5-7.5 and add trypsin. The amount of trypsin is 0.5-2.0% of the mass of American cockroaches. The enzymatic hydrolysis time is more than 1 hour. Then raise the temperature to 65-80℃ and hold for 30-120 minutes to inactivate and obtain the water-extracted enzymatic hydrolysate mixture.

[0011] (2) Filter the water-extracted enzymatic hydrolysis mixture obtained in step (1) to obtain filtrate, and then filter the filtrate to obtain filtrate.

[0012] (3) The filtrate obtained in step (2) is mixed with petroleum ether at a volume ratio of 1:(1-3), and reacted for 30-60 minutes under stirring. After standing and allowing the solution to separate into layers, the lower layer is retained to obtain defatted American cockroach water extract.

[0013] (4) The defatted American cockroach water extract obtained in step (3) is concentrated to obtain a concentrate, and the concentrate is freeze-dried to obtain American cockroach water extract.

[0014] According to a preferred embodiment of the present invention, the method for preparing pulverized American cockroaches in step (1) includes the following steps:

[0015] American cockroaches under two months old were freeze-dried, and then the freeze-dried cockroaches were crushed.

[0016] Further preferred, the particle size is between 80 and 200 mesh.

[0017] Further preferred methods involve fasting American cockroaches under two months of age from food and water for 1 to 3 days before treatment, followed by freeze-drying.

[0018] According to a preferred embodiment of the present invention, in step (1), the crushed American cockroaches are mixed with deionized water at a mass ratio of 1:10.

[0019] According to a preferred embodiment of the present invention, in step (1), the stirring conditions are 50 to 400 r / min.

[0020] According to a preferred embodiment of the present invention, in step (2), the water-extracted enzymatic hydrolysis mixture obtained in step (1) is filtered through a 60-200 mesh to obtain a filtrate, and then the filtrate is subjected to vacuum filtration with a pore size of 1-15 μm to obtain a vacuum filtrate.

[0021] According to a preferred embodiment of the present invention, in step (3), the stirring conditions are 50 to 100 r / min.

[0022] According to a preferred embodiment of the present invention, in step (4), the concentration conditions are a temperature of 40-55°C and a rotary evaporation rate of 100-200 r / min to obtain a concentrate, and the concentrate is subjected to vacuum freeze-drying to obtain an aqueous extract of American cockroach.

[0023] According to a preferred embodiment of the present invention, a method for preparing an aqueous extract of American cockroach includes the following steps:

[0024] ① Mix crushed American cockroaches with deionized water at a mass ratio of 1:10; extract by stirring at 150 r / min for 180 min at a reaction temperature of 40℃; then adjust the temperature to 37℃ and pH to 7.0, add trypsin at a mass ratio of 1% of American cockroaches, and perform enzymatic hydrolysis for 2 h; then raise the temperature to 65℃ and hold for 30 min to inactivate; after inactivation, stop heating to obtain the water-extracted enzymatic hydrolysate mixture;

[0025] ② The water-extracted enzymatic hydrolysis mixture obtained in step ① is filtered through an 80-mesh filter, and the filtrate is collected. The filtrate is then filtered by vacuum filtration with a pore size of 10μm to obtain the filtrate.

[0026] ③ Mix the filtrate obtained in step ② with petroleum ether at a volume ratio of 1:1, stir and react at room temperature for 30 minutes, then let stand at room temperature until the solution separates into layers, retain the lower layer liquid to obtain defatted American cockroach water extract;

[0027] ④ The defatted American cockroach aqueous extract obtained in step ③ is concentrated by rotary evaporation at a reaction temperature of 40℃ and then freeze-dried under vacuum to obtain freeze-dried powder of American cockroach aqueous extract.

[0028] Step ①, the method for preparing pulverized American cockroaches, includes the following steps:

[0029] American cockroaches under two months old were fasted from food and water for three days before treatment, then freeze-dried. The freeze-dried cockroaches were then pulverized to a mesh size of 150.

[0030] According to a preferred embodiment of the present invention, the aqueous extract of American cockroach prepared by the above method has a total sugar content of 10.62–38.51 mg / g, a monosaccharide content of 3.79–18.07 mg / g, a protein content of 59.84–89.59 mg / g, a total flavonoid content of 7.50–31.14 mg / g, and a polyphenol content of 0.10–1.0 mg / g.

[0031] Application of the water extract of American cockroach prepared by the above method in the preparation of soothing, anti-allergic and repair products.

[0032] Application of the aqueous extract of American cockroach prepared by the above method in the preparation of cosmetic products.

[0033] According to a preferred embodiment of the present invention, the water extract of American cockroach prepared by the above method is used in the preparation of soothing, anti-allergic and repairing cosmetic products.

[0034] A composition comprising an aqueous extract of American cockroaches prepared by the above method.

[0035] A cosmetic product comprising an aqueous extract of American cockroaches prepared by the above method.

[0036] The American cockroach (Periplaneta americana) is rich in polysaccharides, proteins, total phenols, and flavonoids, all of which play important physiological functions in organisms. American cockroach polysaccharides regulate immune function, helping to enhance the body's immunity. Polysaccharides can scavenge free radicals, improve antioxidant stress, maintain cell health, and inhibit inflammatory responses, thus promoting wound healing. American cockroach proteins also exhibit significant antioxidant and anti-inflammatory effects, not only aiding in cell repair and regeneration but also effectively soothing and reducing allergies, maintaining skin health. Total phenols and flavonoids further enhance its antioxidant and anti-inflammatory effects, protecting organisms from oxidative stress and inflammation. The effective components of the American cockroach water extract include total sugars, monosaccharides, proteins, total flavonoids, and polyphenols. The molecular structure of American cockroach polysaccharides possesses water-locking and water-absorbing properties, effectively moisturizing and improving the skin's barrier function. In cosmetic research, American cockroach extract shows great application potential. Firstly, polysaccharides possess excellent moisturizing and anti-allergic properties, forming a protective film on the skin's surface to prevent moisture loss, thus effectively hydrating the skin, making them particularly suitable for people with dry and sensitive skin. American cockroach proteins have gained widespread attention in anti-aging skincare products due to their superior antioxidant and anti-inflammatory properties. They can neutralize free radicals, reduce oxidative stress damage to the skin, delay skin aging, and promote cell repair and regeneration, improving skin elasticity and radiance.

[0037] The total phenols and flavonoids further enhance the antioxidant and anti-inflammatory effects of the American cockroach. They not only protect skin cells from oxidative stress but also inhibit inflammatory responses, alleviating redness and swelling. These properties make American cockroach extract a promising candidate for soothing and repairing skincare products. In summary, the application of American cockroach extract in cosmetics, with its significant moisturizing, antioxidant, anti-inflammatory, and soothing effects, provides a novel solution for skincare formulation design. These ingredients not only significantly improve the skincare efficacy of products but also meet consumers' demand for natural, safe, and effective skincare products.

[0038] The beneficial effects of the present invention include at least the following:

[0039] 1. Compared with the prior art, the method for preparing water extract of American cockroaches provided by the present invention has simple extraction and separation operation, high extraction rate of effective active ingredients, and high content of effective ingredients in the final product, which is of great significance for improving product quality.

[0040] 2. The water extract of American cockroaches prepared by the preparation method provided by the present invention can be used as a soothing, anti-allergic and repairing agent, and can be applied in the preparation of cosmetics, especially in the preparation of cosmetics with soothing, anti-allergic and repairing effects.

[0041] 3. The preparation method provided by this invention greatly improves the extraction rate and dissolution rate of active ingredients in water extracts of American cockroaches. The increased content of active ingredients makes the soothing, anti-allergic and repairing effects more significant. Attached Figure Description

[0042] Figure 1 Experimental diagrams of different enzymatic hydrolysis of water extract of American cockroach provided by the present invention;

[0043] In the figure: A is the filtrate obtained from the blank control, B is the filtrate obtained from trypsin hydrolysis, C is the filtrate obtained from fig protease hydrolysis, D is the filtrate obtained from cellulase hydrolysis, E is the filtrate obtained from elastase hydrolysis, F is the filtrate obtained from papain hydrolysis, and G is the filtrate obtained from pepsin hydrolysis.

[0044] Figure 2 Image of freeze-dried powder of water extract of American cockroach provided by the present invention.

[0045] Figure 3 The diagram shows the cytotoxicity experiment of the water extract of American cockroach provided by this invention.

[0046] Figure 4 The experimental diagram of hyaluronidase inhibition of water extract of American cockroach provided by the present invention.

[0047] Figure 5 Experimental diagram of IL-6, an anti-inflammatory factor from the aqueous extract of American cockroaches, provided by this invention.

[0048] Figure 6 Experimental diagram of TNF-α, an anti-inflammatory factor from the aqueous extract of American cockroaches, provided by this invention.

[0049] Figure 7 Experimental diagram of IL-1β, an anti-inflammatory factor from the aqueous extract of American cockroaches, provided by this invention.

[0050] Figure 8 The experimental diagram of DPPH of water extract of American cockroach provided by the present invention.

[0051] Figure 9 The diagram shows an experimental pattern of free radical scavenging from the water extract of American cockroaches provided by this invention.

[0052] Figure 10 Experimental diagram of superoxide anion scavenging of water extract of American cockroach provided by the present invention.

[0053] Figure 11 The diagram shows a human skin allergy test of water extract of American cockroach provided by this invention.

[0054] Figure 12 The graph shows the experimental results of the change rate of human body moisture content and water loss rate of water extract of American cockroach provided by the present invention.

[0055] Figure 13 The image shows the facial redness and heme index of the water extract of the American cockroach provided by this invention.

[0056] Figure 14 The diagram shows an experimental study on the improvement of human skin by water extract of American cockroach provided by this invention.

[0057] Figure 15 Human skin test diagrams for products containing the water extract of American cockroaches provided in this invention. Detailed Implementation

[0058] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0059] Unless otherwise described in the embodiments, all contents are based on existing conventional techniques in the art; experimental materials and reagents not described in detail are all common commercially available products.

[0060] Source of materials:

[0061] Trypsin: Source cultured trypsin EDTA solution, 0.25% Jiangsu Bomeida Life Science Co., Ltd.

[0062] Pepsin: Beijing Solarbio Technology Co., Ltd.

[0063] Fig protease: Shandong Xinxiong Biotechnology Co., Ltd.

[0064] Papain: Nanning Pangbo Biotechnology Co., Ltd.

[0065] Cellulase: Shandong Longkete Enzyme Preparation Co., Ltd.

[0066] Elastase: Shanghai Sangon Biotech Co., Ltd.

[0067] The following describes a method for preparing pulverized American cockroaches: This invention uses two-month-old American cockroach adults, which are fasted from food and water for 3 days before treatment, and then freeze-dried directly. After that, the freeze-dried American cockroaches are pulverized to a mesh size of 150.

[0068] During their experiments, the inventors discovered that when drying American cockroaches, whether by natural sun drying or oven drying, the dried cockroaches had a putrid odor, and the extracted cockroach extract also had an unpleasant smell. The inventors also found that when using older (over 2 months old) American cockroach adults to prepare the extract, the oil content increased, which was detrimental to the preparation of the water extract. The increased oil content led to a decrease in the extraction efficiency of water-soluble components, possibly because oil is hydrophobic and easily interacts with water-soluble components, affecting the extraction of the water extract.

[0069] The inventors discovered that using American cockroach adults under 2 months old results in lower oil content and relatively richer water-soluble components, which is beneficial for the extraction of water extracts. Furthermore, the use of low-temperature drying to dry the American cockroach adults effectively avoids the generation of odor and facilitates the extraction of water extracts.

[0070] Experimental Example 1

[0071] A method for preparing an aqueous extract of the American cockroach includes the following steps:

[0072] Different enzymes—trypsin, pepsin, papain, cellulase, figase, and elastase—were used for water extraction and enzymatic hydrolysis, as detailed below:

[0073] Weigh 10 kg of crushed American cockroaches and add 100 kg of deionized water; extract by stirring at a reaction temperature of 40℃ and a stirring speed of 50 r / min for 120 min.

[0074] (a) The enzymatic hydrolysis conditions for each group are detailed in Table 1. In Table 1, A: blank control, B: trypsin, C: figin, D: fibrinolytic enzyme, E: elastase, F: papain, and G: pepsin. Papain (1% by weight of American cockroach) was added at 37℃ and pH=7, and the enzymatic hydrolysis reaction was carried out for 2 hours. Trypsin (1% by weight of American cockroach) was added at 37℃ and pH=7, and the enzymatic hydrolysis reaction was carried out for 2 hours. Pepsin (1% by weight of American cockroach) was added at 37℃ and pH=2, and the enzymatic hydrolysis reaction was carried out for 2 hours. h; Adjust the temperature to 37℃ and pH=7, add 1% (w / w) of American cockroach fibrinolytic enzyme, and perform enzymatic hydrolysis for 2 hours; Adjust the temperature to 37℃ and pH=7, add 1% (w / w) of American cockroach elastase, and perform enzymatic hydrolysis for 2 hours; Adjust the temperature to 37℃ and pH=7, add 1% (w / w) of American cockroach fig protease, and perform enzymatic hydrolysis for 2 hours; Raise the temperature to 65℃ and stir for 30 minutes to inactivate the enzyme; Stop heating and filter coarsely using 60-mesh gauze; Take the filtrate and perform vacuum filtration using a Buchner funnel (10μm pore size) to obtain the filtrate, see [link to filtrate description]. Figure 1 ;

[0075] The blank control was prepared without the addition of enzyme preparations, using water extracts of American cockroaches.

[0076] (b) The filtrate obtained in step (a) was mixed with petroleum ether at a volume ratio of 1:1 and stirred at room temperature for 30 min. After standing at room temperature until the solution separated into layers, it was separated using a separatory funnel. The lower layer was retained to obtain a defatted American cockroach aqueous extract. The defatted American cockroach aqueous extract was rotary evaporated at a reaction temperature of 40°C and a rotary evaporation rate of 100 r / min. The extract was concentrated to less than one-third of the volume of the defatted American cockroach aqueous extract and then freeze-dried under vacuum to obtain the final freeze-dried powder of the American cockroach aqueous extract.

[0077] The water extract of American cockroach corresponding to trypsin is a clear light brown liquid. After shaking, the water sample to be tested is clear and transparent in a smooth glass container, without any impurities, particles, or unknown colloids. Light can pass through directly without any reflected light, and it has no pungent odor and a faint natural smell of American cockroach.

[0078] The water extract of American cockroaches corresponding to fig protease was more turbid and darker in color than the blank control, and had a slightly pungent odor.

[0079] The water extracts of American cockroaches corresponding to cellulase and papain are dark brown in color, relatively clear, and have a foul odor reminiscent of the insect itself.

[0080] The water extract of American cockroaches corresponding to elastase is clearer in color, leaning towards reddish-brown, and is a clear liquid with a faint natural insect smell.

[0081] The water extract of American cockroaches corresponding to pepsin is a brownish-yellow opaque liquid with a turbid state and a pungent American cockroach odor.

[0082] The protein content of the American cockroach water extracts obtained using different proteases is as follows:

[0083] The protein content was determined by the BCA protein quantification method. The experimental results are detailed in Table 1. The protein content shown in Table 1 is the protein content in the filtrate obtained after the above enzymatic hydrolysis.

[0084] The protein contents of trypsin, figase, cellulase, elastase, papain, and pepsin after enzymatic hydrolysis were 26.85 mg / mL, 23.69 mg / mL, 21.66 mg / mL, 25.67 mg / mL, 20.27 mg / mL, and 17.33 mg / mL, respectively. Trypsin and elastase showed better hydrolysis and higher extraction efficiency. When using aqueous solution as the solvent, polysaccharides and small peptides showed high dissolution rates, and the aqueous extract mainly consisted of water-soluble substances. Through comparison of enzymatic extraction with trypsin, pepsin, papain, cellulase, figase, and elastase, trypsin was ultimately determined to be the most effective and yielded the highest extraction rate.

[0085] Protein content experiments confirmed that the main active ingredients of the American cockroach are water-soluble substances. Therefore, in the extraction process, the American cockroach is first added to an aqueous solvent, followed by trypsin enzymatic hydrolysis. This method significantly improves the extraction rate and dissolution rate of the active ingredients of the American cockroach.

[0086] Table 1. Orthogonal design table of different proteases in American cockroaches

[0087]

[0088]

[0089] Experiment Example 2

[0090] This experiment used different conditions to extract American cockroach extract. First, single-factor experiments were conducted to determine the value ranges of various factors (enzymatic hydrolysis temperature, pH, enzyme dosage, substrate concentration, and hydrolysis time) for the enzymatic hydrolysis of American cockroach water extract. Based on this, orthogonal experiments were used to optimize the extraction process. Trypsin, which showed good results in Experiment 1, was selected to obtain the American cockroach water extract.

[0091] The specific method is as follows:

[0092] (a) Weigh 10 kg of crushed American cockroaches and add 100 kg of deionized water; extract by stirring at a reaction temperature of 40 °C and a stirring speed of 100 r / min for 60 min;

[0093] (b) Effect of feed-to-liquid ratio on yield

[0094] Using the yield of water extract of American cockroach as an indicator, under the conditions of fixed enzymatic hydrolysis temperature of 37℃, pH=7.0, enzymatic hydrolysis time of 1h, and the same amount of enzyme added, trypsin at a mass ratio of 1.0% of American cockroach was added to investigate the effect of different material-liquid ratios of American cockroach to deionized water (1:10, 1:20, 1:30 g / mL) on the yield of water extract of American cockroach.

[0095] Effect of extraction time on yield

[0096] Using the yield of water extract of American cockroach as an indicator, under the conditions of fixed enzymatic hydrolysis temperature of 37℃, pH=7.0, and the same amount of enzyme added, and with the addition of trypsin at a mass ratio of 1.0% of American cockroach, the effect of different extraction times of 1, 2, and 3 h on the yield of water extract of American cockroach was investigated.

[0097] Effect of extraction temperature on yield

[0098] Using the yield of water extract of American cockroach as an indicator, under the conditions of fixed enzymatic hydrolysis time of 1 h, pH = 7.0, and the same amount of enzyme added, and with the addition of trypsin at a mass ratio of 1.0% of American cockroach, the effect of different extraction temperatures of 30, 40, and 50 °C on the yield of water extract of American cockroach was investigated.

[0099] Effect of enzymatic hydrolysis time on yield

[0100] Using the yield of water extract of American cockroach as an indicator, under the conditions of fixed enzymatic hydrolysis temperature of 37℃, pH=7.0, and the same amount of enzyme added, the effects of different enzymatic hydrolysis times of 1, 2, and 3 h on the yield of water extract of American cockroach were investigated.

[0101] Orthogonal experiments were used to optimize the mixed enzyme hydrolysis process. Based on the single-factor experiments, the yield of water extract from American cockroaches was used as the indicator. An orthogonal experiment with four factors and three levels was designed using the L9(3”) orthogonal array (Table 2).

[0102] (c) The samples obtained from the above orthogonal experiments were heated to 70℃ and stirred for 60 min for inactivation; heating was stopped, and the samples were coarsely filtered using 100-mesh gauze; the filtrate was collected and filtered using a Buchner funnel (filter plate pore size 10 μm) to obtain a filtrate; the same volume of petroleum ether was added to the filtrate, and the mixture was reacted at 100 r / min for 30 min at room temperature, then allowed to stand at room temperature until the solution separated into layers, which were then separated using a separatory funnel, retaining the lower layer to obtain a defatted American cockroach aqueous extract; the defatted American cockroach aqueous extract was rotary evaporated at a reaction temperature of 45℃ and a rotary evaporation rate of 120 r / min; the resulting concentrate was freeze-dried under vacuum to obtain the final American cockroach aqueous extract lyophilized powder. The yield of the American cockroach aqueous extract is the ratio of the mass of the freeze-dried sample to the mass of the original sample.

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107] Table 3 above shows that the effects of enzymatic hydrolysis time, extraction time, extraction temperature, and solid-liquid ratio on the yield were studied using a four-factor, three-level ANOVA, with the solid-liquid ratio included as a covariate in the model. Table 3 indicates that the solid-liquid ratio has a significant impact on the yield (p<0.05). The effects of enzymatic hydrolysis time, extraction temperature, and extraction time on the yield are not significant (p>0.05). These results indicate that adjusting the solid-liquid ratio can significantly affect the yield of the aqueous extract of *Periplaneta americana*, while the effects of enzymatic hydrolysis time, extraction temperature, and extraction time are relatively small. In this experiment, the solid-liquid ratio significantly affected the enzymatic hydrolysis process of *Periplaneta americana* powder, indicating that it is a key factor. The optimal extraction process was pH=7, extraction temperature 40℃, enzymatic hydrolysis time 2h, solid-liquid ratio 1:10, extraction time 3h, and enzyme dosage of 1% of the mass of *Periplaneta americana*, achieving a maximum yield of 20.873% (see Table 2). The optimal extraction process was used to prepare the aqueous extract of *Periplaneta americana*.

[0108] Example 1

[0109] Preparation of water extracts from American cockroaches

[0110] (1) Weigh 10 kg of crushed American cockroaches and add 100 kg of deionized water; extract by stirring at 150 r / min for 180 min at a reaction temperature of 40℃; then adjust the pH to 7.0 and the temperature to 37℃ and add trypsin at a mass ratio of 1% of American cockroaches, and perform enzymatic hydrolysis for 2 h; then raise the temperature to 65℃ and hold for 30 min to inactivate; after inactivation, stop heating to obtain a water-extracted enzymatic hydrolysis mixture;

[0111] (2) The water-extracted enzymatic hydrolysis mixture obtained in step (1) is coarsely filtered with 80-mesh gauze, the filtrate is collected, and then the filtrate is filtered by vacuum using a 10μm filter plate to obtain the vacuum filtrate.

[0112] (3) The filtrate obtained in step (2) was mixed with petroleum ether at a volume ratio of 1:1 and reacted at 100 r / min for 30 min at room temperature. After standing at room temperature for 12 h, the solution was separated into layers. The upper layer was petroleum ether and the lower layer was American cockroach liquid. The solution was then separated using a separatory funnel, and the lower layer was retained to obtain defatted American cockroach aqueous extract. The defatted American cockroach aqueous extract was evaporated at a reaction temperature of 40℃ and a rotary evaporation rate of 100 r / min to concentrate it to less than one-third of the volume of the defatted American cockroach aqueous extract. The extract was then freeze-dried under vacuum to obtain freeze-dried powder of American cockroach aqueous extract. The yield of the American cockroach extract was 21.073%.

[0113] The resulting freeze-dried powder of the water extract of American cockroach was grayish-white. Figure 2 It has no odor, a fine texture, and can be used in the cosmetics industry.

[0114] Comparative Example 1

[0115] The preparation of aqueous extracts from American cockroaches includes the following steps:

[0116] The difference from Example 1 is that the pulverized American cockroaches were first defatted, and then subjected to water extraction and enzymatic hydrolysis. The specific method is as follows:

[0117] (a) Weigh 10 kg of crushed American cockroaches and add petroleum ether at a mass-to-volume ratio of 1:1 g / mL to the American cockroaches. Let the mixture stand at room temperature for 12 h. Pour the mixture of American cockroaches and petroleum ether into filter paper, place the filter paper on a funnel, and connect the funnel to a vacuum device. Turn on the vacuum pump, and the petroleum ether will be rapidly filtered through the filter paper, leaving the American cockroaches on the filter paper. Add deionized water at a mass-to-volume ratio of 1:10 g / mL to the American cockroach feed solution, and extract by stirring at 40℃ and 150 rpm for 180 min. Then adjust the temperature to 37℃ and pH=7.0, add trypsin at 1% of the mass of the American cockroach, and perform enzymatic hydrolysis for 2 h. Inactivate the enzyme by heating to 65℃ and 100 rpm for 30 min. Stop heating and filter coarsely using 80-mesh gauze. Collect the filtrate and filter it using a Buchner funnel (10 μm filter plate pore size) to obtain the defatted American cockroach aqueous extract.

[0118] (b) The defatted American cockroach water extract was rotary evaporated at a reaction temperature of 40°C and a rotary evaporation rate of 100 r / min until the volume of the defatted American cockroach water extract was less than one-third. The resulting concentrate was then freeze-dried under vacuum to obtain the final freeze-dried powder of American cockroach water extract.

[0119] The yield of the obtained water extract of American cockroach was only 10.69%, which was far lower than the yield of the water extract of American cockroach prepared in Example 1.

[0120] Example of effect 1

[0121] Determination of effective components in water extract of American cockroach

[0122] Taking the aqueous extract of *Periplaneta americana* prepared in Example 1 as an example, the effective components were determined, and the results are as follows: Using the phenol-sulfuric acid method, the total sugar content of the aqueous extract of *Periplaneta americana* prepared in Example 1 was determined to be 28.51 mg / g; using the 3,5-dinitrosalicylic acid (DNS) method, the monosaccharide content of the aqueous extract of *Periplaneta americana* prepared in Example 1 was determined to be 8.07 mg / g; using the Bradford method, the protein content of the aqueous extract of *Periplaneta americana* prepared in Example 1 was determined to be 79.84 mg / g; using the sodium nitrite-aluminum nitrate method, the total flavonoid content of the aqueous extract of *Periplaneta americana* prepared in Example 1 was determined to be 31.14 mg / g; and using the Folin-Ciocalteu colorimetric method, the total polyphenol content of the aqueous extract of *Periplaneta americana* prepared in Example 1 was determined to be 0.89 mg / g.

[0123] Example 2

[0124] American cockroach water extract cytotoxicity

[0125] (1) Weigh 0.02 g of the water extract prepared in Example 1, add an appropriate amount of PBS (pH 7.2-7.4) to dissolve it completely, prepare a 20 mg / mL stock solution, filter it through a 0.22 μm microporous membrane, store it in a refrigerator at 4℃, and prepare the required concentration for the experiment. Prepare a cell suspension, seed it in a 96-well plate, add 100 μL of cell solution to each well, and the cell density per well is 6-8 × 10⁶ cells / well. 3 .

[0126] (2) Human immortalized keratinocytes HaCaT (purchased from Wuhan Pronos Company) were used as a cell model. After the epidermal cell density was appropriate, the cells were digested with trypsin and incubated at 37°C and 5% CO2 for 24 hours.

[0127] (3) The cytotoxicity experiment included a blank control group (a blank control group without any sample) and experimental groups (experimental groups containing water extracts of American cockroaches at different concentration gradients). Concentration gradients of the analytes were set up, with at least three parallel controls for each concentration. The sample concentration was set at 400-1500 μg / mL. After the cells adhered firmly overnight, samples containing different concentrations were added. The cells were then incubated for 24 hours.

[0128] (4) Remove the 96-well plate from the incubator, add 10 μL of CCK-8 detection solution to each well, and incubate at 37°C and 5% CO2 for 3 hours. After incubation, measure the absorbance at 450 nm.

[0129] (5) Results analysis: Using the measured absorbance values, the cell viability was calculated using the following formula: Cell proliferation rate (%) = (A0 / Ax) × 100% (Ax represents the absorbance of the blank control group at 450nm, and A0 represents the absorbance of the sample group at 450nm).

[0130] The cytotoxicity results of the water extract of American cockroach obtained in Example 1 are shown in [Figure 1]. Figure 3 .like Figure 3 As shown, the addition of the aqueous extract of the American cockroach significantly promoted cell proliferation. This indicates that the HaCaT proliferation rate of the aqueous extract of the American cockroach obtained in Example 1 was above 100% at different concentrations, showing significant proliferation compared to the control group. This experiment concludes that the extract has virtually no toxic effects on cells. It can promote cell proliferation, thus providing a good soothing and anti-allergic effect.

[0131] Example 3

[0132] Hyaluronidase activity inhibition experiment

[0133] Studies have found a close link between hyaluronidase and inflammation and allergies. This enzyme can degrade hyaluronic acid in the body, converting it into low-molecular-weight acidic irritants, thereby inducing allergic symptoms. This method uses an in vitro hyaluronidase inhibition assay to determine the inhibitory effect of water extracts from the American cockroach on hyaluronidase activity, with experimental concentrations of 100 μg / mL, 1 mg / mL, and 5 mg / mL.

[0134] Preparation of solutions required for the experiment:

[0135] Acetic acid buffer solution: Measure 1.155 mL of glacial acetic acid, dilute to 100 mL and mix well, take 4.8 mL of it as solution A; weigh 2.72 g of sodium acetate crystals, dissolve in water and make up to 100 mL and mix well, take 45.2 mL of it as solution B; mix solutions A and B, make up to 100 mL with water and mix well.

[0136] Hyaluronidase solution: Weigh 10 mg of hyaluronidase into a beaker and add 4 mL of acetate buffer solution.

[0137] Sodium hyaluronate solution: Weigh 5 mg of sodium hyaluronate into a beaker and add 10 mL of acetic acid buffer solution.

[0138] Ehrlich reagent: Weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol.

[0139] Acetylacetone solution: Dissolve 3.5 mL of acetylacetone in 50 mL of 1.0 mol / L sodium carbonate solution. This solution should be prepared before use.

[0140] Experimental steps:

[0141] Take 0.1 mL of 0.25 mmol / L CaCl2 solution and 0.5 mL of hyaluronidase solution, and incubate at 37 °C for 20 min; add 0.5 mL of water extract of American cockroach prepared in Example 1 at different concentrations, and continue incubation at 37 °C for 20 min; add 0.5 mL of sodium hyaluronate solution and incubate at 37 °C for 30 min, then place at room temperature for 5 min; add 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 15 min, and immediately cool with ice water for 5 min; add 1.0 mL of Ehrlich reagent and dilute with 3.0 mL of anhydrous ethanol, allow to develop color for 20 min, and measure its absorbance value with a spectrophotometer.

[0142] Formula for calculating the inhibition rate of hyaluronidase on a sample:

[0143] Hyaluronidase inhibition rate (%) = [(A1-A2)-(B1-B2)] / (A1-A2)×100%

[0144] Wherein, A1 is the absorbance of the reference solution, with deionized water used instead of the sample obtained in Example 1; A2 is the absorbance of the reference blank solution, with deionized water used instead of the sample obtained in Example 1, and acetate buffer solution used instead of hyaluronidase and sodium hyaluronate solution; B1 is the absorbance of the sample solution; B2 is the absorbance of the sample blank solution, with acetate buffer solution used instead of hyaluronidase and sodium hyaluronate solution.

[0145] During the experiment, the sample was first scanned in the wavelength range of 450-700nm to determine the maximum absorption wavelength, and the measurement was performed at the maximum absorption wavelength of 530nm.

[0146] The hyaluronidase inhibition rate of the water extract of *Periplaneta americana* obtained in Example 1 is shown in the figure below. Figure 4 .like Figure 4 As shown, the inhibitory effect on hyaluronidase activity gradually increased with the increase of the concentration of the aqueous extract of *Periplaneta americana*. This indicates that the aqueous extract of *Periplaneta americana* obtained in Example 1 can effectively inhibit hyaluronidase activity, thereby playing a good role in soothing and relieving allergies.

[0147] Example of effect 4

[0148] Anti-inflammatory test

[0149] Taking the aqueous extract of *Periplaneta americana* prepared in Example 1 above as an example, the in vitro anti-inflammatory effects of the aqueous extract were determined using mouse IL-6 ELISA, IL-1β ELISA, and TNF-α ELISA kits. Mouse macrophages (RAW246.7) were prepared at a concentration of 2.6 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well into 12-well plates and incubated for 24 hours in an incubator (37°C, 5% CO2). The experiment included a blank control group, a positive control group, and experimental groups. 1 mg / mL and 10 mg / mL of American cockroach water extract were added to the experimental groups. After 2 hours, 1 μg / mL of LPS was added again for stimulation to construct a cellular inflammation model. The supernatant was collected, centrifuged at 10,000 rpm for 5 min, and the levels of various inflammatory factors in the supernatant were detected using an ELISA kit. Each group was set up in triplicate. The concentrations of IL-6, TNF-α, and IL-1β in each group were detected using a mouse IL-6, IL-1β, and TNF-α ELISA kit. Specific results are shown below. Figure 5 , Figure 6 , Figure 7 .

[0150] Depend on Figure 5 , Figure 6 , Figure 7As can be seen, the positive control group with added LPS resulted in a significant increase in intracellular inflammatory factors, proving that LPS successfully induced the inflammatory response. The experimental group with added American cockroach aqueous extract showed a significant anti-inflammatory effect, manifested by a significant reduction in the release of inflammatory factors. This indicates that American cockroach aqueous extract can effectively inhibit LPS-induced inflammatory responses and has potential anti-inflammatory application value.

[0151] Example 5

[0152] Antioxidant experiment

[0153] 1. DPPH is a relatively stable free radical, appearing deep purple in methanol or ethanol solutions, with a maximum absorption peak at 520 nm. The ability to scavenge DPPH is essentially a test of the antioxidant capacity of a sample. Antioxidants can donate H+, converting DPPH to DPPH-H, causing the solution color to disappear or lighten. This can be analyzed by measuring the absorbance. The change in absorbance before and after the addition of DPPH free radicals can evaluate the ability of the antioxidant in the sample to scavenge DPPH free radicals. A greater change in absorbance indicates a stronger antioxidant capacity.

[0154] Prepare 0.1 mM DPPH solutions. Dissolve 0.002 g of DPPH in 50 mL of ethanol each time and store in the dark. The experiment can be performed in 96-well plates with three replicates per group to reduce experimental error. Zero the plate with anhydrous ethanol. Sample group: Dissolve an appropriate amount of the American cockroach water extract in 180 μL of distilled water to achieve final concentrations of 100 μg / mL, 1 mg / mL, and 5 mg / mL, respectively. Add 90 μL of DPPH solution. Control group: Add 180 μL of the corresponding concentration of dimethyl sulfoxide (DMSO) to 90 μL of DPPH solution. Blank group: Add 90 μL of anhydrous ethanol to 180 μL of sample solution. Shake in the dark for 10 min and measure the absorbance. Scavenging rate % = [A control group - (A sample group - A blank group)] / A control group * 100%.

[0155] Depend on Figure 8 The data show that the aqueous extracts of American cockroaches prepared in Example 1 all have a good scavenging effect on DPPH free radicals, indicating that the extract has good antioxidant activity. Furthermore, as the sample concentration increases, the absorbance of the solution decreases, and the scavenging effect on DPPH free radicals also increases.

[0156] 2. This experiment utilizes the salicylic acid method and the Fenton reaction. The principle of this reaction is that the hydroxyl radicals generated in the reaction readily react with salicylic acid. The product obtained from the reaction has a specific absorption peak at 510 nm. The hydroxyl radical scavenging ability of the test substance is evaluated by detecting the absorbance of the substance.

[0157] Figure 9 The results showed that the aqueous extract of American cockroach in Example 1 had a certain scavenging effect on hydroxyl radicals. Within a certain concentration range, the scavenging effect of the American cockroach extract on hydroxyl radicals increased with increasing concentration.

[0158] 3. Ability to scavenge superoxide anion free radicals

[0159] Under weakly alkaline conditions, pyrogallol undergoes an oxidation reaction, producing a colored intermediate and O. 2- As the reaction continues, the solution color changes continuously over time. The experiment measured the linear relationship between the amount of intermediate formed and time during the initial stage of pyrogallol auto-oxidation. When the extract has the ability to scavenge O... 2- When it has the ability to catalyze the combination of hydrogen ions and oxygen ions to generate oxygen and hydrogen peroxide, it can inhibit the formation of colored intermediates and reduce the absorbance of the solution at 320 nm.

[0160] Therefore, by measuring the change in absorbance of the tested solution per minute, the inhibition rate of the extract against superoxide anion free radicals can be calculated.

[0161] Experimental reagents:

[0162] (1) 10 mmol / L HCl: Take 0.122 g of 30% concentrated hydrochloric acid solution into a 100 ml volumetric flask and dilute to 100 ml to prepare 10 mmol / L HCl.

[0163] (2) 5 mmol / L pyrogallol: Weigh 0.63 g of pyrogallol and dissolve it in 100 ml of 10 mmol / L HCl solution.

[0164] (3) 0.1 mol / L Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride: Weigh 1.211 g of Tris and dissolve it in 90 ml of distilled water. Adjust the pH to 8.2 with HCl and bring the volume to 100 ml.

[0165] Experimental steps:

[0166] (1) Solution A: Add 600 μl of 0.1 mol / L Tris-HCl buffer solution and 580 μl of distilled water, mix well, keep in water at 25℃ for 20 min, and add 200 μl of the sample from Example 1 with concentrations of 100 μg / ml, 1 mg / mL and 10 mg / mL;

[0167] (2) Solution B: 0.02 ml of 5 mmol / L pyrogallol solution that has been preheated;

[0168] (3) After preparing solutions A and B, mix them quickly and shake well. Measure the absorbance of the sample solution at 320 nm every minute for four consecutive times (4 min in total). Record the experimental data and use the regression method to determine the absorbance slope k (use 10 mmol / L HCl instead of pyrogallol solution to zero the absorbance).

[0169] (4) Sample addition, see Table 4;

[0170] Table 4

[0171]

[0172] Inhibition rate (%) = (△A0 - △A) / △A0 * 100%

[0173] △A0 represents the auto-oxidation rate of pyrogallol; △A represents the auto-oxidation rate of pyrogallol after the sample solution is added.

[0174] Figure 10 Data analysis concluded that the aqueous extract of American cockroach in Example 1 has a good scavenging ability against superoxide anion free radicals, thus it can be considered that American cockroach has a strong effect in scavenging superoxide anion free radicals.

[0175] Example 6

[0176] Human sensitivity testing

[0177] Thirty volunteers, half male and half female, were selected. Before the experiment, the volunteers' left forearms were cleaned with water and they sat quietly in an environment with a temperature of 20-22℃ and a relative humidity of 50-60%. The subjects did not apply any cosmetics and underwent a skin sensitivity test.

[0178] The product, formulated with water extract of American cockroach prepared in Example 1 (see Table 6), was applied to the left forearm of the subject. It was applied once every half hour for a total of 6 applications, covering an area of ​​3×3 cm. 2 The reactions of the subjects' arms were observed, and the statistical results of the sensitization rate are shown in Table 5. From Figure 11 As can be seen from Table 5, after applying the aqueous extract of the American cockroach prepared in Example 1, there was no stinging sensation or redness on the left forearm, indicating that the aqueous extract of the American cockroach is safe and reliable for human use. (See Table 5 for experimental results.) Figure 11 It can be seen that none of the 30 subjects showed a positive reaction. According to the 2022 Cosmetic Safety Technical Specifications, this product did not cause adverse reactions on human skin. Therefore, we can conclude that this product did not cause adverse reactions on human skin, and the closed patch test was successful.

[0179] Table 5 Results of skin patch experiment

[0180]

[0181] Experimental Example 10

[0182] Human body moisturizing experiment

[0183] Human moisturizing experiments involve a specific experimental population as the test subjects. The subjects are tested for changes in skin moisture and water loss before and after using cosmetics (and their active ingredients) to determine the moisturizing effect of the cosmetics (or active ingredients).

[0184] Experimental method: Volunteers aged 15 to 60 years old were selected, with 15 males and 15 females.

[0185] (1) Select the left and right arms of the subject and mark them in turn: The test area is prepared using the product made from the water extract of American cockroach prepared in Example 1, the formula of which is shown in Table 6;

[0186] (2) Technicians used a Corneometer CM825 skin moisture meter and a Tewameter TM300 skin moisture loss meter to measure the moisture content and take the average value as the blank value.

[0187] (3) Apply the test sample to the test site of the subject after it has been washed with water.

[0188] (4) After the subjects used the sample for 14 and 28 consecutive days, technicians measured the skin moisture content 5 times using a Corneometer CM825 and a Tewameter TM300 skin moisture loss tester, and the average value was taken.

[0189] (5) Statistically analyze the measured values ​​and the changes in skin moisture content and moisture loss.

[0190] The curves showing the changes in average skin surface moisture content and average percentage of moisture loss over time for the subjects are as follows: Figure 12 As shown in the figure. The results indicate that the aqueous extract of American cockroaches prepared in Example 1 has a good long-lasting moisturizing effect. It also shows that it has good properties for enhancing the skin barrier function.

[0191] Experimental Example 11

[0192] Soothing and anti-allergic effects on human skin

[0193] Before the test, the test area was cleansed with a standardized facial cleanser. Images of the subjects before product use (e.g., left and right sides of the face or left and right corners of the eyes) were taken and rated (visual evaluation and photo evaluation), which were recorded as the initial values. When taking the photos, the subjects closed their eyes and were in a relaxed state.

[0194] The test included an essence formula containing water extract of American cockroaches. The subjects were instructed on how to use it according to the instructions. After cleansing their faces for 30 minutes, the subjects' facial images were taken using the VISIA skin image analyzer to observe their facial texture and redness differentiation values. A before-and-after treatment comparison analysis was also conducted.

[0195] The facial redness values ​​and spot measurement results are shown in Tables 7 and 8. Figure 13 , Figure 14 Compared to baseline values, facial redness scores and spots decreased significantly. The average facial redness score decreased by 6.31% and 7.59% on day 14 and day 28, respectively, showing significant differences on both days (0.001≤P<0.01). The average heme content decreased by 6.10% and 5.11% on day 14 and day 28, respectively, showing a significant difference on day 28 (0.01≤P<0.05).

[0196] It is evident that the essence formula containing water extract of American cockroach (Table 6) has a good soothing and repairing effect.

[0197] Formulation process: Turn on the emulsification pot, add water, add phase A raw material in sequence, turn on homogenization until the material is uniform, and heat to 85℃; add phase B to the beaker of the water bath, heat to 85℃ until the material is dissolved and transparent, transfer phase B into the emulsification pot, turn on homogenization until the material is uniform, and cool down to 45℃; add phase C and phase D in sequence to the emulsification pot, turn on stirring until the material is uniform, and remove from the pot after passing the test.

[0198] Table 6. Formula of American cockroach water extract

[0199]

[0200] Table 7 Comparative Analysis of Data for Various Indicators at Different Time Periods (X±s, n=30)

[0201]

[0202]

[0203] Table 8. Improvement rate and differences of various indicators in different periods.

[0204]

[0205] Note: *Significance notation method: "ns" indicates no statistical difference, P>0.05; "*" indicates a significant difference, 0.01≤P<0.05; "**" indicates a significant difference, 0.001≤P<0.01; "***" indicates an extremely significant difference, P<0.001.

[0206] According to the formula provided in Table 6, but without adding the American cockroach extract prepared in Example 1, all other raw material components were the same, and the product was prepared according to the above method. Corresponding experimental tests were conducted according to the above method, and it was found that the product had no significant effect on reducing facial redness and heme content. (See...) Figure 15 .

[0207] Experimental results show that products containing the American cockroach extract prepared in this invention have significantly reduced facial redness and heme content, indicating that the American cockroach extract prepared in this invention can effectively optimize skin condition.

[0208] The extraction method provided by this invention prepares an extract of American cockroaches that is simple to extract and separate, has a high extraction rate of effective active ingredients, and has moisturizing, anti-inflammatory, antioxidant, and anti-allergic effects. It can be used as a soothing, anti-allergic, and repairing agent and can be applied in the preparation of cosmetics, especially in the preparation of cosmetics with soothing, anti-allergic, and repairing effects.

[0209] The American cockroach extract prepared by this invention is odorless, has a fine texture, and is also suitable for use in the cosmetics field.

Claims

1. A method for preparing an aqueous extract of the American cockroach, characterized in that, Includes the following steps: (1) Mix the crushed American cockroaches with deionized water at a mass ratio of 1:(5-20) and extract at 40-60℃ for more than 60 minutes under stirring conditions; then adjust the temperature to 20-50℃ and the pH to 6.5-7.5 and add trypsin. The amount of trypsin is 0.5-2.0% of the mass of American cockroaches. The enzymatic hydrolysis time is more than 1 hour. Then raise the temperature to 65-80℃ and hold for 30-120 minutes to inactivate and obtain the water-extracted enzymatic hydrolysate mixture. (2) Filter the water-extracted enzymatic hydrolysis mixture obtained in step (1) to obtain filtrate, and then filter the filtrate to obtain filtrate. (3) The filtrate obtained in step (2) is mixed with petroleum ether at a volume ratio of 1:(1-3), and reacted for 30-60 minutes under stirring. After standing and allowing the solution to separate into layers, the lower layer is retained to obtain defatted American cockroach water extract. (4) The defatted American cockroach water extract obtained in step (3) is concentrated to obtain a concentrate, and the concentrate is freeze-dried to obtain American cockroach water extract.

2. The preparation method according to claim 1, characterized in that, In step (1), the method for preparing crushed American cockroaches includes the following steps: taking American cockroach adults under two months old, then freeze-drying them, and then crushing the freeze-dried cockroaches; Preferably, the particle size is between 80 and 200 mesh; Preferably, American cockroaches under two months old are fasted from food and water for 1 to 3 days before treatment, and then freeze-dried.

3. The preparation method according to claim 1, characterized in that, In step (1), the crushed American cockroaches are mixed with deionized water at a mass ratio of 1:

10. Preferably, the stirring conditions are 50–400 r / min.

4. The preparation method according to claim 1, characterized in that, In step (2), the water-extracted enzymatic hydrolysis mixture obtained in step (1) is filtered through a 60-200 mesh to obtain a filtrate, and then the filtrate is vacuum filtered with a pore size of 1-15 μm to obtain a vacuum filtrate.

5. The preparation method according to claim 1, characterized in that, In step (3), the stirring conditions are 50–100 r / min; Preferably, in step (4), the concentration conditions are a temperature of 40-55℃ and a rotary evaporation speed of 100-200 r / min to obtain a concentrate, and the concentrate is subjected to vacuum freeze drying to obtain a water extract of American cockroach.

6. The preparation method according to claim 1, characterized in that, Includes the following steps: ① Mix crushed American cockroaches with deionized water at a mass ratio of 1:10; extract by stirring at 150 r / min for 180 min at a reaction temperature of 40℃; then adjust the temperature to 37℃ and pH to 7.0, add trypsin at a mass ratio of 1% of American cockroaches, and perform enzymatic hydrolysis for 2 h; then raise the temperature to 65℃ and hold for 30 min to inactivate; after inactivation, stop heating to obtain the water-extracted enzymatic hydrolysate mixture; ② The water-extracted enzymatic hydrolysis mixture obtained in step ① is filtered through an 80-mesh filter, and the filtrate is collected. The filtrate is then filtered by vacuum filtration with a pore size of 10μm to obtain the filtrate. ③ Mix the filtrate obtained in step ② with petroleum ether at a volume ratio of 1:1, stir and react at room temperature for 30 minutes, then let stand at room temperature until the solution separates into layers, retain the lower layer liquid to obtain defatted American cockroach water extract; ④ The defatted American cockroach aqueous extract obtained in step ③ is concentrated by rotary evaporation at a reaction temperature of 40℃ and then freeze-dried under vacuum to obtain freeze-dried powder of American cockroach aqueous extract. Step ①, the method for preparing pulverized American cockroaches, includes the following steps: American cockroaches under two months old were fasted from food and water for three days before treatment, then freeze-dried. The freeze-dried cockroaches were then pulverized to a mesh size of 150.

7. The aqueous extract of American cockroach prepared by the method according to any one of claims 1-6, wherein the total sugar content is 10.62-38.51 mg / g, the monosaccharide content is 3.79-18.07 mg / g, the protein content is 59.84-89.59 mg / g, the total flavonoid content is 7.50-31.14 mg / g, and the polyphenol content is 0.10-1.0 mg / g.

8. The use of the water extract of American cockroach prepared by the method of any one of claims 1-6 in the preparation of soothing, anti-allergic and repair products.

9. The use of the aqueous extract of American cockroach prepared by the method according to any one of claims 1-6 in the preparation of cosmetic products; Preferably, the use of the water extract of American cockroach prepared by the method of any one of claims 1-6 in the preparation of soothing, anti-allergic and repairing cosmetic products.

10. A composition, characterized in that, Aqueous extracts of American cockroaches prepared by the method according to any one of claims 1-6; Preferably, a cosmetic product comprises an aqueous extract of American cockroaches prepared by the method according to any one of claims 1-6.

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