Preparation method and application of periplaneta americana ground powder

Abstract

The invention discloses a preparation method and application of American cockroach ground powder, and belongs to the technical field of medicinal material processing. After being degreased, the periplaneta americana medicinal material is crushed and sieved to obtain the periplaneta americana ground powder. The content of chitin in the periplaneta americana raw medicinal material is about 15-20%, the content of chitin in the periplaneta americana powder after partial chitin is removed by a physical method is less than or equal to 6%, the removal rate of chitin is greater than 50%, the yield of the powder is greater than 70%, and the problem of stomach fullness and discomfort of a patient after the patient takes the periplaneta americana powder is solved. The content of effective components in the American cockroach ground powder and the adaptability of patients are improved, and the effects of reducing toxicity and enhancing efficiency of traditional Chinese medicine processing are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal material processing, and in particular to a preparation method and application of American cockroach powder. Background Art

[0002] The American cockroach is an insect belonging to the genus Periplaneta in the order Blattaria of the family Blattaria of the class Insecta. It is commonly known as cockroach. It is the largest insect in the family Blattaria and is native to South America. The American cockroach is a traditional Chinese medicinal material. It was first recorded in the "Shennong Bencao Jing" as "salty and cold. It is used to treat blood stasis, hard symptoms, cold and heat, break up accumulation, throat and pharynx, internal cold, and infertility". It is used to treat pediatric malnutrition, furuncle, carbuncle, throat moth, unknown swelling, syphilis, and poisonous snake and centipede bites. In modern clinical practice, the American cockroach slices are ground into fine powder and taken orally for the treatment of immune regulation, gastrointestinal diseases, tumor diseases, etc. However, some patients are prone to stomach fullness and discomfort after taking the American cockroach powder.

[0003] Research has found that the cause of the discomfort is that the powder of American cockroaches contains a large amount of insect chitin, which is a polymer of poly-N-acetylglucosamine, easy to bend, elastic, and very tough. Chitin consumes a large amount of gastric acid and enzymes during the decomposition process, causing stomach discomfort, especially for patients with poor stomach function who are more likely to experience stomach fullness and discomfort after oral administration. Therefore, it is crucial to provide a preparation method for removing chitin from American cockroach powder to "reduce toxicity and increase efficacy". Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of American cockroach powder to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing American cockroach powder. The American cockroach powder can be obtained by defatting the American cockroach medicinal material, crushing it and sieving it.

[0007] The second technical solution of the present invention is the American cockroach powder prepared by the preparation method.

[0008] The third technical solution of the present invention is the use of the American cockroach powder in the preparation of immunomodulatory products.

[0009] A fourth technical solution of the present invention is a medicine or health product for enhancing immunity, comprising the American cockroach powder.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] The present invention provides a preparation method and application of American cockroach powder. The content of chitosan in the original American cockroach medicinal material is about 15% to 20%. After removing part of the chitosan by a physical method, the content of chitosan in the American cockroach powder is less than or equal to 6%, the chitosan removal rate is greater than 50%, and the powder yield is greater than 70%. Before removing part of the chitosan, the adverse reaction rate of temporary stomach distension and discomfort after oral administration of American cockroach powder is close to 10%. The powder obtained by removing part of the chitosan by the present invention does not cause adverse reactions of stomach distension and discomfort after oral administration. This aspect solves the problem of stomach distension and discomfort after patients take American cockroach powder, increases the content of effective ingredients in American cockroach powder and the adaptability of patients, and achieves the effect of "reducing toxicity and increasing efficacy" of Chinese medicine processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 A gravel map.

[0014] Figure 2 The effect of chitin powder from American cockroach on the blood routine of mice. A is white blood cells, B is lymphocytes, C is hemoglobin, D is red blood cells, and E is platelets.

[0015] Figure 3 The effect of removing part of the chitin powder from American cockroach on the organ indexes of immune organs (spleen and thymus). Among them, A is the spleen index and B is the thymus index.

[0016] Figure 4 Effects of partially chitin-removed powder from American cockroach on mouse serum hemolysin (A) and antibody (B) producing cells.

[0017] Figure 5 Effects of partially chitin-removed powder from the American cockroach on delayed-type hypersensitivity in mice.

[0018] Figure 6 The effect of chitosan powder from American cockroach on the transformation of mouse spleen lymphocytes.

[0019] Figure 7 It is the visible light spectrum scanning curve of the reference and test samples.

[0020] Figure 8 Alanine standard curve.

[0021] Fig. 9It is the HPLC chromatogram of nucleoside. Among them, A is uracil, hypoxanthine, xanthine reference solution; B is the powder extract solution. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0028] The embodiment of the present invention provides a method for preparing American cockroach powder. The American cockroach powder can be obtained by defatting the American cockroach medicinal material, crushing it and sieving it.

[0029] The method for removing part of chitin in the American cockroach medicinal material of the present invention adopts the method of physical homogenization, crushing and screening, and the obtained product is the American cockroach ultrafine powder with a mesh size of more than 500.

[0030] In some specific embodiments, the pulverizing rotation speed is 7000 rpm.

[0031] In some specific embodiments, the sieve has an aperture of 500 mesh or above.

[0032] The embodiment of the present invention also provides American cockroach powder prepared by the preparation method.

[0033] In some specific embodiments, the American cockroach powder has a chitosan content of ≤6%, a total amino acid content in terms of alanine of ≥25%, a total amount of uracil, hypoxanthine and xanthine of ≥2 mg / g, a protein content of ≥65%, a total peptide content of ≥12%, an extract content of ≥7.0%, a fat content of ≤5%, and a moisture content of ≤5%.

[0034] The embodiment of the present invention also provides the use of the American cockroach powder in preparing an immunomodulatory product.

[0035] The embodiment of the present invention further provides a medicine or health product for enhancing immunity, comprising the American cockroach powder.

[0036] The invention utilizes the difference in physical properties such as mechanical strength and toughness between chitin and other effective components (proteins, peptides, amino acids, nucleosides) in American cockroaches, and adopts a physical crushing method to separate the part of chitin with high mechanical strength and strong toughness that is not easy to break from other effective components. The innovation of the invention is that it avoids the disadvantages of using a large amount of hydrochloric acid, sodium hydroxide and other chemical agents to separate chitin by traditional chemical separation methods, and strong acids and alkalis will destroy the effective components in American cockroaches.

[0037] The present invention adopts a homogenizing crushing method, and by investigating the relationship between the centrifuge speed, the sieve mesh diameter, the chitosan content in the powder, and the powder yield during the crushing process, a method for removing part of the chitosan in the American cockroach by a physical method is invented. The chitosan of the American cockroach is a polymer of N-acetylglucosamine, and its physical properties are: easy to bend, elastic, and very tough. The hardness and mechanical strength of the chitosan in the American cockroach are greater than those of other effective ingredients such as proteins, peptides, amino acids, and nucleosides in the American cockroach, and it is not easy to be mechanically crushed during the crushing process. The present invention utilizes the difference in physical properties between chitosan and other active ingredients such as proteins, peptides, amino acids, and nucleotides to separate them through physical crushing and sieving. The speed of the pulverizer and the sieve aperture during the crushing process affect the removal rate of chitosan in the American cockroach powder.

[0038] Compared with the model group, the ratio of spleen / body weight and thymus / body weight of immunocompromised mice increased to varying degrees after the chitin-free powder of American cockroach. Compared with the normal group, the SRBC antibody (hemolysin) in the serum of the American cockroach de-chitin powder HC 50The values ​​were all increased; the degree of transformation of mouse spleen lymphocytes gradually increased, and the above immune indicators were significantly different from those in the model group (P < 0.01), indicating that the chitosan powder of American cockroach can significantly improve the immune function of immunocompromised mice and has a good immunoregulatory effect.

[0039] The contents of main active ingredients in the American cockroach powder are: total amino acids calculated as alanine ≥ 25%; total nucleotides calculated as the total amount of uracil, hypoxanthine and xanthine ≥ 2 mg / g; total protein ≥ 65%; total peptides ≥ 12%; and alcohol-soluble extracts ≥ 7%.

[0040] The content of limited ingredients in American cockroach powder: oil content ≤5%; impurity content ≤1.0%; moisture content ≤5%; acid insoluble ash ≤2.0%; total ash ≤8%; sulfur dioxide ≤150mg / kg; aflatoxin ≤10μg / kg.

[0041] The main heavy metal contents in American cockroach powder are: arsenic ≤1mg / kg, mercury ≤0.2mg / kg, lead ≤1mg / kg, cadmium ≤0.3mg / kg, and copper ≤20mg / kg.

[0042] Pesticide residues in American cockroach powder: total BHC content ≤ 0.2 mg / kg, total DDT content ≤ 0.2 mg / kg, pentachloronitrobenzene content ≤ 0.1 mg / kg.

[0043] Microbial limit in American cockroach powder: Total aerobic bacteria ≤ 10 3 cfu / g, total number of molds and yeasts ≤ 10 2 cfu / g, bile-resistant Gram-negative bacteria ≤10 4 cfu / g, Salmonella was not detected in 10g.

[0044] Example 1

[0045] 1 Physical separation and powder preparation process of American cockroach chitin

[0046] Weigh the American cockroach medicinal material, use edible oil-grade extraction oil (n-hexane, cyclohexane or a mixture of the two in any proportion) to remove the American cockroach grease, dry it, and obtain the defatted American cockroach.

[0047] The chitin of the American cockroach is poly-N-acetylglucosamine, which is easy to bend, elastic, and very tough. It has a greater hardness and mechanical strength than the effective ingredients such as amino acids, nucleosides, proteins, and total peptides in the American cockroach, and is not suitable for mechanical crushing during the crushing process. The present invention combines grinding and homogenizing crushing to investigate the relationship between the centrifuge speed, the sieve mesh size, the chitin content in the ground powder, and the powder yield during the crushing process.

[0048] 1.1 Screen aperture inspection

[0049] Weigh about 1000g of defatted powder of American cockroach and place it in a grinder. Set the grinder speed to 7000rpm, and place sieves with pore sizes of 100μm (160 mesh), 60μm (250 mesh), 40μm (400 mesh), 25μm (500 mesh), 18μm (800 mesh), and 13μm (1000 mesh) for grinding to obtain chitosan (on the sieve) and dechitosan powder (under the sieve). Record the weight and calculate the dechitosan powder yield and chitosan removal rate.

[0050] The calculation results are shown in Table 1. The mesh size has a great influence on the yield, chitin removal rate and chitosan content of the chitosan powder. The principal component analysis is used to perform single factor multi-index process optimization.

[0051] Table 1 Effect of sieve aperture on separation process

[0052]

[0053] 1.2 Inspection of the speed of the pulverizer

[0054] Take an appropriate amount of defatted American cockroach medicinal material, put it in a grinder, set the grinder speed to 6000, 7000, 8000, 9000 rpm, place a sieve with an aperture size of 25 μm (500 mesh) for grinding, obtain chitosan (on the sieve) and dechitosan fine powder (under the sieve), record the weight and calculate the yield. Determine the chitosan content by the method of Reference Example 3, and calculate the chitosan removal rate.

[0055] Chitosan removal powder yield: X = W1 / W2 × 100%;

[0056] Wherein, W1: weight of sample under the sieve / g, W2: total weight of sample / g.

[0057] Chitosan removal rate: Y = (C2-C1) / C2×100%;

[0058] Wherein, C1: content of chitosan below the sieve / %, C2: content of chitosan not passed through the sieve / %.

[0059] The calculation results are shown in Table 2. The mill speed has little effect on the yield of chitosan-free powder and the chitosan content in each part, and the RSD values ​​are all less than 3.0. According to the design speed range of the mill in industrial production and the chitosan removal rate, 7000 rpm is selected as the optimal milling speed.

[0060] Table 2 Effect of mill speed on separation process

[0061]

[0062] 1.3 Principal component analysis and process optimization

[0063] 1.3.1 KMO and Bartlett's sphericity test

[0064] The maximum variance method of SPSS24.0 was used to perform factor analysis on the results of component content determination, and the principal component factors were determined. The three indicators of partial chitosan powder removal rate, chitosan removal rate, and chitosan content in chitosan powder were selected for KMO (Kaiser-Meyer-Olkin) and Bartlett's sphericity tests. The KMO test coefficient was 0.644>0.5; Bartlett's sphericity test P=0.000<0.05. This shows that the data structure is reasonable and the principal component analysis can be continued.

[0065] 1.3.2 Principal component screening and contribution rate analysis

[0066] From the factor loading matrix, eigenvalue and variance contribution table (Table 3, Table 4), we can see that the first principal component reflects the information of the three indicators. Figure 1 ) It can be seen that the eigenvalue of the first principal component is 2.962>1, which can explain 98.728% of the total data variation and represents 98.728% of the information content of the three indicator components. The eigenvalue of the first principal component is also much higher than the others. Therefore, the first principal component is selected for subsequent analysis.

[0067] Table 3 Factor loading matrix

[0068]

[0069] Table 4 Eigenvalues ​​and variance contribution rates

[0070]

[0071]

[0072] 1.3.3 Comprehensive score and weight calculation

[0073] According to the above analysis, the principal component analysis extracts 1 principal component, whose variance contribution rate reaches 98.728%, covering most of the information. First, the linear combination coefficient matrix is ​​obtained by dividing the load coefficient by the square root of the corresponding characteristic root. Then, the linear combination coefficients are multiplied by the variance explanation rate and added up, and divided by the cumulative variance explanation rate to obtain the comprehensive score coefficient. After normalization, the weight coefficients of each indicator are shown in Table 5.

[0074] Table 5 Linear combination coefficients and weight results

[0075]

[0076] 1.4 Process Optimization

[0077] According to the weight coefficients of different components, the comprehensive evaluation index of chitosan separation process is calculated:

[0078] Evaluation index Y = 0.9870X1 + 0.9960X2 + 0.9910X3;

[0079] Among them, X1 is the chitosan powder yield, X2 is the chitosan powder removal rate, and X3 is the chitosan powder removal rate.

[0080] The results of comprehensive evaluation indicators showed that the separation process of American cockroach chitin was optimal when the pulverizer speed was 7000rpm and the sieve mesh size was 25μm.

[0081] 1.5 Process Validation

[0082] Three batches of samples were taken and processed at a pulverizer speed of 7000 rpm and a sieve mesh size of 25 μm, and the speed was selected to be 7000 rpm and the sieve mesh size was selected to be 25 μm (500 mesh) to prepare the American cockroach chitin powder. The results are shown in Table 6, which proves that the process is stable and reliable.

[0083] Table 6 Process Validation

[0084]

[0085] 1.6 The preparation method of the American cockroach powder of the control group is: take the American cockroach medicinal material that has not been defatted, the pulverizer speed is 2000rpm, and the aperture of the sieve is 60 meshes. The obtained powder has high chitin and oil content, the color is yellow-brown to dark brown, the smell of the American cockroach powder is very strong, and the American cockroach oil solution oxidizes and becomes rancid during storage. The composition of the main components in the control group is: oil content 20% to 35%, chitin content 15% to 20%, total amino acid content in terms of alanine ≤15%, nucleotides into uracil, hypoxanthine and xanthine total amount ≤1.3mg / g, protein content ≤55%, total peptide content ≤12%, extract content ≥7.0%, moisture content 5% to 8%.

[0086] The powder prepared by the present invention with batch number 20240906 was used as a sample, and the American cockroach powder prepared by the control group was used as a control to conduct oral experimental verification. The results showed that the adverse reaction rate of temporary stomach fullness and discomfort after oral administration of the American cockroach powder of the control group was close to 10%, and the powder obtained by removing part of the chitosan by the present invention did not cause adverse reactions of stomach fullness and discomfort after oral administration.

[0087] The powder obtained by the preparation method of the original powder of American cockroach in the control group has the following problems: 1. It can only be crushed to obtain powder below 80 meshes. Since the oil content in the medicinal material exceeds 20%, it is easy to agglomerate on the screen during the crushing process. The smaller the screen aperture, the easier it is to agglomerate and clog, so it can only be crushed to obtain powder below 80 meshes; 2. The chitosan in the American cockroach cannot be separated by physical crushing. During the crushing process, due to the large sieve aperture, the chitosan cannot be separated from the effective ingredients such as protein, amino acid, glycoprotein, etc.; 3. Due to the high content of chitosan and oil in the original powder, the gastrointestinal burden of the patient is increased, and the adverse reaction rate of the patient after taking it exceeds 10%. 4. The content of effective ingredients such as protein, amino acid, glycoprotein, nucleotide, etc. in the original powder is lower than that of the American cockroach powder obtained by the present invention without part of the chitosan.

[0088] Example 2

[0089] 2 Evaluation of the immune enhancement effect of chitin powder from American cockroaches

[0090] 2.1 Modeling and grouping and drug administration

[0091] A total of 72 mice were randomly divided into three immunization groups (immunization group I, immunization group II, and immunization group III), with 24 mice in each group. The mice in each immunization group were randomly divided into a normal control group (Con group), a low-dose group, a medium-dose group (L), and a high-dose group (H) of American cockroach chitosan powder according to body weight, with 6 mice in each group. The mice were given feed supplemented with different doses of American cockroach chitosan powder at 4 g / mouse / day (the low-dose group took in 0.2 g / mouse / day of American cockroach chitosan, the medium-dose group took in 0.4 g / mouse / day of American cockroach chitosan, and the high-dose group took in 0.8 g / mouse / day of American cockroach chitosan) according to 10, 20, and 30 times the daily recommended amount of human body, respectively. The normal control group was given an equal amount of ordinary feed.

[0092] Group I performed humoral immunity test: serum hemolysin [half value of hemolysin, HC 50 )], antibody-producing cell detection; Group II was subjected to delayed-type hypersensitivity test (plantar thickening method), and Group III was subjected to mouse spleen lymphocyte transformation test.

[0093] 2.2 Index detection

[0094] 2.2.1 Serum hemolysin assay and antibody-producing cell detection

[0095] 2.2.1.1 Serum hemolysin determination

[0096] On the 26th day of administration, mice in the immunization group I were intraperitoneally injected with 0.2 mL of 2% sheep red blood cells (SRBC) for immunization. On the 30th day, blood was collected from mice and centrifuged at 3000 rpm for 15 minutes to separate the serum. After separating the serum, it was diluted 50 times with SA buffer, and 50 μL of the diluted serum was taken into a 96-well plate as a sample well, and SA buffer was used as a control well. Then 25 μL of 10% SRBC and 50 μL of complement were added to mix well, and incubated at 37°C for 30 minutes. After incubation, the reaction was terminated in an ice bath. After centrifugation at 2500 rpm for 10 minutes, 50 μL of the supernatant from each well was transferred to a new 96-well plate, and 12.5 μL of 10% SRBC was added to make a half-hemolysis well. 150 μL of Dulbecco's reagent was added to the sample well and the control well, and 187.5 μL of Dulbecco's reagent was added to the half-hemolysis well. Mix and place for 10 minutes, and measure the OD value at 540 nm with an enzyme reader. Calculate the half-hemolysis value (HC 50 ).

[0097] HC 50 =OD 样 / OD 半 × dilution factor;

[0098] Among them, OD 样 Represents the OD value of the sample, OD 半 Represents the OD value of half of the hemolyzed wells.

[0099] 2.2.1.2 Antibody-producing cell detection

[0100] (1) On the 30th day after administration, the spleens of mice in group I were removed and placed on a plate. 2 mL of 5% FBS-PBS buffer solution was added and ground. The spleen cell suspension was filtered through a 200-mesh cell sieve and collected in a 15 mL centrifuge tube. The tube was marked and centrifuged at 1200 rpm / min for 5 min. The supernatant was discarded. The spleen cells were washed twice with 5% FBS-PBS buffer solution. Finally, the precipitated spleen cells were resuspended in 1 mL of 1640 culture medium and the cell concentration was adjusted to about 1×10 7 / mL spleen cell suspension.

[0101] (2) Take two clean glass slides (75 mm × 25 mm) and use double-sided tape to stick a strip at each end and in the middle of the slides, and stick the two identical slides together tightly to form two small chambers. Immerse one long side of the double-layer glass slide in the melted paraffin pool to seal one side of the small chamber (no need to immerse too deep, just enough to seal the edge of the small chamber).

[0102] (3) Take 1×10 7 / mL spleen cell suspension 50μL, 15% SRBC 50μL, complement 50μL, 1640 culture medium 250μL are placed in a test tube as the experimental tube, and a blank tube without cells is set up. After mixing, take the mixed solution from the above two test tubes to perfuse the two chambers (try to avoid bubbles), seal with wax, mark well, place flat on a glass plate, incubate in a 37℃ incubator for 45 minutes, and observe the results under a microscope.

[0103] The results showed that after mice were immunized with SRBC, SRBC antibodies (hemolysin) appeared in the serum. Under the participation of complement, incubation with SRBC could cause hemolysis and release hemoglobin. The hemoglobin content could quantitatively reflect the amount of hemolysin antibodies generated in the mouse serum. Compared with the normal group, the low, medium and high dose groups of HC 50 The values ​​were all increased, and the differences were significant (P < 0.01), indicating that the experiments in the three dose groups were all positive.

[0104] When spleen lymphocytes come into contact with SRBC (antigen), they stimulate B cells to activate and secrete specific antibodies. The antigen-antibody complex dissolves red blood cells with the participation of complement, forming a hemolytic plaque. A plaque represents an antibody-producing cell (plasma cell). Therefore, the number of hemolytic plaques can reflect the strength of humoral immunity. With the increase of dose, the number of plaques gradually increases. There is no significant difference in the number of plaques between the low-dose group and the normal group (P>0.05). The difference between the medium and high-dose groups is significant (P<0.01), and the experimental results can be judged as positive. HC 50 The value and the number of plaques reflect the changes in the humoral immunity of mice. Both are positive, indicating that the chitin powder of American cockroach can significantly improve the humoral immunity function of experimental mice and has a good immunoregulatory effect. The experimental results are shown in Table 7. Figure 4 .

[0105] Table 7 Effects of partial chitin removal and powdering of American cockroach on mouse serum hemolysin and antibody-producing cells ( n=6)

[0106]

[0107] 2.2.2 Blood routine index detection

[0108] On the 30th day, 20 μL of whole blood was collected from mice in immunization group I and added into 700 μL of blood cell diluent for the detection of routine blood indexes.

[0109] The results of blood routine test showed that there was no significant difference in the blood routine levels of mice in each drug-treated group (P>0.05). The chitosan powder of American cockroach had no significant effect on blood routine, indicating that it had no obvious toxic side effects and maintained all indicators at normal physiological levels. The results are shown in Table 8. Figure 2 .

[0110] Table 8 Effect of chitin-free powder of American cockroach on blood routine of mice (x±s, n=6)

[0111]

[0112] 2.2.3 Organ coefficient determination

[0113] On the 30th day after administration, the mice in immunization group I were killed, and the spleen and thymus were removed and weighed. The organ coefficient was calculated according to the formula: organ index (mg / g) = organ weight / body weight.

[0114] The spleen is rich in lymphocytes and macrophages, with a relatively large proportion of B lymphocytes, which are closely related to humoral immunity. The main function of the thymus is to produce T lymphocytes and secrete thymosin, and it is mainly involved in cellular immunity; the level of thymus and spleen indexes depends on the degree of lymphocyte proliferation, which can reflect the strength of the body's immune function to a certain extent. Compared with the normal group, the spleen / body weight ratio and thymus / body weight ratio of the American cockroach dose group increased to varying degrees, and the spleen index of the high-dose group was significantly different from that of the normal group (P < 0.05). See Table 9, Figure 3 . This indicates that American cockroaches have a certain regulatory effect on the immune function of mice.

[0115] Table 9 Effects of partial chitin removal and powdering of American cockroach on the organ indexes of immune organs (spleen, thymus) n=6)

[0116]

[0117] 2.2.4 Cellular immune function test (delayed hypersensitivity reaction-plantar thickening method)

[0118] On the 26th day of administration, mice in group II were immunized by intraperitoneal injection of 0.2 mL of 2% SRBC. 4 days after immunization, on the 30th day, the baseline thickness of the left hind foot pad of the mice was measured with a vernier caliper, and then 20 μL of 20% (v / v) SRBC was subcutaneously injected into the left hind foot pad of each mouse. The thickness of the left hind foot pad was measured again 24 hours later. The difference in the thickness of the left hind foot pad was used as the degree of swelling.

[0119] After mice were immunized with SRBC, SRBC could stimulate T lymphocytes to proliferate into sensitized lymphocytes. When the toes were attacked with SRBC again 4 days later, swelling would occur in the toes. The degree of swelling could reflect the degree of delayed-type hypersensitivity and thus the proliferation capacity of T lymphocytes. The results are shown in Table 10. Figure 5With the increase of the dose, the thickness of the left hind toe of the mouse gradually thickened. Compared with the normal group, the delayed allergic reaction in the high-dose group was significantly different (P < 0.05), while there was no significant difference in the low- and medium-dose groups (P > 0.05), and the results were negative.

[0120] Table 10 Effects of partial chitosan powder of American cockroach on delayed-type hypersensitivity in mice ( n=6)

[0121]

[0122] 2.2.5 Mouse spleen lymphocyte transformation experiment

[0123] After continuous administration for 30 days, the spleen of mice in group III was removed aseptically, placed on a plate, and ground with 2 mL of 5% FBS-PBS buffer solution. The spleen cell suspension was filtered through a 200-mesh cell sieve and collected in a 15 mL centrifuge tube. The suspension was marked and centrifuged at 1200 rpm / min for 5 min, and the supernatant was discarded. 3 mL of red blood cell lysis buffer was added, mixed, and fully lysed for 4 min, not exceeding 5 min. 3 mL of 3% FBS-PBS buffer solution was added to wash twice (centrifuged at 1200 rpm / min at 4 °C for 5 min) to terminate the lysis, the supernatant was discarded, and 2 mL of 1640 complete medium was added to resuspend the cells; the cell density was adjusted to 1×10 5 / mL. Each spleen cell suspension was added to two wells of a 96-well culture plate, 100 μL per well. One group was added with concanavalin (ConA) solution (final mass concentration of 7.50 μg / mL) as the test well, and the other group was set up without ConA solution as the control group. Incubate in a constant temperature incubator with 5% CO2 and 37°C for 48 hours. 4 hours before the end of the culture, add 20 μL / well of MTT (5 mg / mL) to each well and continue to culture for 4 hours. After the culture is completed, the supernatant is aspirated, 150 μL of acidic isopropanol is added to each well, and the mixture is blown to mix well to completely dissolve the purple crystals. The optical density value is measured by an enzyme marker at a wavelength of 570 nm.

[0124] Lymphocytes are a type of white blood cell and an important cell component of the body's immune response function. When lymphocytes are stimulated by ConA, the mother cells undergo a proliferation reaction. The mitochondrial hydrolase in the proliferating cells can decompose MTT (tetrazolium bromide) into blue-purple crystals (formazan), and the difference in optical density can reflect the body's cellular immunity level. As the dose of American cockroach de-chitin powder increases, the degree of transformation of mouse spleen lymphocytes gradually increases. Compared with the normal control group, the low, medium, and high dose groups of American cockroach all showed significant differences (P < 0.01), and the results were positive. The results are shown in Table 11. Figure 6 .

[0125] Table 11 Effect of partial chitin powder of American cockroach on mouse spleen lymphocyte transformation experiment ( n=5)

[0126]

[0127] This example refers to the test method for enhancing immunity in the "Technical Specifications for Inspection and Evaluation of Health Foods" to study the enhancement of mouse immune function by grinding some chitin from American cockroaches. 50 The results showed that compared with the normal group, the low, medium and high dose groups had significantly higher levels of HC 50 The values ​​of the two groups were significantly different (P < 0.01), and the medium and high dose groups had significant differences in antibody-producing cells (P < 0.01), which can be judged as positive humoral immunity results. The American cockroach de-chitin powder has a regulatory effect on the humoral immune function of mice. In cellular immunity, through the mouse spleen lymphocyte transformation experiment and delayed-type hypersensitivity experiment, it was found that the American cockroach de-chitin powder has a regulatory effect on ConA-induced cellular immune function of mice. Among them, the experimental results of the low, medium and high dose groups have significant differences (P < 0.01), which can be judged as positive cellular immunity results. According to the standards for the determination of the results of enhancing immunity in the "Technical Specifications for Inspection and Evaluation of Health Foods", it can be determined that the American cockroach de-chitin powder has the effect of enhancing immunity.

[0128] Example 3

[0129] 3. Chitosan content determination

[0130] 3.1 Preparation of reference solution

[0131] Accurately weigh 10 mg of glucosamine hydrochloride reference substance, place it in a 10 mL volumetric flask, add distilled water to dissolve and dilute to the scale to obtain a 1 mg / mL reference substance stock solution. Accurately take 1 mL of the above stock solution, add water to dilute to 10 mL, and obtain a 0.1 mg / mL reference substance solution.

[0132] 3.2 Preparation of test solution

[0133] Weigh 0.25 g of the chitosan powder of American cockroach prepared in Example 1 into an ampoule, add 5 mL of 9 mol / L hydrochloric acid solution, seal and shake well, hydrolyze at 100 ° C in an oven for 50 min, cool, neutralize to neutrality with 40% sodium hydroxide solution, make up to 100 mL, centrifuge at 4000 rpm for 10 min, accurately pipette 10 mL of the supernatant, transfer to a 25 mL volumetric flask, dilute to the scale with water, shake well, and obtain the test solution.

[0134] 3.3 Preparation of acetylacetone solution

[0135] Take 2.0 mL of acetone acetate, add sodium carbonate solution to 50 mL, and place in the refrigerator for later use. It should be prepared one day before use.

[0136] 3.4 Preparation of p-dimethylbenzaldehyde solution

[0137] Weigh 0.8 g of p-dimethylbenzaldehyde, add 15 mL of aldehyde-free ethanol and 15 mL of hydrochloric acid, and shake well.

[0138] 3.5 Selection of measurement wavelength

[0139] Take 1 mL of 0.1 mg / mL glucose standard solution and 1 mL of sample solution in a volumetric flask, dilute with water to 5 mL, add 1 mL of acetylacetone solution to each, shake well, let stand in a boiling water bath for 25 minutes, take out, quickly cool in an ice water bath, add 3.0 mL of anhydrous ethanol, and then add 1.0 mL of p-diaminobenzaldehyde solution, shake vigorously, let stand in a 70°C water bath for 50 minutes, take out, and cool to room temperature with cold water. Scan the wavelength in the range of 400-800 nm to obtain the absorption spectrum curve. The results show that both the reference and test solutions have maximum absorption at 525 nm. Figure 7 .

[0140] 3.6 Display Conditions

[0141] 3.6.1 Study on the reaction time of acetylacetone

[0142] Accurately measure 1 mL of the test solution in step "3.2" and place it in a 10 mL volumetric flask. Add 1 mL of acetylacetone solution and boil in a boiling water bath for 10, 15, 20, 25, 30, 35, 40, and 45 min. Then, treat according to the method under "3.5". The results show that the absorbance value increases with the increase of boiling water bath time. However, after 20 min, the absorbance increase is small and the RSD value is less than 3.0. Therefore, the acetylacetone reaction time is selected as 25 min.

[0143] 3.6.2 Study on the reaction temperature of acetylacetone

[0144] Accurately measure 1 mL of the test solution in step "3.2" and place it in a 10 mL volumetric flask. Add 1 mL of acetylacetone solution and react in a boiling water bath at 60, 70, 80, and 90 °C for 25 min. Then, treat according to the method under "3.5". The results show that the absorbance value increases with the increase of reaction temperature, but after 90 °C, the absorbance increase is small. Therefore, the acetylacetone reaction temperature is selected as a boiling water bath.

[0145] 3.6.3 Investigation of p-diaminobenzaldehyde reaction time

[0146] Accurately measure 1 mL of the test solution in step "3.2" and place it in a 10 mL volumetric flask. Add 1 mL of p-diaminobenzaldehyde solution and react at 70°C for 10, 20, 30, 40, 50, and 60 min. Then, treat according to the method under "3.5". The results show that the absorbance value increases with the increase of reaction temperature. However, after 40 min, the absorbance increase is small and the RSD value is less than 3.0. Therefore, the p-diaminobenzaldehyde reaction time is selected as 50 min.

[0147] 3.6.4 Study on reaction temperature of p-diaminobenzaldehyde

[0148] Accurately measure 1 mL of the test solution in step "3.2" and place it in a 10 mL volumetric flask. Add 1 mL of p-diaminobenzaldehyde and react in a boiling water bath at 60, 70, 80, and 90 °C for 50 min. Then, treat according to the method under "3.5". The results show that the absorbance is the highest at 70 °C, so the temperature of p-diaminobenzaldehyde is selected to be 70 °C.

[0149] 3.7 Investigation of hydrolysis conditions

[0150] 3.7.1 Investigation of hydrochloric acid concentration

[0151] Accurately weigh 7 portions of the test sample of the same batch number, each portion is about 0.25g, put them into ampoules, add 5ml of 2, 4, 6, 8, 9, 10, 12mol / L hydrochloric acid, prepare the test sample solution according to step "3.2", develop the color according to the color development method of step "3.5", and measure the absorbance A at 525nm. The results show that with the increase of hydrochloric acid concentration, the content of glucosamine hydrochloride also increases, but after the concentration of 8mol / L, the content of glucosamine hydrochloride increases less, and the RSD value is less than 3.0, so the hydrochloric acid concentration is selected as 9mol / L.

[0152] 3.7.2 Liquid-to-solid ratio investigation

[0153] Weigh 6 samples of the same batch number accurately, each weighing about 0.25g, put them into ampoules, add 9mol / L hydrochloric acid solution at a ratio of 1:5, 1:10, 1:15, 1:20, 1:25, and 1:30, and treat them according to the method of step "3.2". The results show that when the material-liquid ratio is 1:20, the content of glucosamine hydrochloride in American cockroaches is the highest. Therefore, the selected liquid-to-material ratio is 1:20.

[0154] 3.7.3 Investigation of hydrolysis time

[0155] Accurately weigh 5 portions of the test sample of the same batch number, each portion is about 0.25g, put them into an ampoule, add 5mL of 9mol / L hydrochloric acid solution, hydrolyze at 100℃ for 10, 20, 30, 40, 50, 60, 70, and 80min, and then treat according to the method of step "3.2". The results show that with the increase of hydrolysis time, the content of glucosamine hydrochloride also increases, but after 40min, the content of glucosamine hydrochloride increases less, and the RSD value is less than 3.0, so the hydrolysis time is selected as 50min.

[0156] 3.7.4 Hydrolysis temperature investigation

[0157] Accurately weigh 5 portions of the test sample of the same batch number, each portion is about 0.25g, put them into an ampoule, add 5mL of 9mol / L hydrochloric acid solution, hydrolyze them at 80, 90, 100, 110, and 120℃ for 50min respectively, and then treat them according to the method of step "3.2". The results show that with the increase of hydrolysis temperature, the content of glucosamine hydrochloride also increases, but after 90min, the content of glucosamine hydrochloride increases less, and the RSD value is less than 3.0, so the hydrolysis time is selected as 100℃.

[0158] 3.8 Methodological Review

[0159] 3.8.1 Drawing of standard curve

[0160] Accurately pipette 0.00, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml of the reference solution in step "2.1" into 10 mL volumetric flasks respectively, and process according to the method in step "3.5". Perform linear regression with absorbance A as the ordinate and concentration c (mg / mL) of the reference solution as the abscissa. The regression equation is y=3.3314x+0.0017, r=0.9995. The results show that glucosamine hydrochloride has a good linear relationship in the range of 0.005-0.03 mg / mL.

[0161] 3.8.2 Precision test

[0162] Accurately pipette 1 mL of the reference solution from step "3.1" and place it in a 10 mL volumetric flask and process it according to the method in step "3.5". Measure 6 times in a row, record the absorbance value, and calculate the RSD value. The RSD value is 0.08%, which proves that the precision of the instrument is good.

[0163] Table 12 Precision investigation results

[0164]

[0165] 3.8.3 Stability test

[0166] Accurately transfer 1 mL of the test solution from step "3.2" into a 10 mL volumetric flask and process according to the method in step "3.5". Measure the absorbance once every 30 minutes for 2 hours. The RSD value is 2.5%. The results show that the test solution is stable within 2 hours.

[0167] Table 13 Stability test results

[0168]

[0169] 3.8.4 Repeatability test

[0170] Accurately weigh about 0.25g of American cockroach powder, prepare the test solution according to step "3.2", then color according to step "3.5", and measure the absorbance at 525nm. The results showed that the average content of glucosamine hydrochloride in American cockroach was 54.08mg / g, and the RSD was 2.84% (n=6).

[0171] Table 14 Repeatability test results

[0172]

[0173] 3.8.5 Sample recovery test

[0174] Accurately weigh about 0.17 g of American cockroach powder (defatted) with a known content, add 1 mL of the reference solution in step "3.1" respectively, prepare the test solution according to the method in step "3.2", and then develop the color according to the method in step "3.5", measure the absorbance at 525 nm, and calculate the average recovery rate of glucosamine hydrochloride in American cockroach. See Table 15.

[0175] Table 15 Recovery determination of glucosamine hydrochloride (n=6)

[0176]

[0177] 3.8.6 Chitosan content

[0178] According to the optimal process of Example 1, 11 batches of American cockroach powder were prepared. The chitosan content in the American cockroach chitosan-free powder was determined according to the method of step "3.8.4". The results are shown in Table 16. The chitosan content ranged from (4.33% to 5.65%).

[0179] Table 1611 Determination results of chitosan content in the powder of 1611 batches of American cockroaches without chitosan

[0180]

[0181]

[0182] Example 4

[0183] Determination of protein content in ground flour

[0184] Referring to the first method Kjeldahl nitrogen determination method in GB5009.5-2016 "National Food Safety Standard Determination of Protein in Food", the protein content in 11 batches of American cockroach chitosan powder was determined. The determination was repeated 3 times, and the results are shown in Table 17. The protein content range is (68.67% to 76.93%).

[0185] Table 1711 batches of American cockroach chitosan powder protein content determination results (n = 3)

[0186]

[0187] Example 5

[0188] Determination of extractable content

[0189] According to the thermal method under the 2020 edition of the Chinese Pharmacopoeia, the extract in the sample was determined by the determination method of alcohol-soluble extracts (2201), using dilute ethanol as the extraction solvent, repeated 3 times, and the determination results are shown in Table 18. The extract content range is (7.04% to 9.08%).

[0190] Table 1811 batches of American cockroach chitosan powder extract test results (n = 3)

[0191]

[0192] Example 6

[0193] Total amino acid content determination

[0194] 6.1 Preparation of reference solution

[0195] Accurately weigh 10 mg of alanine reference substance, place it in a 10 mL volumetric flask, add distilled water to dissolve and dilute to the scale to obtain a 1 mg / mL reference substance stock solution. Accurately take 1 mL of the above stock solution, add water to dilute to 10 mL, and obtain a 0.1 mg / mL reference substance solution.

[0196] 6.2 Preparation of test solution

[0197] Weigh 0.25 g of the sample into an ampoule, add 5 mL of 9 mol / L hydrochloric acid solution, seal and shake well, hydrolyze at 100 ° C in an oven for 24 h, cool, neutralize with sodium hydroxide solution to neutrality, make up to 100 mL, centrifuge twice at 4000 rpm, 10 min each time, accurately pipette 5 mL of the supernatant, transfer to a 100 mL volumetric flask, dilute to the scale with water, shake well, and the test solution is obtained.

[0198] 6.3 Preparation of citrate buffer

[0199] Take 4.0 g of citric acid, add 40 mL of distilled water to dissolve, adjust the pH to 6.0 with sodium hydroxide, transfer to a 100 mL volumetric flask, dilute to the scale, and shake well.

[0200] Preparation of 6.41% ninhydrin ethanol solution

[0201] Weigh 0.5 g of ninhydrin, add 50 mL of anhydrous ethanol, and shake well.

[0202] 6.5 Drawing of standard curve

[0203] Accurately measure 1mL of the test solution under "6.2" and place it in a 10mL volumetric flask. Accurately transfer 0.00, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0mL of the reference solution in step "6.1" to a 10mL volumetric flask. Add 1mL of pH 6.0 citric acid buffer solution and 1mL of 1% ninhydrin ethanol solution. After boiling in a water bath for 30 minutes, measure the absorbance at 570nm. With absorbance A as the ordinate and concentration c (mg / mL) of the reference solution as the abscissa, perform linear regression. The regression equation is y=13.663x+0.0061, R 2 =0.9983, the results show that glucosamine hydrochloride has a good linear relationship in the range of 1-20 μg / mL. Figure 8 .

[0204] 6.6 Testing of test products

[0205] Accurately measure 1 mL of the test solution in step "6.2" and place it in a 10 mL volumetric flask. Add 1 mL of citrate buffer and 1% ninhydrin ethanol solution. React in a boiling water bath for 30 min. Add 1 mL of pH 6.0 citrate buffer solution and 1 mL of 1% ninhydrin ethanol solution. After boiling in a boiling water bath for 30 min, measure the absorbance at 570 nm and substitute it into the standard curve equation in step "6.5" to calculate the amino acid content.

[0206] 6.7 Test results

[0207] Table 1911 batches of American cockroach chitin powder total amino acid content determination results (n = 3)

[0208]

[0209] Example 7

[0210] Nucleoside content determination

[0211] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; methanol-0.01 mol / L potassium dihydrogen phosphate solution (1:99) as the mobile phase; and the detection wavelength was 260 nm.

[0212] Preparation of the test solution Take about 1g of the powder of this product, weigh it accurately, place it in a stoppered conical flask, accurately add 50mL of water, weigh it, ultrasonicate it for 60 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it. Accurately weigh an appropriate amount of uracil reference substance and hypoxanthine reference substance, add water to make a solution containing 0.2mg of uracil and hypoxanthine per 1mL; accurately weigh an appropriate amount of xanthine reference substance, add water to make a solution containing 60μg of xanthine per 1mL; accurately measure 1mL of each of the above three reference substance solutions, place them in the same 10mL volumetric flask, dilute them to the scale with water, shake it well, and obtain it.

[0213] Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0214] The results of the test are shown in Fig. 9 and Table 20.

[0215] Table 2011 batch American cockroach chitosan powder nucleoside content determination results (n = 3)

[0216]

[0217] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing American cockroach powder, characterized in that: The American cockroach medicinal material is defatted, crushed and sieved to obtain the American cockroach powder.

2. The preparation method according to claim 1, characterized in that: The rotation speed of the pulverization is 7000 rpm.

3. The preparation method according to claim 1, characterized in that: The sieve has an aperture of 500 mesh or more.

4. The American cockroach powder prepared by the preparation method as claimed in claim 1.

5. The American cockroach powder according to claim 4, characterized in that The American cockroach powder has a chitin content of ≤6%, a total amino acid content of ≥25% in terms of alanine, a total amount of uracil, hypoxanthine and xanthine of ≥2 mg / g, a protein content of ≥65%, a total peptide content of ≥12%, an extract content of ≥7.0%, a fat content of ≤5% and a water content of ≤5%.

6. Use of the American cockroach powder as claimed in claim 4 or 5 in the preparation of immunomodulatory products.

7. A medicine or health product for enhancing immunity, characterized in that: The invention comprises the American cockroach powder as claimed in claim 4 or 5.

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