阅读说明：本技术 一种蜚蠊糖蛋白及其制备方法与应用 (Cockroach glycoprotein and preparation method and application thereof ) 是由 杨永寿 肖培云 郝芳芳 何正春 顾婷 和英 陈怡� 袁丽萍 王宝宇 周永芳 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种蜚蠊糖蛋白及其制备方法与应用,该制备方法包括以下步骤：原料处理：将蜚蠊成虫进行杀灭,将成虫中的杂质分筛出,蜚蠊用温水清洗干净,烘干,得药材,粉碎,备用；蜚蠊糖蛋白的提取；蜚蠊糖蛋白的纯化。本发明从蜚蠊体内提取出一种用于调整免疫活性、抗氧化活性的糖蛋白物质,该糖蛋白物质是一种动物来源的天然免疫调节剂和抗氧化剂,可应用于治疗免疫低下、免疫紊乱及与脂质过氧化相关的慢性疾病。(The invention discloses cockroach glycoprotein and a preparation method and application thereof, wherein the preparation method comprises the following steps: raw material treatment: killing adult cockroach, screening out impurities in the adult cockroach, washing the adult cockroach with warm water, drying to obtain medicinal materials, and crushing for later use; extracting cockroach glucoprotein; purification of cockroach glycoprotein. The present invention extracts a glycoprotein substance for regulating immunological activity and antioxidant activity from blattaria, which is a natural immunomodulator and antioxidant derived from animals and is useful for treating hypoimmunity, immune disorders and chronic diseases associated with lipid peroxidation.)

1. A preparation method of cockroach glycoprotein is characterized by comprising the following steps:

step 1, raw material treatment: killing adult cockroach, screening out impurities in the adult cockroach, washing the adult cockroach with warm water, drying to obtain medicinal materials, and crushing for later use;

step 2, extracting cockroach glucoprotein;

and 3, purifying the cockroach glycoprotein.

2. The method for preparing the compound of claim 1, wherein the killing in the step 1 is performed by: raising the temperature of a culture workshop of adult cockroaches to 40-50 ℃ and maintaining for 20-30 minutes to kill the adult cockroaches, or pouring the adult cockroaches into warm water at 40-50 ℃ to kill the adult cockroaches, wherein the drying conditions are as follows: drying with hot air at a temperature below 80 deg.C until the water content is below 5%.

3. The method according to claim 1, wherein the extraction of cockroach glycoprotein in step 2 is specifically:

step 2.1, placing the crushed medicinal materials into an extraction tank, adding water, heating and refluxing under the condition of stirring, filtering, collecting filtrate, and repeatedly extracting for 3-4 times;

step 2.2, concentration: concentrating the obtained filtrate under reduced pressure to 1/5-2/5 of the original volume to obtain a concentrated solution with the specific gravity of 1.1-1.2, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding ether or extraction oil with the volume of 1-2 times (V/V), extracting and degreasing, standing, discharging a water layer, collecting filtrate, and filtering to obtain a degreased cockroach extract;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the ethanol concentration to 85% -90%, standing overnight, centrifuging, removing supernatant, recovering ethanol from the supernatant under reduced pressure, recycling, and collecting the precipitate after centrifugation to obtain glycoprotein crude extract.

4. The preparation method according to claim 1, wherein the ratio (g/ml) of the pulverized herbs to water in step 2.1 is 1: 7-1: 9, the temperature of heating reflux is 80-90 ℃, and the time is 2-3 hours.

5. The method according to claim 1, wherein the temperature of the concentration under reduced pressure in the step 2.2 is 60 to 70 ℃.

6. The preparation method according to claim 1, wherein the temperature of the standing overnight in the step 2.4 is 0-5 ℃, the centrifugal speed is 3000-4000 rpm, and the centrifugal time is 7-10 minutes.

7. The method according to claim 1, wherein the purification of cockroach glycoprotein in step 3 is specifically:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring until the crude glycoprotein extract is completely dissolved, preparing a 5% (W/V) aqueous solution, adding the solution into an activated AB series resin column, statically adsorbing for more than 8-12 hours, eluting with pure water with the volume of 3-5 times of the column volume, and collecting the eluent;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 50-70 ℃, and freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, thereby preparing the cockroach glycoprotein.

8. A cockroach glycoprotein produced by the production method according to any one of claims 1 to 7, wherein the cockroach glycoprotein is a mixture of an extraction active site consisting of a glycoprotein group, and the extract contains 25 to 30% of polysaccharides in terms of glucose and 65 to 70% of proteins in terms of bovine serum albumin.

9. Use of the cockroach glycoprotein according to claim 8 for the production of a drug or health product for regulating hypoimmunity, immune disorders and chronic diseases associated with lipid peroxidation.

10. The use according to claim 9, wherein the pharmaceutical dosage form of the medicament or nutraceutical is a tablet, a capsule, a granule or an oral liquid.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to cockroach glycoprotein, and a preparation method and application thereof.

Background

The glycoprotein is a glycosylation combined protein formed by connecting oligosaccharide and polypeptide or protein through covalent bond, and the structure mainly comprises polypeptide chain and sugar, which are connected through covalent bond. Glycoproteins are widely present in animals, plants and microorganisms in various forms as a class of binding proteins, and play roles such as cell signal recognition, growth regulation, intercellular information transfer, and regulation of immune activity in organisms. Researches show that the glycoprotein has the effects of enhancing immunity regulation, inhibiting tumors, reducing blood sugar and blood fat, resisting oxidation, preventing aging and the like.

Blatta Seu Periplaneta (Periplaneta americana L.) is one of the insects of the genus Periplaneta of the family Blattaceae, called Blatta Seu Periplaneta, Veronica nigricana, horse-range chicken, etc., and is considered as one of the four pests. However, as an important part of the traditional Chinese medicine cockroach in treating diseases, more than 2000 years ago, the book "Shen nong Ben Cao Jing" recorded that blatta is cheap and mainly causes blood stasis, hardness, chills and fever, accumulation and pharyngitis ". The compendium of materia medica, insect department, blattaria records its main indications of blood stasis, hard mass, cold and heat, descending qi, promoting blood circulation. Modern Chinese medicine researches find that the cockroach component contains polysaccharide, protein, polypeptide, amino acid, some small molecular substances and the like, and the cockroach extract has the effects of protecting the liver, promoting wound healing, resisting oxidation, resisting tumors, resisting inflammation and the like.

The research on extraction and purification of cockroach glucoprotein in cockroach medicinal materials and the immunoregulation and antioxidation effects of cockroach glucoprotein has not been reported. Therefore, there is a need for a novel process for producing cockroach glycoprotein.

Disclosure of Invention

In view of the above, the present invention provides a cockroach glycoprotein, and a preparation method and applications thereof.

In order to solve the technical problem, the invention discloses a preparation method of cockroach glycoprotein, which comprises the following steps:

step 1, raw material treatment: killing adult cockroach, screening out impurities in the adult cockroach, washing the adult cockroach with warm water, drying to obtain medicinal materials, and crushing for later use;

step 2, extracting cockroach glucoprotein;

and 3, purifying the cockroach glycoprotein.

Optionally, the killing method in step 1 is: raising the temperature of a culture workshop of adult cockroaches to 40-50 ℃ and maintaining for 20-30 minutes to kill the adult cockroaches, or pouring the adult cockroaches into warm water at 40-50 ℃ to kill the adult cockroaches, wherein the drying conditions are as follows: drying with hot air at a temperature below 80 deg.C until the water content is below 5%.

Optionally, the extraction of cockroach glycoprotein in the step 2 is specifically:

step 2.1, placing the crushed medicinal materials into an extraction tank, adding water, heating and refluxing under the condition of stirring, filtering, collecting filtrate, and repeatedly extracting for 3-4 times;

step 2.2, concentration: concentrating the obtained filtrate under reduced pressure to 1/5-2/5 of the original volume to obtain a concentrated solution with the specific gravity of 1.1-1.2, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding ether or extraction oil with the volume of 1-2 times (V/V), extracting and degreasing, standing, discharging a water layer, collecting filtrate, and filtering to obtain a degreased cockroach extract;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the ethanol concentration to 85% -90%, standing overnight, centrifuging, removing supernatant, recovering ethanol from the supernatant under reduced pressure, recycling, and collecting the precipitate after centrifugation to obtain glycoprotein crude extract.

Optionally, the ratio (g/ml) of the pulverized herbs to water in step 2.1 is 1: 7-1: 9, the temperature of heating reflux is 80-90 ℃, and the time is 2-3 hours.

Optionally, the temperature of the reduced pressure concentration in the step 2.2 is 60-70 ℃.

Optionally, the temperature of the standing overnight in the step 2.4 is 0-5 ℃, the centrifugal speed is 3000-4000 rpm, and the centrifugal time is 7-10 minutes.

Optionally, the purification of cockroach glycoprotein in the step 3 is specifically as follows:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring until the crude glycoprotein extract is completely dissolved, preparing a 5% (W/V) aqueous solution, adding the solution into an activated AB series resin column, statically adsorbing for more than 8-12 hours, eluting with pure water with the volume of 3-5 times of the column volume, and collecting the eluent;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 50-70 ℃, and freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, thereby preparing the cockroach glycoprotein.

The present invention also discloses a cockroach glycoprotein produced by the above production method, wherein the cockroach glycoprotein is a mixture and is an extraction active site consisting of a glycoprotein group, and the extract contains 25 to 30% by glucose and 65 to 70% by bovine serum albumin.

The invention also discloses application of the cockroach glycoprotein in preparing medicines or health products for regulating low immunity, immune disorder and chronic diseases related to lipid peroxidation.

Optionally, the pharmaceutical dosage form of the medicine or health care product is tablets, capsules, granules or oral liquid.

Compared with the prior art, the invention can obtain the following technical effects:

the present invention relates to a glycoprotein substance extracted from blattaria in order to regulate immune activity and antioxidant activity, which is a natural immunomodulator and antioxidant derived from animals, and can be used for treating diseases such as hypoimmunity, immune disorder and lipid peroxidation, such as: the recovery of the tumor patients after the damage of the immune system after radiotherapy and chemotherapy; patients with immune imbalance due to the large amount of hormone-based drugs required for the disease; susceptible people due to low immunity; delay the occurrence and development of chronic diseases caused by lipid peroxidation, and the like.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a HE staining pattern (100X) of spleen tissue according to the present invention;

FIG. 2 is a graph showing an amplification curve and a dissolution curve of the GAPDH gene of the present invention;

FIG. 3 is a graph of the amplification curve and lysis curve for the TLR4 gene of the invention;

FIG. 4 is a graph showing the amplification curve and dissolution curve of the MyD88 gene of the invention;

FIG. 5 is a graph showing the amplification curve and dissolution profile of the TRAF-6 gene of the present invention;

FIG. 6 is a graph showing an amplification curve and a dissolution curve of the NF- κ B p65 gene of the invention;

FIG. 7 is a graph showing the amplification curve and dissolution profile of the p-I.kappa.B.alpha.gene of the present invention;

FIG. 8 is a graph showing an amplification curve and a dissolution curve of the p-NF- κ B p65 gene of the invention.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

The invention discloses a preparation method of cockroach glycoprotein, which comprises the following steps:

step 1, raw material treatment: raising the temperature of an adult cockroach cultivation workshop to 40-50 ℃ and maintaining for 20-30 minutes to kill the adult cockroach, or pouring the adult cockroach into warm water at about 40-50 ℃ to kill the adult cockroach, screening out impurities in the adult cockroach, cleaning the cockroach with warm water, drying the cockroach with hot air at the temperature lower than 80 ℃ until the moisture content is lower than 5%, obtaining medicinal materials, and crushing the medicinal materials for later use;

step 2, extracting cockroach glycoprotein:

step 2.1, placing the crushed medicinal materials into an extraction tank, adding water according to a material-liquid ratio (g/ml) of 1: 7-1: 9, stirring, heating and refluxing at 80-90 ℃ for 2-3 hours, filtering, collecting filtrate, and repeatedly extracting for 3-4 times;

step 2.2, concentration: concentrating the obtained filtrate at 60-70 ℃ under reduced pressure to 1/5-2/5 of the original volume to obtain a concentrated solution with the specific gravity of 1.1-1.2, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding ether or extraction oil with the volume of 1-2 times (V/V), extracting and degreasing, standing, discharging a water layer, collecting filtrate, and filtering to obtain a degreased cockroach extract;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the concentration of the ethanol to 85% -90%, standing overnight at 0-5 ℃, centrifuging for 7-10 minutes at 3000-4000 rpm, removing supernatant, recovering the ethanol from the supernatant under reduced pressure, recycling, collecting the centrifuged precipitate, and obtaining glycoprotein crude extract;

wherein, the temperature of heating reflux is lower than 80 ℃, the extraction rate of glycoprotein is too low, when the temperature of heating reflux reaches 90 ℃, the extraction rate of glycoprotein is the highest and is 4.68%, and then the extraction rate of glycoprotein is reduced along with the rise of temperature, so that the glycoprotein is easy to decompose under the high temperature condition, thereby reducing the extraction rate.

And 3, purifying cockroach glycoprotein:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring until the crude glycoprotein extract is completely dissolved, preparing a 5% (W/V) aqueous solution, adding the solution into an activated AB series resin column, statically adsorbing for more than 8-12 hours, eluting with pure water with the volume of 3-5 times of the column volume, and collecting the eluent;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 50-70 ℃, and freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, thereby preparing the cockroach glycoprotein.

The invention also discloses the cockroach glucoprotein prepared by the preparation method, which is a mixture and is an extraction active site consisting of glucoprotein group, in the extract, the content of polysaccharide is 25-30% calculated by glucose, and the content of protein is 65-70% calculated by calf serum albumin.

The invention also discloses application of the cockroach glycoprotein in preparing a medicine or health-care product for regulating the hypoimmunity of a human body, wherein the medicine dosage form of the medicine or health-care product is tablets, capsules, granules or oral liquid.

Example 1

A preparation method of cockroach glycoprotein comprises the following steps:

step 1, raw material treatment: raising the temperature of an adult cockroach cultivation workshop to 40-50 ℃ and maintaining for 20-30 minutes to kill the adult cockroach, or pouring the adult cockroach into warm water at about 40-50 ℃ to kill the adult cockroach, screening out impurities in the adult cockroach, cleaning the cockroach with warm water, drying the cockroach with hot air at the temperature lower than 80 ℃ until the moisture content is lower than 5%, obtaining medicinal materials, and crushing the medicinal materials for later use;

step 2, extracting cockroach glycoprotein:

step 2.1, weighing 100g of crushed medicinal materials, putting the crushed medicinal materials into an extraction tank, adding water according to a material-liquid ratio (g/ml) of 1: 9, stirring, heating and refluxing at 90 ℃ for 2.25 hours, filtering, collecting filtrate, and repeatedly extracting for 4 times;

step 2.2, concentration: concentrating the filtrate at 65 deg.C under reduced pressure to 1.5/5 of the original volume to obtain concentrated solution with specific gravity of 1.16, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding ether or extraction oil with the volume of 1-2 times (V/V), extracting and degreasing, standing, discharging a water layer, collecting filtrate, and filtering to obtain a degreased cockroach extract;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the ethanol concentration to 88%, standing overnight at 0-5 ℃, centrifuging for 8 minutes at 3500 rpm, removing the supernatant, recovering ethanol from the supernatant under reduced pressure, recycling, and collecting the centrifuged precipitate to obtain a glycoprotein crude extract;

and 3, purifying cockroach glycoprotein:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring to dissolve completely, preparing into 5% (W/V) water solution, adding the solution into activated AB series resin column, statically adsorbing for more than 10 hr, eluting with 4 times column volume of pure water, and collecting eluate;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 60 ℃, freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, and preparing to obtain the cockroach glycoprotein, wherein the extraction rate of the obtained glycoprotein is 6.10%.

Example 2

A preparation method of cockroach glycoprotein comprises the following steps:

step 1, raw material treatment: heating the temperature of adult cockroach cultivation workshop to 40 deg.C, maintaining for 30min to kill, or pouring adult cockroach into warm water of about 40 deg.C to kill, sieving out impurities in adult cockroach, cleaning cockroach with warm water, drying with hot air of below 80 deg.C until water content is below 5%, to obtain medicinal material, and pulverizing;

step 2, extracting cockroach glycoprotein:

step 2.1, placing the crushed medicinal materials into an extraction tank, adding water according to a material-liquid ratio (g/ml) of 1: 7, stirring, heating and refluxing at 80 ℃ for 3 hours, filtering, collecting filtrate, and repeatedly extracting for 3 times;

step 2.2, concentration: concentrating the filtrate at 60 deg.C under reduced pressure to 2/5 to obtain concentrated solution with specific gravity of 1.1, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding 2 times volume (V/V) of ether or extraction oil, extracting for degreasing, standing, discharging water layer, collecting filtrate, and filtering to obtain degreased extract of Blatta Seu Periplaneta;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the ethanol concentration to 85%, standing overnight at 0-5 ℃, centrifuging for 7 minutes at 4000rpm, removing supernatant, recovering ethanol from the supernatant under reduced pressure, recycling, and collecting the centrifuged precipitate to obtain glycoprotein crude extract;

and 3, purifying cockroach glycoprotein:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring to dissolve completely, preparing into 5% (W/V) water solution, adding the solution into activated AB series resin column, statically adsorbing for more than 8 hr, eluting with 3 times column volume of pure water, and collecting eluate;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 70 ℃, freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, and preparing to obtain the cockroach glycoprotein, wherein the extraction rate of the obtained glycoprotein is 5.85%.

Example 3

A preparation method of cockroach glycoprotein comprises the following steps:

step 1, raw material treatment: heating the temperature of adult cockroach cultivation workshop to 50 deg.C, maintaining for 20min to kill, or pouring adult cockroach into 50 deg.C warm water to kill, sieving out impurities in adult cockroach, cleaning cockroach with warm water, drying with 80 deg.C hot air until water content is less than 5%, to obtain medicinal material, and pulverizing;

step 2, extracting cockroach glycoprotein:

step 2.1, placing the crushed medicinal materials into an extraction tank, adding water according to a material-liquid ratio (g/ml) of 1: 9, stirring, heating and refluxing at 90 ℃ for 2 hours, filtering, collecting filtrate, and repeatedly extracting for 4 times;

step 2.2, concentration: concentrating the filtrate at 70 deg.C under reduced pressure to 1/5 to obtain concentrated solution with specific gravity of 1.2, and cooling to room temperature;

step 2.3, degreasing: adding the concentrated solution obtained in the previous step into an oil-water separation device, adding 1 volume (V/V) of ether or extraction oil, extracting for degreasing, standing, discharging water layer, collecting filtrate, and filtering to obtain degreased extract of Blatta Seu Periplaneta;

step 2.4, precipitation: adding 95% ethanol into the degreased water layer, slowly stirring while adding, adjusting the ethanol concentration to 90%, standing overnight at 0-5 ℃, centrifuging for 10 minutes at 3000 r/min, removing the supernatant, recovering ethanol from the supernatant under reduced pressure, recycling, and collecting the centrifuged precipitate to obtain a glycoprotein crude extract;

and 3, purifying cockroach glycoprotein:

step 3.1, decoloring and removing impurities: adding water into the crude glycoprotein extract, stirring to dissolve completely, preparing into 5% (W/V) water solution, adding the solution into activated AB series resin column, statically adsorbing for more than 12 hr, eluting with 5 times column volume of pure water, and collecting eluate;

and 3.2, filtering the obtained eluent, concentrating under reduced pressure at 50 ℃, freeze-drying to obtain freeze-dried fine powder of the cockroach glycoprotein, and preparing to obtain the cockroach glycoprotein, wherein the extraction rate of the obtained glycoprotein is 5.92%.

The technical effects of the invention are illustrated below with reference to specific experimental data:

effect of cockroach glycoprotein (PAG extract) on proliferation potency of RAW264.7

Selecting RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to 2 × 10⁴one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO₂The culture chamber was incubated for 24 hours, and then the supernatant was discarded, 3.125, 12.5, 50, 200, and 800. mu.g/. mu.L of PAG solution prepared in example 1 was added thereto, 5 wells were set for each concentration, and a blank control group and a normal group were set. After administration, the cells were placed in an incubator and incubated for 24 hours, and 20. mu.L of MTT solution (5mg/mL) was added to each well, and incubation was continued for 4 hours. Sucking out the culture solution, adding 150 μ L DMSO into each well, placing on a shaker at 37 deg.C, shaking at low speed for 10min,the crystals were fully dissolved. The OD value of each well was measured at 490nm, and the proliferation rate of each group of cells was calculated according to the following formula, and the experiment was repeated 3 times.

Proliferation rate (%) - (drug group OD mean-blank OD mean)/(normal group OD mean-blank OD mean) × 100%

PAG has a proliferation capacity of 140.6% for RAW264.7 cells when administered at a concentration of 800 ug/mL.

TABLE 1 Effect of cockroach glycoprotein on RAW264.7 proliferation potency (n ═ 5)

Second, cockroach glucoprotein (PAG extract) for regulating immunity of mice with hypoimmunity

Cyclophosphamide (CTX) is an important chemotherapeutic drug in tumor therapy, belonging to the alkylating agent class. Use of CTX results in myelosuppression and immunosuppression, destroys DNA structure, kills immune cells, interferes with proliferation and differentiation of B and T cells, and inhibits humoral and cellular immunity.

In the experiment, a mouse model with low immunity is established by carrying out intraperitoneal injection on 40mg/kg of CTX (cytotoxic T lymphocyte) to Balb/c mice for 3 consecutive days, the change condition of the weight of the mice is monitored in the experimental process, the daily activity state of the mice is observed, and after the mice die, the indexes of the organ index, the proliferation of splenic lymphocytes, splenic T lymphocytes and subgroups, serum immunoglobulin, serum cytokines, whole blood and the like of the mice are measured to prove whether the PAG has immunoregulatory activity on the mice with low immunity. The immunoregulatory mechanism of PAG was initially explored by measuring mRNA expression of genes associated with NF- κ B signaling pathway in spleen tissue.

1 method of experiment

The method comprises the following steps of (1) taking an immunocompromised mouse as a research object, measuring the content of RBC, WBC, HGB and PLT of the mouse by adopting a full-automatic animal blood cell analyzer, and researching the influence of PAG on the blood index of the immunocompromised mouse; determining the influence of PAG on the proliferative capacity of splenic lymphocytes of mice by adopting an MTT method; flow cytometry analysis for determination of PAG on splenocytesCD₃ ⁺、CD₄ ⁺、CD₈ ⁺The influence of (a); measuring the contents of IgG, IgA, IgM, IL-1 beta, IL-6 and TNF-alpha in serum by adopting an ELISA method; and detecting the expression of the NF-kB signal channel related factor mRNA in the mouse splenocytes by adopting an RT-qPCR method. The grouping and dose are shown in table 2.

Normal group (Normal): physiological saline;

model group (Model): CTX and saline;

LNT positive group (LNT): CTX + Lentinan (LNT);

LMS positive group (LMS): CTX + LMS;

PAG low dose group (PAG-L): CTX + PAG-L (prepared in example 1);

dose group in PAG (PAG-M): CTX + PAG-M (prepared in example 1);

PAG high dose group (PAG-H): CTX + PAG-H (prepared in example 1);

table 2 methods of administration for each group of mice (n ═ 10)

2 results of the experiment

2.1 effects on visceral indices: PAG increased spleen and thymus indices in immunocompromised mice (P <0.05, P <0.01), see table 3;

TABLE 3 influence of PAG on organ index in immunocompromised mice: (n＝10)

Note: comparison with the normal group: p <0.05, P < 0.01; comparison with model groups: # P <0.05, # P < 0.01.

2.2 Effect on Whole blood RBC, WBC, HGB, PLT: PAG can improve blood index of mice with low immunity, WBC and RBC content of administration group are increased, and difference has statistical significance (P <0.05, P < 0.01);

the effect of PAG prepared in example 1 on WBC, RBC, HGB, PLT levels in CTX-induced immunocompromised mice is shown in table 4. Compared with the normal group, the contents of WBC, RBC, HGB and PLT in the model group are obviously reduced, and the differences have statistical significance (P <0.05 and P <0.01), which indicates that the CTX molding is successful; compared with the model group, the PAG administration group has different increases of WBC, RBC, HGB and PLT content, and the WBC and RBC content have statistical significance (P <0.05 and P <0.01), wherein the HGB and PLT content increase obviously, and the difference has statistical significance (P < 0.05).

TABLE 4 influence of PAG on four indicators of whole blood in immunocompromised mice: (n＝10)

Note: comparison with the normal group: p <0.05, P < 0.01; comparison with model groups: # P <0.05, # P < 0.01.

2.3 Effect on splenic lymphocyte proliferation: PAG can promote proliferation of spleen lymphocyte; compared with the model group, the proliferation capacity of the PAG-M, PAG-H, PAG-L dose on B lymphocytes is obviously enhanced (P <0.05, P < 0.01); PAG significantly increases the number of T and B lymphocytes. Compared with the normal group, the spleen lymphocyte proliferation capacity of the model group is the weakest, and the difference has obvious statistical significance (P < 0.01). See table 5.

TABLE 5 Effect of PAG on splenic lymphocyte proliferation in immunocompromised mice: (n＝10)

Note: comparison with the normal group: p <0.05, P < 0.01; comparison with model groups: # P <0.05, # P < 0.01.

2.4PAG vs CD₃ ⁺、CD₄ ⁺And CD₈ ⁺Modulation of cells: the results of the effect of PAG on splenic T lymphocyte subpopulations in immunocompromised mice are shown in table 6. (III) CD₃ ⁺: compared with the normal group, the model group and the administration group can enable CD₃ ⁺The amount is increased and the difference is statistically significant (P)<0.01); the administration group caused T-lymphocytosis and the PAG-M and PAG-H groups increased significantly (P) compared to the model group<0.05，P<0.01)；②CD₄ ⁺: compared with the normal group, the model group and the administration group can enable CD₄ ⁺Increase, the difference has significant statistical significance (P)<0.01)；③CD₈ ⁺: compared with the normal group, the model group and the administration group can enable CD₈ ⁺An increase in the number of cells; CD₄ ⁺/CD₈ ⁺: model group CD compared to Normal group₄ ⁺/CD₈ ⁺The ratio is obviously increased, and the difference has statistical significance (P)<0.01); PAG-L, PAG-M, PAG-H group CD compared to model group₄ ⁺/CD₈ ⁺The obvious reduction and the difference have statistical significance (P)<0.01), indicating that PAG can gradually recover the body of the mouse with the immune disorder and the immune regulation has an amount-effect relationship.

Table 6 effect of PAG on splenic T lymphocyte subpopulations in immunocompromised mice: (n＝10)

Note: comparison with the normal group: p <0.05, P < 0.01; comparison with model groups: # P <0.05, # P < 0.01.

2.5 Effect on the immunoglobulin content in the serum of mice: the effect of PAG on serum immune factors of immunocompromised mice is shown in table 7. Compared with the normal group, the IgA, IgG and IgM of the model group are obviously reduced, and the difference has statistical significance (P is less than 0.01), which indicates that the CTX molding is successful; compared with the model group, the IgA, IgG and IgM expressions of the drug group are all increased, and the differences of the groups except the PAG-L group have statistical significance (P <0.05, P < 0.01).

Table 7 effect of PAG on serum immunoglobulin in hypoimmunity mice: (n＝10)

Note: compared with the normal group: p <0.05, P < 0.01; and (3) comparing with the model group: # P <0.05, # P < 0.01.

2.6 Effect on cytokine levels in mouse sera: the effect of PAG on serum cell factor of mice with low immunity is shown in Table 8, compared with the normal group, the model group IL-1 beta, IL-6 and TNF-alpha are obviously reduced, and the difference has statistical significance (P <0.01), which indicates that the cyclophosphamide molding is successful; compared with the model group, the PAG-L, PAG-M, PAG-H group has obviously increased content of IL-1 beta, IL-6 and TNF-alpha, and the difference has statistical significance (P is less than 0.05 and P is less than 0.01).

Table 8 effect of PAG and PAGW on serum cytokines in immunocompromised mice ((ii))n＝10)

Note: compared with the normal group: p <0.05, P < 0.01; and (3) comparing with the model group: # P <0.05, # P < 0.01.

3 Observation of spleen histopathology

The spleen tissue HE staining results are shown in fig. 1. The dark blue is white marrow of spleen tissue and is scattered in red marrow; the red area is the red marrow of the spleen tissue, and is distributed between the white marrow or between the white marrow and the spleen trabecula. The red marrow and the white marrow of normal spleen tissues are clearly demarcated; the boundary between the red marrow and the white marrow of the model group is fuzzy; PAG (prepared in example 1) low, medium and high dose groups, the boundary between white marrow and red marrow becomes gradually clear with increasing dosage; in the PAGW low/medium/high dose group, the boundary between red marrow and white marrow is clear, and the pathological change of spleen tissues is obvious. The PAG and the PAWGW are shown to have immunoregulation effect on mice with low immunity.

Third, study on mechanism of regulation of cockroach glycoprotein (PAG extract) prepared in example 1 on immunity of mice with low immunity

1, the expression of NF-kB signal channel related factor mRNA in mouse spleen tissues is detected through RT-qPCR, so as to research the regulating mechanism of PAG extract on the immunity of mice with low immunity.

The expression level of PAG on mRNA of NF-kB signal channel related gene in mouse spleen tissue is shown in Table 9. RT-qPCR results show that compared with a normal group, the expression of model groups of TLR4, MyD88, TRAF-6, NF-kappa Bp65, P-I kappa B alpha and P-NF-kappa B P65 is obviously increased, and the difference has statistical significance (P is less than 0.01); compared with the model group, the expression of the mRNA of TLR4, MyD88, TRAF-6, NF-kappa B P65, P-I kappa B alpha and P-NF-kappa Bp65 of each PAG group is obviously reduced (P < 0.01).

TABLE 9 expression of TLR4, MyD88, TRAF-6, p65, p-I κ B α, p-p65 mRNA in spleen (R) ((R))n＝10)

Note: comparison with the normal group: p <0.05, P < 0.01; comparison with model groups: # P <0.05, # P < 0.01.

CTX can increase the expression of TLR4, MyD88, TRAF-6, NF-kappa B P65, P-IKB alpha and P-NF-kappa B P65 mRNA in an NF-kappa B channel, and compared with a model group, the expression of TLR4, MyD88, TRAF-6, NF-kappa B P65, P-IKB alpha and P-NF-kappa B P65 mRNA in an administration group is obviously reduced (P <0.01), which indicates that PAG can regulate the immune function of an organism through an NF-kappa B signal channel.

2 spleen tissue related gene amplification and dissolution curve results in NF-kB signal channel.

The expression level of related cytokines in a NF-kB signal channel of a mouse spleen tissue detected by an RT-qPCR method is shown in figures 2-8, amplification curves of various groups of target genes and internal reference genes are S-shaped, the amplification curves have obvious platform periods, the amplification efficiency of various target genes is similar, a dissolution curve presents a single peak, and the method indicates that non-specific amplification and dimer formation do not exist and indicates that the gene expression is good.

The experiment discovers that the PAG and the PAWGW can increase the content of IL-1 beta, IL-6 and TNF-alpha in the serum of a mouse with low immunity by measuring the content of IL-1 beta, IL-6 and TNF-alpha in the serum, and indicates that the PAG and the PAWGW can regulate the immune function of an organism by activating an NF-kappa B signal channel. Experiments select a plurality of key factors of TLR4, MyD88, TRAF-6, NF-kappa B p65, p-I kappa B alpha and p-NF-kappa Bp65 in an NF-kappa B signal channel to carry out PCR experiments, and preliminarily explore the action mechanism of PAG immune regulation by measuring the expression of mRNA of the factors. The experimental results show that CTX can increase the expression of TLR4, MyD88, TRAF-6, NF-kappa B P65, P-I kappa B alpha and P-NF-kappa B P65 mRNA in NF-kappa B pathway (P <0.01), compared with a model group, the expression of TLR4, MyD88, TRAF-6, NF-kappa Bp65, P-I kappa B alpha and P-NF-kappa B P65 mRNA in an administration group is obviously reduced, PAG can down-regulate the expression of NF-kappa B signal pathway related factor mRNA, and the PAG is possible to realize the regulation of the immune activity of an organism through NF-kappa B signal pathway.

Fourth, measurement of antioxidant Activity of cockroach glycoprotein (PAG extract) obtained in example 1

The in vitro antioxidant activity is adopted to research the scavenging rate of PAG on DPPH free radical, total reduction rate, hydroxyl free radical and ABTS free radical.

1 determination of the ability to scavenge DPPH free radicals

Respectively refining 2mL of PAG and vitamin C (Vc) solutions with different concentrations (0.01, 0.02, 0.04, 0.06, 0.08 and 0.10mg/mL), finely adding 2mL of DPPH solution, mixing uniformly, standing at room temperature for 30min, centrifuging at 4000rpm for 10min, and taking the supernatant to measure the light absorption value at 517 nm.

Wherein A is₀: the light absorption value of 2mL of absolute ethyl alcohol and 2mL of DPPH solution; a. the₁: the light absorption value of 2mL of sample solution +2mL of DPPH solution; a. the₂: absorbance of 2mL sample solution +2mL absolute ethanol.

2 determination of Total reducing Capacity

2mL of 0.025mol/L phosphate buffer solution with pH 6.86 and 2mL of PAG and Vc solutions with different concentrations (0.04, 0.08, 0.12, 0.16, 0.20 and 0.24mg/mL) are respectively extracted from a 10mL centrifuge tube, 2mL of 1% potassium ferricyanide is added, and the mixture is mixed uniformly and reacted for 20min at 50 ℃. After the reaction solution is taken out, 2mL of 10% trichloroacetic acid is added to stop the reaction, and the reaction solution is centrifuged at 4000rpm for 10 min. 2mL of the supernatant was extracted, and 2mL of distilled water and 0.5mL of 0.1% FeCl were added to the extract₃Standing for 10min after mixing uniformly, and detecting the light absorption value at 700 nm.

Reducing power of A₁-A₂

Wherein A is₁: absorbance of the sample set; a. the₂: sample background absorbance (equal volume of distilled water instead of FeCl)₃A solution).

Determination of 3-hydroxy radical scavenging Rate

Refining 9mmol/L FeSO₄1mL of 9mmol/L salicylic acid-ethanol solution and 1mL of PAG Vc solution with different concentrations (0.4, 0.8, 1.2, 1.6 and 2.0 mg/mL). Refined 1mL of 1% H₂O₂The reaction was started and carried out at 37 ℃ for 30min, and the absorbance of each concentration sample was measured at 510nm with distilled water as a reference.

In the formula, A₀: 1mL salicylic acid +1mL FeSO₄+1mL of water +1mL of H₂O₂；A₁: 1mL salicylic acid +1mL FeSO₄+1mL sample +1mL H₂O₂；A₂: 1mL salicylic acid +1mL FeSO₄+1mL sample +1mL H₂O。

Determination of the scavenging Capacity of 4ABTS free radicals

Mixing ABTS with the same volume of 7.4mmol/L and potassium persulfate solution with the same volume of 2.6mmol/L, and standing for 12-16 h at room temperature in the dark to prepare ABTS stock solution. The stock solution was diluted with a phosphate buffer solution of pH 7.4 to give an absorbance at 734nm (0.70. + -. 0.02) to prepare an ABTS working solution. PAG and Vc solutions with different concentrations (0.01, 0.02, 0.04, 0.08, 0.16, 0.32mg/mL) are extracted and added with 6mL ABTS working solution, mixed evenly, kept stand for 6min at room temperature and then measured for absorbance at 734 nm.

Wherein A is₀: absorbance of 1mL of distilled water +6mL of ABTS working solution; a. the₁: absorbance of 1mL of sample solution +6mL of ABTS working solution; a. the₂: absorbance of 1mL of sample solution +6mL of distilled water.

5 results of the experiment

5.1 measurement Table of DPPH radical scavenging ability of PAG

As a result of scavenging DPPH free radicals by PAG and Vc, as shown in Table 10, the scavenging ability of PAG Vc for DPPH free radicals increased with increasing administration concentration, and when the concentration was 008mg/mL, PAG clearance is 91.67%, Vc clearance is 96.43%. IC with PAG for DPPH radical scavenging₅₀The values were 0.028mg/mL, respectively, and the results showed that PAG had a good ability to scavenge DPPH radicals.

TABLE 10 DPPH radical scavenging Rate for PAG and Vc

5.2 determination of Total reducing Capacity of PAG

The results of the reducing abilities of PAG and Vc are shown in table 11, and the reducing abilities both increased significantly with increasing concentrations of PAG and Vc. When the concentration of Vc reaches 0.16mg/mL, the absorbance value is basically stable; when the concentration of PAG was 0.16mg/mL, the absorbance was 0.593, respectively, and the reducing ability was not as good as Vc, but the good reducing ability of PAG was still shown.

TABLE 11 Total reducing power of PAG and Vc

5.3 measurement of hydroxyl radical scavenging Rate

As can be seen from Table 12, the OH radical scavenging rate increases with the increase of the glycoprotein concentration at a glycoprotein concentration of 0.4-1.6 mg/mL, and when the PAG concentration reaches 2.0mg/mL, the scavenging rate is 96.25%, the Vc scavenging rate is 99.91%, and the scavenging capacities are relatively close. IC for scavenging OH free radical by PAG₅₀The value was 0.748mg/mL, indicating that PAG has a good ability to scavenge OH radicals.

TABLE 12 hydroxy radical scavenging rates for PAG and Vc

5.4 determination of the scavenging Capacity of ABTS free radicals

The results are shown in Table 13. When PAG is 0.01-0.16 mg/mL, ABTS free radical scavenging ability of the medicine is strong; when the concentration of the medicine is 0.32mg/mL, the clearance rate of PAG to ABTS free radicals is 91.03%. IC for scavenging ABTS free radical by PAG and Vc₅₀The values were 0.075 and 0.035mg/mL, respectively, indicating that PAG has a certain ability to scavenge ABTS free radicals.

TABLE 13 PAG and Vc clearance of ABTS

6 conclusion of antioxidant Activity detection

Biological oxidation produces reactive free radicals, which are associated with the onset and progression of various diseases. These diseases include cardiovascular disease, gastrointestinal digestive system disease, cancer, diabetes, cataract, aging, etc. almost all chronic diseases are closely related to the accumulation of excessive free radicals in the human body. Eliminating excessive free radicals in body can reduce their damage to body. In the invention, IC of PAG for scavenging DPPH free radical is found through experiments₅₀A value of 0.025 mg/mL; when the concentration of PAG is 0.16mg/mL, the absorbance is 0.593; IC for scavenging OH free radical by PAG₅₀The value was 0.748 mg/mL; IC for scavenging ability of PAG to ABTS free radical₅₀The value was 0.075 mg/mL.

The research shows that PAG has the functions of eliminating DPPH free radical, hydroxyl free radical and ABTS free radical, certain total reducing capacity and high antioxidant activity.

Fifth, experiment for measuring Total sugar and Total protein content in cockroach glycoprotein (PAG extract) prepared in example 1

Respectively establishing a content determination method for determining total sugar and total protein in extract PAG. The total protein content of the glycoprotein extract was determined by Folin-phenol method (Fowry method) with bovine serum albumin as a control and at a wavelength of 750 nm. And (3) determining the total sugar content in the glycoprotein extract by adopting a phenol-sulfuric acid method and taking glucose as a reference substance, wherein the detection wavelength is 490 nm.

1 polysaccharide content measurement results

As in table 14, the polysaccharide content of PAG was 21.24%.

TABLE 14 PAG polysaccharide assay results

2PAG protein content determination results

The measurement results of the protein content are shown in Table 15, and the protein content of PAG is 65.99%.

TABLE 15 protein content determination results for PAG and PAWGW

In conclusion, in vitro activity experiments show that the value increment rate of PAG on RAW264.7 is 140.6%, and the proliferation capacity is strong.

PAG has good scavenging ability to DPPH free radical, hydroxyl free radical and ABTS free radical, and has good antioxidant activity.

PAG can recover the weight, organ index, blood index, spleen tissue and immune globulin and cell factor in blood serum of hypoimmunity mice, increase spleen lymphocyte and accelerate proliferation capacity, which shows that PAG has stronger immunoregulation activity.

PAG can down-regulate the expression of related factors with high expression in NF-kB signal channel caused by CTX, and the regulation mechanism may be related to NF-kB signal channel.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.