Periplaneta americana extract as well as preparation method and application thereof

Abstract

The invention discloses a periplaneta americana extract as well as a preparation method and application thereof. The preparation method comprises the following steps that periplaneta americana nymphs are sequentially subjected to extraction treatment, degreasing treatment and purification treatment, and the periplaneta americana extract is prepared; wherein the extraction treatment comprises a step of extracting with an alcoholic solution A; the purification treatment comprises the step of eluting with macroporous adsorption resin, and the elution comprises the step of eluting with an alcoholic solution B; the volume concentration of the alcoholic solution B is 1%-20%, and% is the volume ratio of alcohol in the alcoholic solution B. According to the preparation method disclosed by the invention, the periplaneta americana nymphs do not need to be subjected to any treatment operation, extraction operation is directly carried out, the yield of the extract is improved while the growth cycle of medicinal materials is shortened, and the obtained periplaneta americana extract is relatively low in molecular weight and rich in micromolecule polypeptide.

Description

Technical Field

[0001] This invention relates to an extract of the American cockroach, its preparation method, and its application. Background Technology

[0002] The American cockroach (Periplaneta Americana Linnaeus) is an insect belonging to the order Blattodea, family Blattodea, and genus Periplaneta. Commonly known as the "cockroach," it is the largest insect in the Blattodea family. The cockroach's life cycle consists of three stages: egg, nymph, and adult, and it is a type of insect that undergoes incomplete metamorphosis.

[0003] Modern research has found that the main active ingredients in the extract of American cockroaches include polyols, polypeptides, mucoglycosylated amino acids, etc., which have the effects of promoting wound and mucosal repair, anti-inflammatory and analgesic, anti-tumor, and immune enhancement. It has achieved good results in the repair of traumatic wounds, digestive system diseases such as gastritis, ulcers, upper gastrointestinal bleeding, gastric cancer, esophagitis, etc., as well as cardiovascular and cerebrovascular diseases such as acute and chronic heart failure.

[0004] Patent CN108403723B discloses a method for preparing American cockroach extract for intervention in Alzheimer's disease (AD), which involves pulverizing dried American cockroach bodies to obtain crude American cockroach powder, followed by extraction. Patent CN111728984B discloses a method for preparing defatted American cockroach extract, which involves inactivating, pulverizing, and sieving fresh American cockroaches to obtain crude American cockroach powder, followed by extraction. Patent application CN110742908A discloses a process for preparing American cockroach extract, which involves freezing live American cockroaches at -50 to -20°C to kill them, drying the frozen and inactivated bodies, and then extracting the extract.

[0005] The existing methods for preparing American cockroach extract disclosed above all require the American cockroach body to be crushed, sieved, or freeze-dried before subsequent extraction processes can be carried out, which is complicated and cumbersome. Therefore, there is an urgent need for a simple preparation process for American cockroach extract that does not require extensive treatment of the American cockroach body. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides an extract from the American cockroach (Periplaneta americana), its preparation method, and its applications. This preparation method uses American cockroach nymphs for extraction, eliminating the need for any pretreatment of the nymphs and allowing for direct extraction. This shortens the growth cycle of the medicinal material, increases the extract yield, reduces production costs, and results in an American cockroach extract with a low molecular weight and rich in small molecule peptides.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing an extract from the American cockroach, comprising the following steps:

[0009] The American cockroach nymphs were sequentially extracted, defatted, and purified to obtain the American cockroach extract; wherein...

[0010] The extraction process includes the step of extraction with alcohol solution A;

[0011] The purification process includes elution with a macroporous adsorption resin, and the elution includes elution with an alcohol solution B; the volume concentration of the alcohol solution B is 1%-20%, where % is the volume percentage of alcohol in the alcohol solution B.

[0012] In some embodiments, the American cockroach nymph is a fresh or dried American cockroach nymph; preferably, it is a fresh American cockroach nymph.

[0013] In some embodiments, the nymphs of the American cockroach are 3-10 years old, for example, 6 or 7.

[0014] In some embodiments, the alcohol solution A is an aqueous alcohol solution, preferably an aqueous ethanol solution.

[0015] In some embodiments, the volume concentration of the alcohol solution A is 10%-95%, preferably 50%-95%, more preferably 50%-65%, where % is the volume fraction of ethanol in the aqueous ethanol solution.

[0016] In some implementations, the extraction is a reflux extraction, and the reflux extraction temperature is preferably 70°C-90°C, for example 80°C-85°C.

[0017] The reflux extraction is preferably isothermal reflux extraction. Here, "isothermal reflux" means that the temperature remains constant or substantially constant during the reflux process (e.g., the temperature fluctuates by ±5°C).

[0018] In some embodiments, the extraction is performed three times; wherein, the first extraction preferably takes 1-3 hours, and the amount of alcohol solution A used is preferably 8-12 L of alcohol solution A per 1 kg of American cockroach nymphs; the second extraction preferably takes 1-2 hours, and the amount of alcohol solution A used is preferably... For each 1 kg of American cockroach nymphs, use 6-10 L of alcoholic solution A; The preferred extraction time for the three extractions is: The extraction time is 0.5-2 hours. Preferably, the amount of alcohol solution A used is 6-10 L of alcohol solution A per 1 kg of American cockroach nymphs. More preferably, the type and concentration of alcohol solution A are the same in all three extractions.

[0019] In some embodiments, the extraction process further includes steps of filtering and concentrating the extracted product.

[0020] The concentration is carried out under reduced pressure, and the time for concentration is not particularly limited, until no alcohol odor is detected. The temperature for concentration is preferably 40℃-90℃, such as 50℃, 60℃, 75℃ or 80℃. The pressure for concentration is preferably -0.040Mpa to -0.099Mpa, such as -0.08Mpa, -0.074Mpa, -0.09Mpa or -0.065Mpa.

[0021] In some embodiments, the defatting process can be conventional in the art and generally includes a step of defatting with purified water, such as mixing the product obtained from the extraction process with purified water, allowing it to stand and separate into layers, removing the upper layer of oil and the lower layer of precipitate, and filtering and centrifuging the resulting aqueous phase.

[0022] Preferably, the weight of the purified water is 1-10 times the weight of the product obtained from the extraction process.

[0023] The preferred temperature for the static setting is 0℃-20℃, for example, 8℃, 10℃ or 15℃.

[0024] The settling time is preferably 6-48 hours, for example, 12 hours, 18 hours or 24 hours.

[0025] The centrifugation speed is preferably 5000-20000 rpm, for example, 12000 rpm or 15000 rpm. Centrifugation effectively solves the problem of slow filtration speed of degreasing liquid, improves production efficiency, and removes impurities more effectively.

[0026] In some embodiments, the defatting process further includes a step of concentrating the defatted product.

[0027] The concentration is preferably vacuum concentration, and the vacuum concentration temperature is preferably 40℃-90℃, for example 50℃, 60℃, 75℃ or 80℃; the vacuum concentration pressure is preferably -0.040Mpa to -0.099Mpa, for example -0.08Mpa, -0.072Mpa, -0.09Mpa or -0.065Mpa.

[0028] The concentration is preferably concentrated to a relative density of 1.05-1.20, for example 1.12 or 1.15, of the product obtained from the defatting.

[0029] In this invention, elution with the macroporous adsorption resin can effectively remove ineffective components, enrich active components, and improve sample activity. It is characterized by low price, stable performance, and long service life, which greatly reduces industrial production costs.

[0030] In some embodiments, the macroporous adsorption resin is a nonpolar, weakly polar, or polar macroporous adsorption resin.

[0031] In some embodiments, the macroporous adsorption resin is preferably one or more of the following macroporous adsorption resins: D101, HP20, HPD100, HPD200, HPD300, HPD400, HPD600, AB-8, ADS-7, X-5, NKA-II, and D3520; more preferably, the macroporous adsorption resin is one or more of the following macroporous adsorption resins: D101, HPD600, HP20, AB-8, and NK-2.

[0032] In some embodiments, the alcohol solution B is an aqueous alcohol solution, preferably an aqueous ethanol solution.

[0033] In some embodiments, the volume concentration of the aqueous ethanol solution is 1%-10%, for example 3%, 5% or 8%, where % is the volume fraction of ethanol in the aqueous ethanol solution.

[0034] In some embodiments, the amount of alcohol solution B is 1 to 10 times, for example 6 times or 6.7 times, the column bed volume of the macroporous adsorption resin.

[0035] In some preferred embodiments, the elution includes the steps of first eluting with water and then eluting with the alcohol solution B, wherein the volume of water is preferably 0-10 times, for example 2 times, 2.5 times, 4 times or 5 times, the volume of the macroporous adsorption resin column bed.

[0036] In some embodiments, before elution with macroporous adsorption resin, the macroporous adsorption resin is packed into a column. The column packing steps include: soaking the macroporous adsorption resin in 95% ethanol for more than 6 hours to allow it to fully swell; after wet packing, washing with 4 to 6 column volumes of 95% ethanol, and then washing with purified water until no alcohol odor is detected; then soaking in 2% to 5% hydrochloric acid for 2 to 4 hours and washing with 4 to 6 column volumes of purified water until neutral; finally soaking in 5% to 10% sodium hydroxide solution for 2 to 4 hours and washing with 4 to 6 column volumes of purified water until neutral, to obtain a chromatography column packed with macroporous adsorption resin.

[0037] In some embodiments, the macroporous adsorption resin is packed in a chromatography column, the column diameter to column height ratio of which is 1:5 to 1:12, for example 1:6 or 1:8.

[0038] In some embodiments, the purification process further includes a step of concentrating the eluted product.

[0039] The concentration is preferably vacuum concentration, and the vacuum concentration temperature is preferably 40℃-90℃, such as 60℃, 70℃, 75℃ or 80℃; the vacuum concentration pressure is preferably -0.040Mpa to -0.099Mpa, such as -0.08Mpa, -0.072Mpa, -0.086Mpa or -0.06Mpa.

[0040] The concentration is preferably concentrated to a relative density of 1.0-1.5, for example, 1.20, 1.21, 1.25 or 1.30.

[0041] In some embodiments, the purification process further includes a drying and pulverizing step after the concentration step; the drying is preferably vacuum drying; the drying temperature is preferably 40℃-90℃, for example 60℃, 70℃, 75℃ or 80℃.

[0042] The present invention also provides an extract of American cockroaches, which is prepared by the method for preparing American cockroach extract as described above.

[0043] In some embodiments, the peptide content of the American cockroach extract is 75% or more, where % is the weight percentage of the peptide in the American cockroach extract.

[0044] In some embodiments, the molecular weight of the American cockroach extract is less than 1000.

[0045] The present invention also provides the application of the American cockroach extract as described above in the preparation of wound repair drugs.

[0046] The present invention also provides the use of the American cockroach extract as described above in the preparation of a drug for treating gastric or duodenal ulcers.

[0047] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0048] The reagents and raw materials used in this invention are all commercially available.

[0049] The positive and progressive effects of this invention are as follows:

[0050] The preparation method of this invention uses nymphs of the American cockroach, eliminating the need for further crushing, sieving, freeze-drying, or other operations on the cockroach body; it effectively shortens the growth cycle of the American cockroach, improves production efficiency, and reduces production costs; moreover, the obtained American cockroach extract has a polypeptide content of over 75% and a molecular weight as low as below 1000, meaning it is rich in small molecule peptides and has a good effect on promoting wound repair and improving gastritis and ulcers. Detailed Implementation

[0051] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0052] In the following examples and comparative examples, unless otherwise specified, all ethanol used is an aqueous solution of ethanol, and the percentage concentration refers to the volume concentration of ethanol in the aqueous solution of ethanol.

[0053] In the following examples and comparative examples, unless otherwise specified, the macroporous adsorption resins used were pre-packed into columns before elution. The specific steps are as follows:

[0054] The macroporous adsorption resin was soaked in 95% ethanol for more than 6 hours to allow it to fully swell. After wet packing, the column was washed with 4 to 6 column volumes of 95% ethanol, followed by rinsing with purified water until no alcohol odor was detected. Then, the column was soaked in 2% to 5% hydrochloric acid for 2 to 4 hours and rinsed with 4 to 6 column volumes of purified water until neutral. Finally, the column was soaked in 5% to 10% sodium hydroxide solution for 2 to 4 hours and rinsed with 4 to 6 column volumes of purified water until neutral, yielding a chromatography column packed with macroporous adsorption resin.

[0055] Example 1

[0056] American cockroach extract was prepared using the following steps:

[0057] Extraction process: Take 1 kg of fresh 6th instar nymphs of the American cockroach, add 65% ethanol, and reflux extract three times (extraction temperature 80℃-85℃). For the first extraction, add 10 L of 65% ethanol and reflux for 2 h; for the second extraction, add 10 L of 65% ethanol and reflux for 2 h; for the third extraction, add 10 L of 65% ethanol and reflux for 2 h. Combine the above extracts, filter, and concentrate under reduced pressure at 60℃ (pressure -0.08 MPa) until no alcohol odor remains, to obtain 514 g of American cockroach extract A.

[0058] Degreasing treatment: Add 2 kg of purified water to extract A, let stand at 10℃ for 12 h to remove the upper oil and lower precipitate, filter the aqueous phase, centrifuge the filtrate at 5000 rpm, and concentrate the filtrate under reduced pressure at 60℃ (pressure -0.08 MPa) to obtain 102.4 g of American cockroach extract B with a relative density of 1.12;

[0059] Purification: A chromatography column packed with D101 macroporous adsorption resin (column diameter:height = 1:12, total column bed volume 10L) was used for elution of American cockroach extract B. American cockroach extract B was loaded onto the column, eluted with 40L of purified water, and then eluted with 60L of 8% ethanol. The ethanol eluent was collected and concentrated under reduced pressure (pressure -0.08MPa) at 60℃ to an extract with a relative density of 1.20. The extract was dried under reduced pressure at 60℃ and pulverized to obtain 13.0g of American cockroach nymph extract.

[0060] Example 2

[0061] American cockroach extract was prepared using the following steps:

[0062] Extraction process: Take 1 kg of dried 10th-instar nymphs of the American cockroach, add 75% ethanol, and reflux extract three times (extraction temperature 80℃-85℃). For the first extraction, add 12 L of 75% ethanol and reflux for 3 h; for the second extraction, add 10 L of 75% ethanol and reflux for 2 h; for the third extraction, add 8 L of 75% ethanol and reflux for 1 h. Combine the above extracts, filter, and concentrate under reduced pressure at 80℃ (pressure -0.074 MPa) until no alcohol odor remains, to obtain 552 g of American cockroach extract A.

[0063] Degreasing treatment: Add 4 kg of purified water to extract A, let stand at 15℃ for 24 h to remove the upper layer of oil and the lower layer of precipitate, filter the aqueous phase, centrifuge the filtrate at 20000 rpm, and concentrate the filtrate under reduced pressure at 80℃ (pressure -0.072 MPa) to obtain 75.6 g of American cockroach extract B with a relative density of 1.20;

[0064] Purification: A chromatography column packed with HPD600 macroporous adsorption resin (column diameter:height = 1:5, total column bed volume 12L) was used for elution of American cockroach extract B. American cockroach extract B was loaded onto the column, eluted with 60L of purified water, and then eluted with 72L of 5% ethanol. The ethanol eluent was collected and concentrated under reduced pressure at 80℃ (pressure -0.072MPa) to a relative density of 1.30. The extract was then dried under reduced pressure at 80℃, pulverized, and 26.6g of American cockroach nymph extract was obtained.

[0065] Example 3

[0066] American cockroach extract was prepared using the following steps:

[0067] Extraction process: Take 1 kg of fresh 3rd instar nymphs of the American cockroach, add 50% ethanol, and reflux extract three times (extraction temperature 80℃-85℃). For the first extraction, add 8 L of 50% ethanol and reflux for 2 h; for the second extraction, add 6 L of 50% ethanol and reflux for 1 h; for the third extraction, add 6 L of 50% ethanol and reflux for 1 h. Combine the above extracts, filter, and concentrate under reduced pressure at 50℃ (pressure -0.09 MPa) until no alcohol odor remains, to obtain 545 g of American cockroach extract A.

[0068] Extraction process: Add 3 kg of purified water to extract A, let stand at 20℃ for 18 h to remove the upper oil and lower precipitate, filter the aqueous phase, centrifuge the filtrate at 15000 rpm, and concentrate the filtrate under reduced pressure at 50℃ (pressure -0.09 MPa) to obtain 318 g of American cockroach extract B with a relative density of 1.05;

[0069] Degreasing treatment: A chromatography column packed with HP20 macroporous adsorption resin (column diameter: height = 1:8, total column bed volume of 12L) was used for elution of American cockroach extract B. American cockroach extract B was loaded onto the column, eluted with 24L of purified water, and then eluted with 80L of 10% ethanol. The ethanol eluent was collected and concentrated under reduced pressure at 70℃ (pressure -0.086MPa) to an extract with a relative density of 1.25. The extract was then dried under reduced pressure at 70℃, pulverized, and 11.7g of American cockroach nymph extract was obtained.

[0070] Example 4

[0071] American cockroach extract was prepared using the following steps:

[0072] Extraction process: Take 1 kg of fresh 7th instar nymphs of the American cockroach, add 90% ethanol, and reflux extract three times (extraction temperature 80℃-85℃). For the first extraction, add 10 L of 90% ethanol and reflux for 2 h; for the second extraction, add 9 L of 90% ethanol and reflux for 1 h; for the third extraction, add 8 L of 90% ethanol and reflux for 0.5 h. Combine the above extracts, filter, and concentrate under reduced pressure at 75℃ (pressure -0.065 MPa) until no alcohol odor remains, to obtain 509 g of American cockroach extract A.

[0073] Degreasing treatment: Add 5 kg of purified water to extract A, let stand at 8℃ for 12 h to remove the upper oil and lower precipitate, filter the aqueous phase, centrifuge the filtrate at 12000 rpm, and concentrate the filtrate under reduced pressure at 60℃ (pressure -0.65 MPa) to obtain 80.5 g of American cockroach extract B with a relative density of 1.15;

[0074] Purification: Pretreatment was performed using a chromatography column (column diameter: height = 1:6, total bed volume 4L) packed with NK-2 macroporous adsorption resin. This pretreatment was then used to elute American cockroach extract B. American cockroach extract B was loaded onto the column, eluted with 10L of purified water, and then eluted with 40L of 3% ethanol. The ethanol eluent was collected and concentrated under reduced pressure (pressure -0.06MPa) at 75℃ to a relative density of 1.21. The extract was then dried under reduced pressure at 75℃ and pulverized to obtain 11.2g of American cockroach nymph extract.

[0075] Comparative Example 1

[0076] American cockroach extract was prepared using the following steps:

[0077] Extraction process: Take 1 kg of dried American cockroach adult product, add 65% ethanol, and reflux extract three times (extraction temperature 80℃-85℃). For the first extraction, add 10 L of 65% ethanol and reflux for 2 h; for the second extraction, add 10 L of 65% ethanol and reflux for 2 h; for the third extraction, add 10 L of 65% ethanol and reflux for 2 h. Combine the above extracts, filter, and concentrate under reduced pressure at 60℃ (pressure -0.07 MPa) until no alcohol odor is detected, to obtain 547 g of American cockroach extract A.

[0078] Degreasing treatment: Add 2 kg of purified water to extract A, let stand at 10℃ for 12 h to remove the upper oil and lower precipitate, filter the aqueous phase, centrifuge the filtrate at 5000 rpm, and concentrate the filtrate under reduced pressure at 60℃ (pressure -0.07 MPa) to obtain 236.1 g of American cockroach extract B with a relative density of 1.12;

[0079] Purification: A chromatography column packed with D101 macroporous adsorption resin (column diameter:height = 1:12, total column bed volume 10L) was used for elution of American cockroach extract B. American cockroach extract B was loaded onto the column, eluted first with 40L of purified water, then with 60L of 8% ethanol. The ethanol eluent was collected and concentrated under reduced pressure (pressure -0.07MPa) at 70℃ to an extract with a relative density of 1.25. The extract was dried under reduced pressure at 70℃ and pulverized to obtain 14.9g of American cockroach adult extract.

[0080] Comparative Example 2

[0081] American cockroach extract was prepared using the following steps:

[0082] Extraction process: Take 1 kg of fresh adult American cockroaches, freeze-crush them, then add 70% ethanol and reflux extract three times (extraction temperature 80℃-85℃). The first time, add 10L and extract for 3 hours; the second time, add 10L and extract for 2 hours; the third time, add 10L and extract for 1 hour. Combine the three extracts and concentrate under reduced pressure at 60℃ (pressure -0.085MPa) until there is no alcohol odor. American cockroach extract A: 536g.

[0083] Degreasing treatment: Add 2 kg of purified water to extract A and let it stand at 10°C for 12 h; remove the upper layer of oil and the lower layer of precipitate, and filter the aqueous phase using filter paper; take the filtrate and concentrate it under reduced pressure (pressure -0.085 MPa) at 60°C to obtain 62.7 g of American cockroach extract B with a relative density of 1.15;

[0084] Purification: A chromatography column packed with AB-8 macroporous adsorption resin (column diameter:height = 1:8, total column bed volume 10L) was used for elution of American cockroach extract B. American cockroach extract B was loaded onto the column, eluted with 40L of purified water, and then eluted with 60L of 8% ethanol. The ethanol eluent was collected and concentrated under reduced pressure (pressure -0.055MPa) at 70℃ to an extract with a relative density of 1.25. The extract was then dried under reduced pressure at 70℃, pulverized, and 8.5g of American cockroach adult extract was obtained.

[0085] Example 1: Yield and active ingredient content of American cockroach extract

[0086] 1. Yield

[0087] To standardize the comparison of extract yields from American cockroach nymphs, fresh nymphs were dried under the same conditions as those used for preparing dried products. After calculating the weight loss during drying, the fresh nymphs were converted to dried product yields, and the extract yield was calculated. The yield was calculated based on a 40% conversion between 1 kg of fresh nymphs and dried product. The results are shown in Table 1.

[0088] 2. Determination of polypeptide content

[0089] The protein content was determined according to Method IV of the 2020 edition of the Pharmacopoeia General Chapter 0731, the 2,2'A-quinoline-4,4'-dicarboxylic acid method (BCA method):

[0090] This method is based on the reaction of proteins with Cu in an alkaline solution. 2+ Reduced to Cu + 2,2'-Biquinoline-4,4'-dicarboxylic acid (BCA) and Cu + The proteins combine to form a purple complex. Within a certain range, the intensity of the color is directly proportional to the protein concentration. Using a protein / peptide reference solution as a standard curve, the protein / peptide content in the extract is determined by colorimetry.

[0091] The results are shown in Table 1.

[0092] 3. Molecular weight distribution results

[0093] GPC method

[0094] Chromatographic conditions and system suitability test

[0095] Chromatographic column: PL Multisolvent 20, 7.8×150 mm; mobile phase: acetonitrile - water (containing 50 mM NaCl, 20 mM Na2HPO4, pH adjusted to 7.0 with phosphoric acid) (30:70); detection wavelength: 210 nm; column temperature: 35 °C, differential detector flow cell temperature: 35 °C.

[0096] The results are shown in Table 1.

[0097] Table 1 Yield, polypeptide content and molecular weight distribution of Periplaneta americana extracts in each example and comparative example

[0098] serial number Yield polypeptide content Molecular weight distribution Example 1 3.3％ 84.5％ 314.92 Example 2 2.7％ 77.3％ 326.89 Example 3 3.0％ 82.4％ 325.83 Example 4 2.8％ 80.2％ 336.11 Comparative Example 1 1.5％ 70.9％ 3036.3 Comparative Example 2 2.1％ 72.6％ 2788.1

[0099] As can be seen from Table 1, when using Periplaneta americana nymphs (Examples 1 - 4) for extraction, the extraction process is simple, and the yield of Periplaneta americana extract is higher than that using Periplaneta americana adults (Comparative Examples 1 - 2), at least about 30% higher.

[0100] The molecular weights of Examples 1 - 4 are significantly smaller than those of Comparative Examples 1 and 2, that is, the molecular weight of the nymph extract is significantly smaller than that of the adult extract, indicating that there are significant differences in the polypeptide structures of the two, and among them, the nymph extract is mainly composed of small - molecule peptides.

[0101] Biological experiment verification of Periplaneta americana extract in Effect Example 2

[0102] Periplaneta americana extract for mouse hemorrhagic gastric ulcer test

[0103] 1 Experimental materials

[0104] 1.1 Experimental drugs

[0105] Positive drug control group drug: Esomeprazole Sodium for Injection (AstraZeneca Pharmaceuticals Co., Ltd.);

[0106] Drug for Comparative Test Group 1: Periplaneta americana extract prepared according to the method described in Comparative Example 1;

[0107] Drug for Comparative Test Group 2: Periplaneta americana extract prepared according to the method described in Comparative Example 2;

[0108] Drug for Example 1: Prepared according to the method described in Example 1.

[0109] Drug for Example 2: Prepared according to the method described in Example 2.

[0110] 1.2 Test animals

[0111] SPF - level 12 - 14 g ICR male mice, Shanghai Slake Experimental Animal Co., Ltd., production license: SCXK(Shanghai)2022 - 0004.

[0112] 2 Experimental Methods

[0113] 2.1 Experimental Grouping

[0114] Sixty healthy male ICR mice were randomly divided into six groups: a model group (only water was given during the modeling period, no drugs were given; environment: the animal room environment was maintained at 20-25℃ and 40-70% humidity, with 12-hour light and dark alternation. Five animals were housed in each cage, and the bedding (corn cob bedding) was changed twice a week; food and water: the experimental animals were fed SPF rat growth and reproduction feed, and the animal water was high-pressure sterilized filtered water), a positive drug control group, a control group 1, a control group 2, Example 1, and Example 2, for a total of six groups, with 10 mice in each group.

[0115] 2.2 Model Replication

[0116] The experimental mice were administered the drug solution by gavage once a day for 10 consecutive days. The model group was given the corresponding amount of pure water. The positive control group was given the corresponding amount of pure water by gavage from day 1 to day 9. On day 10, the mice were given the corresponding amount of esomeprazole sodium injection via tail vein injection.

[0117] Before the last administration, all mice were fasted but allowed free access to water for 24 hours. Mice in the positive control group (esomeprazole sodium, G2) were administered an anhydrous ethanol at a dose of 10 mL / kg orally 40 minutes after administration to induce the ulcer model. Mice in the other groups were administered anhydrous ethanol at a dose of 10 mL / kg orally 1 hour after administration to induce the ulcer model. All animals were euthanized by CO2 inhalation 1 hour after model induction. The gastric cardia and pylorus were ligated and clamped, and the entire stomach was harvested. 1 mL of 1% formaldehyde solution was injected into the stomach through the proventriculus, the gastric cardia was ligated, and the stomach was removed and immediately placed in 1% formaldehyde solution for fixation for 25 minutes. The entire stomach was cut open along the greater curvature, and the stomach contents were cleaned with physiological saline. The stomach was then laid flat and the ulcer area and ulcer inhibition rate were calculated.

[0118] 3 Observation Indicators

[0119] Ulcer area calculation: The gastric mucosa of the damaged mouse will appear as cord-like congestion, crisscrossing and of varying thickness. After taking pictures, the ulcer area is calculated using ImageJ software.

[0120] Ulcer inhibition rate % = (Ulcer area in model group - Ulcer area in treatment group) / Ulcer area in model group × 100%.

[0121] 4 Experimental Results

[0122] The results are shown in Table 2. In the surgical group, no bleeding points were found in the stomach of the mice; in the model group, the model was successfully established, and bleeding points and obvious ulcers were found in all mice. Compared with the model group, the area of ​​gastric ulcers in the positive control group, control group 1, control group 2, and mice treated with *Periplaneta americana* nymph extract was significantly reduced (P < 0.05), suggesting that the above drugs have a significant therapeutic effect on acute hemorrhagic gastric ulcers in model mice; the ulcer area treated with *Periplaneta americana* nymph extract was significantly smaller than that in other drug groups, indicating that the therapeutic effect was superior to other drugs.

[0123] Table 2. Effect of continuous administration of the test drug for 10 days on ulcer area in mice with anhydrous ethanol-induced hemorrhagic gastric ulcers (±s, N=10)

[0124]

[0125] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

Claims

1. A method of preparing an extract of Periplaneta americana, characterized in that, It comprises the following steps: The periplaneta americana nymphs are successively subjected to extraction treatment, defatting treatment and purification treatment to obtain the periplaneta americana extract; The extraction treatment comprises the step of extracting with alcohol solution A; The purification treatment comprises the step of eluting with macroporous adsorption resin, and the eluting comprises the step of eluting with alcohol solution B; the volume concentration of the alcohol solution B is 1%-20%, and the % is the volume percentage of alcohol in the alcohol solution B.

2. The method of claim 1, wherein the Periplaneta americana extract is prepared by the steps of: The periplaneta americana nymphs are fresh periplaneta americana nymphs or dried periplaneta americana nymphs; preferably fresh periplaneta americana nymphs; ​ And / or, the instar of the periplaneta americana nymphs is 3-10.

3. The method of claim 1, wherein the Periplaneta americana extract is prepared by the steps of: The alcohol solution A is an alcohol aqueous solution, preferably an ethanol aqueous solution; wherein the volume concentration of the ethanol aqueous solution is preferably 10%-95%, more preferably 50%-95%, and further more preferably 50%-65%, and the % is the volume fraction of ethanol in the ethanol aqueous solution; ​ And / or, the extraction is reflux extraction, preferably constant-temperature reflux extraction; And / or, the temperature of the extraction is 70-90℃; And / or, the extraction is performed three times; wherein the time of the first extraction is preferably 1-3h, and the amount of the alcohol solution A is preferably 8-12L per 1kg of periplaneta americana nymphs; the time of the second extraction is preferably 1-2h, and the amount of the alcohol solution A is preferably 6-10L per 1kg of periplaneta americana nymphs; the time of the third extraction is preferably 0.5-2h, and the amount of the alcohol solution A is preferably 6-10L per 1kg of periplaneta americana nymphs; more preferably, the type and concentration of the alcohol solution A are the same in the three extractions.

4. The method for preparing the American cockroach extract as described in claim 1, characterized in that, The extraction treatment further comprises the steps of filtering and concentrating the extracted product; Preferably, the concentration is reduced-pressure concentration; the temperature of the reduced-pressure concentration is preferably 40-90℃, and the pressure of the reduced-pressure concentration is preferably -0.040Mpa to -0.099Mpa.

5. The method for preparing the American cockroach extract as described in claim 1, characterized in that, The defatting treatment comprises the step of defatting with purified water, preferably comprising the steps of mixing the extracted product with purified water, standing for stratification, removing the upper layer of oil and the lower layer of precipitate, filtering and centrifuging the obtained water phase; The weight of the purified water is preferably 1-10 times the weight of the extracted product; The standing temperature is preferably 0-20℃, and more preferably 10℃; The standing time is preferably 6-48h, and more preferably 12h; The centrifugation speed is preferably 5000-20000rpm; And / or, the defatting treatment further comprises the step of concentrating the defatted product; Preferably, the concentration is reduced-pressure concentration, and the temperature of the reduced-pressure concentration is preferably 40-90℃; the pressure of the reduced-pressure concentration is preferably -0.040Mpa to -0.099Mpa; Preferably, the concentration is concentrated to a relative density of 1.05-1.

20.

6. The method for preparing the American cockroach extract as described in claim 1, characterized in that, The macroporous adsorption resin is non-polar, weakly polar or polar macroporous adsorption resin; the macroporous adsorption resin is preferably one or more of D101, HP20, HPD100, HPD200, HPD300, HPD400, HPD600, AB-8, ADS-7, X-5, NKA-II and D3520 type macroporous adsorption resin; more preferably, the macroporous adsorption resin is one or more of D101, HPD600, HP20, AB-8 and NK-2 type macroporous adsorption resin; And / or, the macroporous adsorption resin is loaded in a chromatography column, and the ratio of column diameter to column height of the chromatography column is 1:5-1:12; And / or, the alcohol solution B is an alcohol aqueous solution, preferably an ethanol aqueous solution, wherein the volume concentration of the ethanol aqueous solution is preferably 1%-10%, and the % is the volume fraction of ethanol in the ethanol aqueous solution; And / or, the volume of the alcohol solution B is 1-10 times the column bed volume of the macroporous adsorption resin; And / or, the elution comprises the steps of eluting with water first and then eluting with the alcohol solution B, and the volume of the water is preferably 0-10 times the column bed volume of the macroporous adsorption resin.

7. The method for preparing the American cockroach extract as described in claim 1, characterized in that, The purification process further comprises the step of concentrating the eluted product; Preferably, the concentration is reduced pressure concentration; the temperature of the reduced pressure concentration is preferably 40°C-90°C; the pressure of the reduced pressure concentration is preferably -0.040Mpa~ -0.099Mpa; Preferably, the concentration is concentrated to a relative density of the purified product of 1.0-1.

5.

8. An extract of Periplaneta americana, characterized in that, It is prepared by the preparation method of the periplaneta americana extract according to any one of claims 1-7.

9. The Periplaneta americana extract of claim 8, characterized in that, The polypeptide content of the periplaneta americana extract is 75% or more, and the % is the weight percentage of polypeptide in the periplaneta americana extract; And / or, the molecular weight of the periplaneta americana extract is less than 1000.

10. Use of the periplaneta americana extract according to claim 8 or 9 in the preparation of a drug for repairing wounds or treating gastric or duodenal ulcers.

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