Periplaneta americana glucan Pa1-B1 as well as preparation method and application thereof

Abstract

The invention provides a periplaneta americana glucan Pa1-B1 as well as a preparation method and application of the periplaneta americana glucan Pa1-B1. Belongs to the technical field of biological medicine. The American cockroach polysaccharide Pa1-B1 is glucan of which the main chain is connected by alpha (1-> 4), glucose on a side chain is connected to the main chain by alpha (1-> 6), and the weight-average molecular weight is 36000-42000 kDa. The periplaneta americana glucan Pa1-B1 is obtained by the following steps: crushing a periplaneta americana scorpion, degreasing with 95% ethanol, carrying out enzyme hydrolysis extraction under an alkaline condition, and separating and purifying through alcohol precipitation, ion exchange column chromatography and gel column chromatography. The American cockroach glucan Pa1-B1 disclosed by the invention is a polysaccharide which is found in American cockroaches for the first time and is novel in structure, and has the activity of enhancing immune regulation. After in-vivo advanced administration, periplaneta americana glucan Pa1-B1 can powerfully train body immunity, significantly inhibit malignant tumor metastasis and prolong the lifetime of tumor-bearing mice, and has an anti-tumor effect for a long time after training and immunization. Therefore, the American cockroach glucan Pa1-B1 disclosed by the invention can be used for preparing anti-tumor or anti-tumor metastasis medicines for enhancing the immune function and training immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to American cockroach glucan Pa1-B1 and a composition thereof and a preparation method and application thereof. Background Art

[0002] The American cockroach (Periplaneta americana) belongs to the class Insecta, order Blattaria, family Blattaria, and is one of the most resilient and ancient insect species. As a traditional Chinese medicine, the American cockroach has the effects of promoting blood circulation, promoting diuresis and reducing swelling, healing wounds and promoting tissue regeneration, and treating injuries and ulcers that have not healed for a long time.

[0003] The chemical substances in American cockroaches include proteins, nucleosides, peptides, sugars, fatty acids, etc. Among them, the chemical structure of American cockroach polysaccharides has not been reported so far, and the biological activity is still unclear. The chemical structure and biological efficacy of American cockroach polysaccharides need to be further studied.

[0004] Tumor is a major human disease that poses a serious threat to human life and health. There is still an urgent need to develop drugs to prevent and treat tumors clinically.

[0005] So far, there is no report on American cockroach glucan Pa1-B1 and its activity in the prior art. Summary of the invention

[0006] In view of the above problems existing in the prior art, the purpose of the present invention is to provide an American cockroach glucan Pa1-B1, a preparation method thereof, a pharmaceutical composition and application. The present invention extracts and purifies an American cockroach glucan Pa1-B1 with a novel structure from the American cockroach, and the American cockroach polysaccharide is reported for the first time. Pharmacological studies have found that the American cockroach glucan Pa1-B1 has significant immunoenhancing activity; further studies have confirmed that the American cockroach glucan Pa1-B1 can train immunity and inhibit tumor growth, significantly prolonging the survival of tumor-bearing mice.

[0007] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0008] An American cockroach glucan, wherein the American cockroach glucan is a glucan with a main chain connected by α (1→4) and glucose on the side chain connected to the main chain by α (1→6), denoted as Pa1-B1, and has a structure shown in the following general formula I:

[0009]

[0010] In Formula I, n represents the number of repeating units and is a natural number, 40000≦n≦47000.

[0011] The weight average molecular weight of the American cockroach glucan Pa1-B1 is about 36000 kDa to 42000 kDa.

[0012] Furthermore, the present invention also discloses a method for preparing the American cockroach polysaccharide, the method comprising:

[0013] The whole dried or fresh American cockroaches are crushed, defatted, and then subjected to enzymatic extraction to obtain a crude polysaccharide solution, which is then precipitated with alcohol to obtain American cockroach glucan.

[0014] The defatting includes extracting with ethanol to remove organic matter such as lipids contained therein; the enzymatic hydrolysis includes hydrolyzing the defatted sample with alkaline protease, centrifuging, precipitating the supernatant with alcohol, and collecting the precipitate.

[0015] Further, the component with a weight average molecular weight of 36000kDa to 42000kDa in the American cockroach glucan is refined to obtain American cockroach glucan Pa1-B1, and the refining method includes: one or more of alcohol precipitation, anion exchange chromatography, gel exclusion chromatography, dialysis and ultrafiltration.

[0016] The preparation method of the American cockroach glucan Pa1-B1, wherein in the degreasing step, the ethanol concentration is 95%, and the degreasing time is 0.5h to 3h;

[0017] The volume concentration of the alkaline protease is 1% to 5%, the enzymatic hydrolysis conditions are pH = 9, the enzymatic hydrolysis temperature is 50 to 65° C., and the enzymatic hydrolysis time is 2h to 24h;

[0018] The preparation method of the American cockroach glucan Pa1-B1, wherein the alcohol precipitation comprises: taking the supernatant for alcohol precipitation to make the alcohol concentration reach 35% to 45%, and collecting the precipitate.

[0019] The American cockroach glucan has the ability to significantly enhance the body's immune activity and has the activity of training immunity against tumors and / or against tumor metastasis.

[0020] A pharmaceutical composition comprises an effective amount of American cockroach glucan Pa1-B1 and a pharmaceutically acceptable carrier.

[0021] The American cockroach glucan Pa1-B1 is used in the preparation of immunomodulatory active drugs, immune anti-tumor, trained immune anti-tumor, anti-tumor metastasis drugs and / or in the preparation of foods that improve immune function.

[0022] The pharmaceutical composition is used in preparing immunomodulatory active drugs, immune anti-tumor drugs, trained immune anti-tumor drugs, and anti-tumor metastasis drugs.

[0023] The method for preparing the pharmaceutical composition comprises the following steps:

[0024] Step 1: Dry and crush the whole American cockroach and defatted it, then extract it by enzymatic method, obtain a crude polysaccharide solution after centrifugation, and then precipitate it with alcohol to obtain crude American cockroach glucan, wherein in the defatting step, the ethanol concentration is 95%, and the defatting time is 0.5h-3h; wherein the enzyme of the enzymatic method is preferably alkaline protease, and its volume concentration is 1%-5%, the enzymatic hydrolysis conditions are pH=9, the enzymatic hydrolysis temperature is 50°C-65°C, and the enzymatic hydrolysis time is 2h-24h;

[0025] Step 2: purifying the component with a weight average molecular weight of 36000 kDa to 42000 kDa in the American cockroach glucan to obtain the American cockroach glucan, wherein the purification step comprises: one or more of an alcohol precipitation method, anion exchange chromatography, and gel exclusion chromatography; the alcohol precipitation method comprises: taking the supernatant and performing alcohol precipitation to make the alcohol concentration reach 35% to 45%, and collecting the precipitate;

[0026] Step 3: Take the American cockroach glucan obtained in the above step and add a pharmaceutically acceptable carrier.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] The structural characteristics of the American cockroach polysaccharide Pa1-B1 of the present invention are that its main chain is a glucan connected by α (1→4) glycosidic bonds, the glucose on the side chain is connected to the main chain by α (1→6), and the weight average molecular weight is 36000kDa to 42000kDa. The American cockroach polysaccharide Pa1-B1 is reported for the first time, filling the gap in the prior art.

[0029] The inventor's research has confirmed that American cockroach polysaccharide Pa1-B1 can significantly promote the release of NO by RAW264.7 macrophages, promote the secretion of IL-6 and TNF-α by macrophages, and has the ability to stimulate macrophages to secrete cytokines. Therefore, the American cockroach glucan Pa1-B1 has good immune activity, can be used to prepare foods that improve immune function, and can also be used to prepare therapeutic drugs for immunodeficiency diseases. After giving mice an effective dose of American cockroach glucan Pa1-B1 in advance to train the body's immunity, it significantly inhibited the lung metastasis of tumor cells, prolonged the survival of tumor-bearing mice, and could play a long-term role in anti-tumor metastasis, confirming that American cockroach glucan Pa1-B1 has excellent training immune anti-tumor activity. Therefore, the present invention discloses for the first time the use of American cockroach glucan Pa1-B1 in the preparation of immune anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solution of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application.

[0031] Figure 1is the structural formula of American cockroach glucan Pa1-B1;

[0032] Figure 2 is the molecular weight distribution curve of American cockroach glucan Pa1-B1;

[0033] Figure 3 HPLC chart of standard monosaccharide mixture of American cockroach glucan Pa1-B1 and its PMP derivative;

[0034] Figure 4 This is the UV spectrum of American cockroach glucan Pa1-B1;

[0035] Figure 5 This is the infrared spectrum of American cockroach glucan Pa1-B1;

[0036] Figure 6 The American cockroach glucan Pa1-B1 1 H-NMR spectrum;

[0037] Figure 7 The American cockroach glucan Pa1-B1 13 C-NMR spectrum;

[0038] Figure 8 The American cockroach glucan Pa1-B1 1 H- 1 H COSY spectrum;

[0039] Fig. 9 The American cockroach glucan Pa1-B1 1 H- 1 H TOCSY spectrum;

[0040] Fig.10 The American cockroach glucan Pa1-B1 1 H- 13 C HSQC spectrum;

[0041] Fig.11 The American cockroach glucan Pa1-B1 1 H- 13 C HMBC spectrum;

[0042] Fig.12 The American cockroach glucan Pa1-B1 1 H- 13 C HSQC-TOCSY spectrum;

[0043] Fig.13 The American cockroach glucan Pa1-B1 1 H- 1 H ROESY spectrum;

[0044] Fig.14 The effect of American cockroach glucan Pa1-B1 on the activity of macrophage RAW264.7 (n=3, * indicates significant difference compared with the blank group, ** indicates p<0.01);

[0045] Fig.15 The effect of American cockroach glucan Pa1-B1 on the phagocytic ability of macrophage RAW264.7 (n=3, * indicates significant difference compared with the blank group, ** indicates p<0.01);

[0046] Fig.16 The effect of American cockroach glucan Pa1-B1 on NO release of macrophage RAW264.7 (n=3, * indicates significant difference compared with the blank group, **** indicates p<0.0001);

[0047] Fig.17 The effect of American cockroach glucan Pa1-B1 on IL-6 secretion by macrophage RAW264.7 (n=3, * indicates significant difference compared with the blank group, **** indicates p<0.0001);

[0048] Fig.18 The effect of American cockroach glucan Pa1-B1 on the secretion of TNF-α by macrophage RAW264.7 (n=3, * indicates significant difference compared with the blank group, **** indicates p<0.0001);

[0049] Fig.19 The American cockroach glucan Pa1-B1 inhibited the lung metastasis of melanoma cells (n=5);

[0050] Fig. 20 To evaluate the inhibitory activity of American cockroach glucan Pa1-B1 on melanoma cell lung metastasis by in vivo imaging (n=5);

[0051] Fig.21 HE staining images of lung tissues of tumor-bearing mice in the American cockroach glucan Pa1-B1 group (2 mg);

[0052] Fig. 22 The American cockroach glucan Pa1-B1 significantly prolonged the survival of tumor-bearing mice (n=10);

[0053] Fig.23 American cockroach glucan Pa1-B1 (2mg) exerts long-term trained immune anti-tumor effects. DETAILED DESCRIPTION

[0054] Below in conjunction with the accompanying drawings, the substantive content of the present invention is further illustrated with embodiments of the present invention, but the present invention is not limited thereto. In the detailed description below, many specific details are proposed to provide a comprehensive understanding of the application. But it is obvious to those skilled in the art that the application can be implemented when some details in these specific details are not needed. In addition, the features, structures or characteristics described hereinafter can be combined in one or more embodiments in any suitable manner.

[0055] In the examples, if the specific conditions are not specified, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer, and the instruments or reagents used, if the manufacturers are not specified, are all conventional products available from the market. The cockroach species used in the present invention is American cockroach, which is all from artificial breeding.

[0056] American cockroach, also known as cockroach, commonly known as "cockroach", has other aliases such as "stone ginger, slippery insect, oil-stealing woman", etc. American cockroach was first recorded as a medicine in "Shennong's Herbal Classic": "Feilian, salty and cold in taste, treats blood stasis, hard cold and heat, breaks up accumulation, throat and pharynx closure, internal cold and infertility", and is listed as a medium-grade product. "Compendium of Materia Medica Supplement" records: "No matter how many cockroaches are on the stove, smash them and apply them, and the carbuncle roots will come out by themselves." American cockroach is a dried whole insect used as medicine, which is salty and cold in nature. It enters the heart, liver, spleen, and kidney meridians, can strengthen the spleen and eliminate malnutrition, promote blood circulation and unblock meridians, promote diuresis and reduce swelling, and heal sores and promote tissue regeneration. In addition, American cockroach is a traditional medicine used by ethnic minorities in the southwest, mainly used to treat trauma and ulcers that have not healed for a long time.

[0057] The chemical components of American cockroaches include polypeptides, nucleosides, polyols, steroids and terpenes, etc., which have the effects of anti-fibrosis, tissue repair, anti-inflammatory and detumescent, myocardial protection, etc. However, there is no report on polysaccharides in American cockroaches, and its chemical structure has not been elucidated so far, and the biological activity of its polysaccharides is even less clear. Based on this, the inventors conducted a systematic chemical and pharmacological activity study on American cockroach polysaccharides, and reported for the first time a novel American cockroach glucan Pa1-B1. After in-depth research, it was surprisingly found that the glucan has significant immunomodulatory activity, and it was also found that it has a long-term anti-tumor pharmacological activity by training the body's immunity.

[0058] American cockroach glucan Pa1-B1

[0059] The first aspect of the present invention provides American cockroach glucan Pa1-B1 having a structure as shown in the following formula I:

[0060]

[0061] In Formula I, n represents the number of repeating units and is a natural number, 40000≦n≦47000.

[0062] The American cockroach glucan Pa1-B1 disclosed in the embodiment of the present invention has a monosaccharide composition of glucose, and the glucose in the main chain is linked by α (1→4) glycosidic bonds, and the side chain is linked by α (1→6) glycosidic bonds.

[0063] The present invention adopts an innovative method to extract and purify American cockroaches, and obtains American cockroach glucan with a novel structure, and the American cockroach glucan with the structure is publicly reported for the first time. Further activity studies have found that the American cockroach glucan Pa1-B1 significantly promotes the release of NO and the secretion of cytokines IL-6 and TNF-α by macrophages RAW264.7. This shows that the American cockroach glucan has a significant ability to regulate the body's immunity. Therefore, the present invention further discloses a new use of the American cockroach glucan in the preparation of drugs and / or foods for improving the body's immunity.

[0064] In some embodiments, the weight average molecular weight of the American cockroach glucan is 36000 kDa to 42000 kDa.

[0065] In some embodiments, the dispersion coefficient of the American cockroach glucan is 3.01.

[0066] In some embodiments, the purity of the American cockroach glucan is greater than 95%.

[0067] According to an embodiment of the present invention, the weight average molecular weight of the American cockroach glucan is about 36000 kDa to 42000 kDa, and the glucose has an α(1→4) glycosidic bond as the main chain and an α(1→6) glycosidic bond as the side chain connection.

[0068] Preparation method of American cockroach glucan

[0069] The second aspect of the present invention provides a method for preparing American cockroach glucan Pa1-B1, comprising the following steps S100 to S200:

[0070] S100: Extraction of crude polysaccharides from the American cockroach;

[0071] S200: Purification to obtain American cockroach glucan Pa1-B1.

[0072] According to an embodiment of the present invention, the American cockroach glucan with novel structure and significant immunomodulatory activity can be obtained by the above-mentioned preparation method, wherein the weight average molecular weight of the American cockroach glucan is about 36000kDa to 42000kDa, and the glucan is a glucan with α(1→4) glycosidic bond as the main chain and α(1→6) glycosidic bond as the side chain. Therefore, the preparation method provided in the second embodiment of the present application can be used to prepare the American cockroach glucan Pa1-B1 of the first embodiment of the present application.

[0073] In the embodiment of the present invention, the American cockroach body can be a fresh body, or a dried body, or a powder obtained by crushing the dried body.

[0074] In some embodiments, the American cockroach body is a powder obtained by crushing the dried American cockroach body.

[0075] In some embodiments, extracting crude polysaccharides from American cockroach bodies comprises: crushing the American cockroach bodies, extracting with warm water, hot water and / or enzymatic hydrolysis to obtain a solution of the crude polysaccharide.

[0076] Specifically, the extraction of crude polysaccharides from the American cockroach body includes: taking dry cockroach body powder, adding 95% ethanol at a material-liquid ratio of 1g:5mL, heating at a water bath temperature of 90°C for 3h, cooling to room temperature, filtering to obtain a 95% ethanol extract, extracting three times, combining the extracts, concentrating and freeze-drying to obtain an American cockroach defatted extract. Then, the extract is added with deionized water at a material-liquid ratio of 1g:5mL, extracting at a constant temperature of 65°C for 24h, extracting three times, centrifuging, and taking the supernatant.

[0077] In some embodiments, extracting the component with a weight average molecular weight of 36000 kDa to 42000 kDa from the crude polysaccharide comprises the following steps S210 to S220:

[0078] S210: extracting by enzymatic hydrolysis and precipitating by alcohol to obtain crude American cockroach glucan.

[0079] Specifically, the enzymolysis temperature is 50-65° C., the enzymolysis time is 2-24 hours, and 24 hours is optional; the volume concentration of alkaline protease is 1%-5%, and 3% is optional.

[0080] In the embodiment of the present invention, the alcohol precipitation comprises: boiling the solution after enzymatic hydrolysis to inactivate the enzyme, centrifuging, taking the supernatant for alcohol precipitation, and collecting the precipitate.

[0081] In the embodiment of the present invention, taking the supernatant and precipitating with alcohol comprises the following steps:

[0082] Add 95% ethanol to the clear solution after enzyme inactivation under stirring until the final concentration of alcohol is 35% to 45%, centrifuge to obtain 35% to 45% alcohol precipitate, add water to reconstitute and then freeze-dry;

[0083] Specifically, the final concentration of ethanol in step S211 may be selected as 40%. The final concentration of ethanol in step S211 may be selected as 40%.

[0084] The components with a weight average molecular weight of 36000 kDa to 42000 kDa in the crude American cockroach glucan were purified to obtain American cockroach glucan Pa1-B1.

[0085] In the embodiment of the present invention, the method for refining crude American cockroach glucan comprises one or more combinations of anion exchange column chromatography, gel exclusion chromatography, dialysis, and ultrafiltration.

[0086] Specifically, the crude American cockroach glucan can be re-dissolved in deionized water, and then the insoluble matter can be removed by centrifugation. The glucan can be purified by alcohol precipitation classification, gel exclusion chromatography, dialysis or ultrafiltration. The fraction or retentate or dialyzate containing the American cockroach glucan can be collected. If the fraction contains salt, it needs to be desalted, and then the fraction is directly freeze-dried in vacuum or concentrated under reduced pressure and then alcohol precipitated and dried under reduced pressure to obtain the purified American cockroach glucan.

[0087] It is understood by those skilled in the art that, for gel exclusion chromatography, it is based on the molecular weight of the glucan substance in the polysaccharide component of American cockroach glucan, and the gel material is reasonably selected, such as the glucan gel Sephadex series, the agarose gel Sepharose series, the polyacrylamide Bio-Gel P series and the gel filler formed by cross-linking each other, and then the column is loaded, the sample is loaded, and the saline or salt-free elution solution is used to elute and collect the flow fractions in sequence according to the actual performance of each filler. Each outflow peak is merged, and the effluent can be concentrated or does not need to be concentrated, and is loaded into a dialysis bag for dialysis or an ultrafiltration membrane for dialysis or ultrafiltration desalination, and the retentate after desalination is collected, and vacuum freeze drying or vacuum decompression drying is performed to obtain purified American cockroach glucan. Or when using methods such as dialysis or ultrafiltration to purify the American cockroach polysaccharide component, an ultrafiltration membrane with an appropriate molecular weight is selected to carry out ultrafiltration interception treatment. For example, the aqueous solution of the American cockroach polysaccharide component is first fully ultrafiltered through an ultrafiltration membrane with a molecular weight smaller than that of the American cockroach glucan Pa1-B1, the permeate is collected, and substances with small molecular weight are removed, and then fully dialyzed or ultrafiltered through a dialysis bag or ultrafiltration membrane with a molecular weight larger than that of the American cockroach glucan Pa1-B1, the retentate is collected, and after concentration, it is vacuum freeze-dried or vacuum-reduced dried to obtain refined American cockroach glucan Pa1-B1.

[0088] The American cockroach glucan provided in the embodiment of the present invention was subjected to structural analysis, and the results showed that the American cockroach glucan showed a chromatographic peak with good symmetry on the HPGPC gel chromatography and had high purity. The methylation analysis results showed that the American cockroach glucan mainly contained α(1→4) glycosidic bonds and α(1→6) glycosidic bonds. The one-dimensional and two-dimensional NMR spectrum data can clearly attribute the nuclear magnetic signals of the American cockroach glucan.

[0089] The American cockroach glucan Pa1-B1 provided in the embodiment of the present invention has Figure 1 The structural features shown in the general formula I are: it is a glucan with α(1→4) glycosidic bonds as the main chain and α(1→6) glycosidic bonds as the side chain. In the formula I, n represents the number of repeating units and is a natural number, 40000≦n≦47000.

[0090] Furthermore, the weight average molecular weight of the American cockroach glucan Pa1-B1 is about 41611 kDa, and the dispersion coefficient is 3.01.

[0091] Application of American cockroach glucan Pa1-B1 in functional foods and / or medicines

[0092] The third aspect of the present invention provides an application of American cockroach glucan in the preparation of food and / or medicine for enhancing immune activity and / or training immune anti-tumor.

[0093] According to the embodiment of the present invention, American cockroach glucan Pa1-B1 can activate macrophages, stimulate macrophages to release NO and immune-related active substances such as cytokines IL-6 and TNF-α, and has significant immunomodulatory activity. Therefore, the present invention discloses the use of American cockroach glucan Pa1-B1 in the preparation of food and / or medicine for regulating immunomodulatory activity.

[0094] According to the embodiment of the present invention, the American cockroach glucan Pa1-B1 was injected into the abdominal cavity of mice in advance, and melanoma cells were inoculated into the tail vein 7 days after training and immunization. Compared with the control group, the American cockroach glucan Pa1-B1 training and immunization can significantly inhibit the lung metastasis of tumor cells, significantly prolong the survival of tumor-bearing mice, and play a long-term anti-tumor metastasis effect, confirming that the American cockroach glucan Pa1-B1 has significant training immune anti-tumor activity. Therefore, the present invention further discloses the use of American cockroach glucan Pa1-B1 in the preparation of training immune anti-tumor drugs.

[0095] Example

[0096] The present invention is described below with reference to specific examples. It should be noted that the examples described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the instruments or reagents used are not specified, they are all conventional products that can be obtained commercially.

[0097] Example 1: Preparation of American cockroach glucan.

[0098] Extraction of Crude Polysaccharide from American Cockroach

[0099] 1) After the American cockroach is dried, the American cockroach is crushed to obtain American cockroach whole insect powder;

[0100] 2) Defatting: Weigh the dried American cockroach powder, add 95% ethanol at a solid-liquid ratio of 1:5 (kg:L), heat and extract at 90°C in a water bath for 3 hours, cool to room temperature, filter to obtain a residue, extract 3 times, filter to obtain a defatted residue sample, and evaporate the ethanol;

[0101] 3) Enzymatic extraction: Weigh 700 g of the defatted and dried residue sample, place it in a 5 L reactor, add 3.5 L of deionized water and 21 g of alkaline protease (3%), react at 65 ° C in the reactor for 24 h, monitor the pH value of the reaction solution during the extraction process to keep it constant at 9, centrifuge (4000 rpm × 15 min), collect the supernatant, and repeat the precipitation extraction 3 times;

[0102] 4) Alcohol precipitation: add 95% ethanol to the supernatant of step 3) under continuous stirring to a final concentration of 40%, let stand at 4°C for 12 h, and centrifuge (4000 rpm×15 min) to obtain a precipitate;

[0103] 5) Wash the precipitate with anhydrous ethanol for 3 times, evaporate the alcohol, and freeze-dry after re-dissolving in water to obtain 22.61 g of crude polysaccharide sample; Refined American cockroach glucan

[0104] 6) Anion exchange column chromatography: After activation and column loading of DEAE-52cellulose, elution was performed with deionized water for 3 column volumes, the pH of the eluate was adjusted to 7, and the crude American cockroach polysaccharide was obtained by freeze-drying.

[0105] 7) Gel column chromatography: Sephadex G100 gel was used. About 200 mg of crude polysaccharide of American cockroach was weighed, dissolved in 10 mL of deionized water, centrifuged at 4000 rpm for 15 min, and the supernatant was slowly and evenly injected into the top of the Sephadex G100 gel column after filtering with a 0.45 μm filter membrane. The flow rate was controlled to be 0.1 mL / min with 0.1 M NaCl solution, and 2 mL was collected in each tube. The polysaccharide content was detected by the phenol-sulfuric acid method with the absorbance value as the ordinate and the number of tubes as the abscissa. The elution curve of the polysaccharide component was drawn, and the eluate belonging to the same elution peak was combined, and desalted with a 500 Da dialysis bag. After dialysis, the sample was freeze-dried and weighed. After purification by Sephadex G100, the component was subjected to liquid phase analysis, which was a single symmetrical peak. No obvious color change was observed when using methylene blue to detect, and purified American cockroach glucan Pa1-B1 was obtained.

[0106] Example 2: Structural analysis of American cockroach glucan.

[0107] The structure of the American cockroach polysaccharide Pa1-B1 obtained in Example 1 was analyzed, specifically including:

[0108] (1) Molecular weight determination:

[0109] Chromatographic conditions: Agilent Technologies 1260 series high performance liquid chromatograph;

[0110] Detectors: Refractive Index Detector (RID) and Diode Array Detector (DAD);

[0111] Chromatographic conditions: OHpak SB-804HQ (8.0×300mm); injection volume: 30μL; column temperature: 35°C; mobile phase: 0.1M sodium chloride solution; flow rate: 0.5mL / min; detection time: 40min.

[0112] Determination process: Accurately weigh 5 mg of American cockroach glucan Pa1-B1 sample or dextran reference substance with known molecular weight, add mobile phase to prepare a 5 mg / mL solution, pass through a 0.2 μm microporous filter membrane, and analyze the filtrate in a high performance liquid chromatograph to record the chromatogram. The data is processed using GPC software, a standard curve is drawn, and the data is substituted into the equation to calculate the molecular weight.

[0113] Experimental results: The weight average molecular weight of Pa1-B1 was 41611 kDa and the polydispersity coefficient was 3.01 as determined by high performance gel permeation chromatography (HPGPC). Figure 2 shown.

[0114] (2) Analysis of monosaccharide composition: 0.5 mL of the prepared sample solution, monosaccharide standard solution, and monosaccharide standard mixed solution (deionized water as blank control) were placed in each COD test tube, and 0.5 mL of 4M trifluoroacetic acid TFA was added and vortexed to mix well. The mixture was then sealed and dried in an oven at 110°C for 4 h for acid hydrolysis. The hydrolyzate was removed and cooled to room temperature, and then evaporated to dryness in a rotary evaporator at 50°C. 1 mL of methanol was added to promote deacidification, and the mixture was repeated 2 to 3 times. Finally, 500 μL of deionized water was added to fully dissolve the evaporated sample, and 200 μL was removed for later use. To the 200 μL solution taken out of the above COD tube, add 200 μL of 0.6 M NaOH solution and 400 μL of 0.5 M PMP-methanol solution, vortex mix, and derivatize in a 70 ° C oven under sealed conditions for 1 h. After the reaction is completed, cool to room temperature, slowly add 0.6 M hydrochloric acid solution for neutralization, add chloroform for thorough extraction 3 times, filter the aqueous phase through a 0.22 μm filter membrane, and use the filtrate as the test solution for liquid chromatography analysis.

[0115] Chromatographic conditions:

[0116] Chromatography: Agilent Technologies 1260series high performance liquid chromatograph;

[0117] Chromatographic column: Hadesil C18-BIO (5 μm, 4.6 × 250 nm); column temperature: 25 °C;

[0118] Mobile phase: phosphate buffer (pH = 6.8)-acetonitrile (83:17), flow rate 1 mL / min; injection volume 20 μL;

[0119] Detection: DAD detector, detection wavelength: 250nm, detection time: 60min.

[0120] like Figure 3 The results showed that the American cockroach polysaccharide mainly contained glucose and was a glucan.

[0121] (3) Methylation analysis:

[0122] Pretreatment: Weigh 3 mg of Pa1-B1 into a 10 mL spiral tube. Add 2 mL of methanol and place in a vacuum oven. Repeat twice. Add 0.5 mL of dimethyl sulfoxide and ultrasonically treat for 20 to 30 minutes.

[0123] Preparation of sodium hydroxide DMSO solution: weigh 1200 mg NaOH and add 10 mL DMSO to dissolve it to obtain 120 mg / mL sodium hydroxide DMSO solution.

[0124] 500 μL of DMSO / NaOH solution was added to the sample and ultrasonicated for 30 min.

[0125] Methylation: Add 200 μL CH 3 After ultrasonic treatment for 10 min, 200 μL CH 3 I was treated with ultrasound for 10 min, and 400 μL CH 3 I. Ultrasonic treatment for 30 min. Add 1 ml of water to terminate the reaction, add 3 ml of chloroform to extract the sample, extract 3 times, combine the organic phase solutions, wash the chloroform phase with an equal volume of water 3 times, and dry the organic layer at 45 °C by rotary evaporation.

[0126] Acid hydrolysis of polysaccharides: Dissolve the dried sample in 1 mL of deionized water, then add 1 mL of 4 M TFA solution to fully dissolve the methylated product. Hydrolyze in an oven at 121 °C for 2 h. After the reaction, add 2 mL of methanol and dry by rotary evaporation at 50 °C. Repeat twice.

[0127] Monosaccharide reduction: Dissolve the dried sample in 1 mL of 2 M NH 4OH and add 1mL of freshly prepared 1M NaBD4. Ultrasonicate the sample for 1min and incubate at room temperature for 2.5h. Slowly drop 400μL of acetic acid to destroy the excess reducing agent. Dissolve in 1mL of 5% (v / v) acetic acid-methanol solution and spin dry twice. Dissolve in 1mL of methanol and spin dry, repeat twice. Dry the spin-dried sample in a vacuum drying oven.

[0128] Sugar alcohol acetylation: Add 1 mL of acetic anhydride and 1 mL of pyridine to the sample, vortex to dissolve, place at 100°C for 1.5 hours, add 1 mL of water to terminate the reaction, add 3 mL of chloroform to extract 3 times, extract the organic phase with an equal volume of water 3 times, spin dry the organic layer, add 2 mL of methanol and spin dry twice. Add 1 mL of chromatographic grade chloroform to dissolve, filter with a 0.22 μm filter membrane, and perform GC-MS analysis.

[0129] GC conditions: Chromatographic column: DB-5MS quartz capillary column (30m×0.25mm×0.25μm); column temperature program: starting temperature 80℃, hold for 11min, program temperature rise 10℃ / min to 195℃, hold for 1min; continue to increase at 2℃ / min to 210℃, hold for 2min; finally increase at 10℃ / min to 250℃, hold for 5min; column flow rate: 1.5mL / min; injection port temperature: 270℃; split mode: no split; carrier gas: high-purity helium, injection volume: 1μL.

[0130] MS conditions: ionization mode: EI; electron energy: 70ev; transfer line temperature: 290℃; ion source temperature: 230℃; quadrupole temperature: 150℃; mass range: 50-600; detection time: 60min.

[0131] Table 1 GC-MS signal attribution of methylated products of glucan Pa1-B1 from American cockroach

[0132]

[0133]

[0134] As shown in Table 1, the main ion peaks were assigned: the ion peak at 20.157 min was assigned to 2,3,4,6-tetra-O-methyl-1,5-diacetyl-D-glucitol, indicating the presence of Glcp-(1→ structural fragment in the sugar chain; the ion peak at 22.343 min was assigned to 2,3,6-tri-O-methyl-1,4,5-diacetyl-D-glucitol, indicating the presence of →4)-Glcp-(1→ structural fragment in the sugar chain; the ion peak at 23.350 min was assigned to 2,3,6-tri-O-methyl-1,4,5-diacetyl-D-glucitol, indicating the presence of →4)-Glcp-(1→ structural fragment in the sugar chain; The sub-peak was assigned to 2,3,4-tri-O-methyl-1,5,6-triacetyl-D-glucitol, indicating the presence of a →6)-Glcp-(1→ structural fragment in the sugar chain; the ion peak at 24.357 min was assigned to 2,3-di-O-methyl-1,4,5,6-tetraacetyl-D-glucitol, indicating the presence of a →4,6)-Glcp-(1→ structural fragment in the sugar chain. It was inferred that the American cockroach glucan contained α(1→4) glycosidic bonds and α(1→6) glycosidic bonds.

[0135] (4) UV spectral analysis: Take 1 mg of American cockroach polysaccharide Pa1-B1 and dissolve it in water to 1 mg / mL. Detect the ultraviolet absorption spectrum of the sample in the range of 190-800 nm.

[0136] UV spectrum Figure 4 As shown, dextran Pa1-B1 has less impurities and higher purity.

[0137] (5) Infrared spectroscopy analysis: 2 mg of American cockroach glucan Pa1-B1 was taken and dried in vacuum at 40°C for 24 h. Then, it was pressed into tablets with potassium bromide. The sample was analyzed by infrared spectrometer at 4000-400 cm -1 Scan and record the spectrum.

[0138] The infrared spectrum of Pa1-B1 is as follows Figure 5 As shown, its characteristic absorption peaks are: 3417cm -1 It is caused by the stretching vibration of the hydroxyl group of the sugar ring alcohol; 2929cm -1 It is the absorption vibration peak of sugar ring methylene CH; 1635cm -1 is the stretching vibration peak of aldehyde group; 1417~1200cm -1 It is the CH angle vibration peak; 1200~1000cm -1 The COC stretching vibrations on the sugar ring; 927 and 848 cm -1 It is the characteristic peak of CH vibration on the pyran ring; 700~500cm -1 The multiple absorption bands are the stretching vibrations of the sugar ring.

[0139] (6) Nuclear magnetic resonance (NMR) spectroscopy analysis: About 10 mg of the American cockroach glucan Pa1-B1 sample was taken and subjected to three D 2 O exchange, D2 O was dissolved to 20 mg / mL and detected by 800 MHz NMR 1 H-NMR / 13 C-NMR spectra and 1 H- 1 H COSY, 1 H- 1 H TCOSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 13 CHSQC-TCOSY, 1 H- 1 H ROESY spectrum.

[0140] The 1D / 2D NMR results of the American cockroach polysaccharide Pa1-B1 are as follows Figure 6 to Figure 13 As shown, the chemical shift values ​​of each C and H of each residue are assigned according to the NMR spectrum. 1 H. 13 The C signal attribution is shown in Table 2.

[0141] Table 2 American cockroach glucan Pa1-B1 1 H and 13 C NMR signal assignment

[0142]

[0143] Depend on 1 H- 13 C HSQC and 1 H- 13 The C HMBC spectrum clearly shows the correlation signals between CH on the sugar ring. 1 H- 1 H ROESY, 1 H- 13 C HSQC and 1 H- 13 C HMBC can determine that the glycosidic bond is α(1→4) and there is also α(1→6). Combined with the methylation results, according to 1 H / 13 C-NMR and two-dimensional NMR spectral data confirmed that Pa1-B1 is a glucan with α(1→4) glycosidic bonds as the main chain and α(1→6) glycosidic bonds as the glucose side chains.

[0144] Example 3: Experiment on enhancing immune activity of American cockroach glucan Pa1-B1.

[0145] The American cockroach glucan Pa1-B1 prepared in the above example was used for activity experiment, and the specific steps and results are as follows:

[0146] (1) Effect of American cockroach glucan Pa1-B1 on RAW264.7 cell viability:

[0147] RAW264.7 cells in the logarithmic growth phase were taken and made into a cell density of 8×10 4 cells / mL of cell suspension was inoculated in a 96-well cell culture plate, 100 μL per well; after culturing for 24 hours, American cockroach glucan Pa1-B1 was added to make the final concentrations of 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively. After culturing in an incubator for 24 hours, 10 μL CCK8 reagent was added to each well, and the plates were returned to the incubator for incubation for 1 to 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the effects of American cockroach glucan Pa1-B1 at different concentrations on the viability of RAW264.7 cells were calculated.

[0148] like Fig.14 It can be seen that American cockroach glucan Pa1-B1 has no cytotoxic effect on RAW264.7 cells in the concentration range of 100 μg / mL to 800 μg / mL.

[0149] (2) Effect of American cockroach glucan Pa1-B1 on the phagocytic ability of RAW264.7 cells:

[0150] RAW264.7 cells in the logarithmic growth phase were taken and made into a cell density of 1×10 5 cells / mL of cell suspension, inoculated in a 96-well cell culture plate, 100 μL per well; after culturing for 24 hours, add American cockroach glucan Pa1-B1 to make its final concentration 2 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL, respectively; after culturing in an incubator for 24 hours, remove the cell culture medium, add 150 μL of 0.075% neutral red solution to each well, continue to culture for 1 hour and then discard the neutral red solution, add 200 μL PBS to each well, wash the cells, repeat the operation 3 times, add 100 μL (ethanol: glacial acetic acid = 1:1) solution, let stand at room temperature for 30 minutes to 1 hour, and use an enzyme marker to measure the absorbance at 540 nm.

[0151] The results of the test are as follows Fig.15 As shown, at concentrations of 50 μg / mL and 100 μg / mL, American cockroach glucan Pa1-B1 can enhance the phagocytic ability of RAW264.7 cells.

[0152] (3) Effect of American cockroach glucan Pa1-B1 on NO release from RAW264.7 cells:

[0153] RAW264.7 cells in the logarithmic growth phase were taken and made into a cell density of 1×10 6 Cell suspensions of 100 μL / well were inoculated in 24-well cell culture plates, 100 μL per well, and medium was added to 600 μL. Polysaccharide samples Pa1-B1 of different concentrations were added to the experimental group, LPS was used as the positive control group, and a blank group was set up. After culturing for 24 hours under the original culture conditions, the cell supernatant was collected as the samples of the experimental group and the control group. Polysaccharide samples of different concentrations and LPS control group were added to 96-well plates at 50 μL / well, and 50 μL of Griess I and Griess II were added successively at room temperature. After 10 minutes of light protection, the plate was shaken and the absorbance was measured at 540 nm using an enzyme reader.

[0154] The results of the test are as follows Fig.16 As shown, polysaccharide Pa1-B1 has a promoting effect on the content of NO secreted by macrophages when the concentration is 12.5-200 μg / mL.

[0155] (4) Effect of American cockroach glucan Pa1-B1 on the secretion of IL-6 and TNF-α by macrophages:

[0156] Polysaccharide samples Pa1-B1 with different concentrations were taken, LPS was used as the positive control group, and a blank group was set up. After 24 hours of cell culture, the cell supernatant was collected as the experimental group samples and the control group samples.

[0157] The IL-6 and TNF-α content detection operation is as follows: add 100 μL of standard dilution and samples of different concentrations, as well as 100 μL of positive control LPS to the ELISA plate, add 50 μL of detection antibody diluted 1 / 100 to each well, seal the plate, and incubate at room temperature at 300 rpm for 2 h. After the incubation, discard the liquid and add 300 μL of washing solution 6 times. After washing, dry on absorbent paper, add 100 μL of horseradish peroxidase-labeled streptavidin diluted 1 / 100 to each well, seal the plate, incubate at room temperature at 300 rpm for 45 min, wash according to the previous step, add 100 μL of chromogenic substrate TMB to each well, protect from light, incubate at room temperature for 30 min, then add 100 μL of stop solution to terminate the reaction. Use an ELISA reader to detect OD450 in a short time.

[0158] The effects of American cockroach glucan Pa1-B1 on the secretion of cytokines IL-6 and TNF-α by macrophage RAW264.7 are shown in Fig.17 and Fig.18 The results showed that the American cockroach glucan Pa1-B1 at a concentration of 2-100 μg / mL could significantly promote the secretion of cytokines IL-6 and TNF-α by macrophages.

[0159] The research results of the present invention show that American cockroach glucan Pa1-B1 promotes the release of NO by macrophages and promotes the secretion of cytokines in a dose-dependent manner. Therefore, American cockroach glucan Pa1-B1 can effectively activate the immune response of macrophages and has the physiological function of increasing immune activity.

[0160] Example 4: Experiment on the anti-tumor metastasis activity of American cockroach glucan Pa1-B1 trained immune system.

[0161] The American cockroach glucan Pa1-B1 prepared in the above example was used to conduct an in vivo immune anti-tumor activity experiment. The animal experiment protocol was approved by the Ethics Committee of the Kunming Institute of Botany, Chinese Academy of Sciences, and was strictly implemented in accordance with the relevant national regulations on animal experiments. The specific steps and results are as follows:

[0162] (1) Experimental procedure: C57BL / 6J mice were intraperitoneally injected with 200 μL PBS or American cockroach glucan Pa1-B1 (1 mg or 2 mg / 200 μL PBS) once and fed with normal feed and deionized water for 7 days.

[0163] C57BL / 6J mouse homologous melanoma cells to be inoculated were cultured in 10 cm culture dishes, namely B16-F10 and B16-F10 / luc with luciferase gene. Melanoma cells in logarithmic growth phase were digested with trypsin for 1-2 min and then terminated with complete medium. After centrifugation (1000 rpm × 3 min), the supernatant was discarded and the cells were resuspended with 1× PBS and pipetted until the cells were single cell suspension. The cells were counted using a cell counter and the cell concentration was adjusted to 3×10 5 / mL~5×10 5 Pieces / mL.

[0164] Seven days after intraperitoneal administration, 100 μL of tumor cells of adjusted concentration were inoculated through the tail vein of mice. 14 to 18 days after inoculation of tumor cells, lung tissues were removed for photographic recording or small animal live imaging was performed after intraperitoneal injection of luciferase in mice. Image J was used to count the gray value of tumor metastasis lesions to represent the degree of metastasis.

[0165] The lung tissues were embedded in paraffin and then sectioned. The tissue sections were stained with HE to evaluate the immune anti-tumor activity of American cockroach glucan Pa1-B1.

[0166] Ten tumor-bearing mice in each of the three treatment groups, PBS, American cockroach glucan Pa1-B1 (1 mg) and Pa1-B1 (2 mg), were used for survival statistics and analysis to evaluate the effect of American cockroach glucan Pa1-B1 on the survival of tumor-bearing mice.

[0167] (2) Experimental results: When the survival statistics of each experimental group were calculated, 7 mice in the Pa1-B1 (2 mg) group survived on the 50th day after tumor inoculation. The lung tissues of two mice were taken for observation, showing no tumor lesions in the lungs, and they were considered tumor-free mice. On the 60th day after the administration, 5 mice in the Pa1-B1 (2 mg) group were inoculated again through the tail vein with 3.5×10 5 melanoma cells, and lung tissue was obtained for observation on the 18th day after inoculation to evaluate whether Pa1-B1 can induce long-term trained immune anti-tumor effects.

[0168] Depend on Fig.19 and Fig. 20 As shown, after 7 days of training and immunization with American cockroach glucan Pa1-B1, melanoma cells were inoculated through the tail vein on the 18th day, the mouse lung tissues were taken and fluorescent small animal live imaging was used to count the melanoma metastases in the mouse lungs. The experimental results showed that a single dose of 1 mg of American cockroach glucan has a tendency to inhibit the lung metastasis of melanoma, and a single dose of 2 mg can significantly inhibit the lung metastasis of melanoma cells.

[0169] like Fig.21 As shown, HE staining of lung tissue showed that a single dose of 2 mg of American cockroach glucan Pa1-B1 significantly inhibited the lung metastasis of tumor cells, indicating that American cockroach glucan Pa1-B1 has significant training immune anti-tumor activity.

[0170] like Fig. 22 As shown, compared with the control group, American cockroach glucan Pa1-B1 at a dose of 1 mg and 2 mg could significantly prolong the survival of tumor-bearing mice, among which 7 mice in the 2 mg administration group were still alive on the 50th day after tumor cell inoculation.

[0171] like Fig.23 As shown in the figure, after autopsy, no melanoma metastasis was found in the lungs of 2 mice, and the 7 surviving mice can be regarded as tumor-free mice. Five of the mice were inoculated with melanoma cells again, and lung tissue was taken for observation 18 days later. Among the 5 mice, 4 had significantly fewer melanoma metastasis in the lungs than the control group.

[0172] Furthermore, the bone marrow cells of mice trained with American cockroach glucan Pa1-B1 and treated with PBS control group for 7 days were transplanted into untrained mice (recipients). After 14 days, melanoma cells were inoculated into the tail vein of the recipient mice. After 18 days, the lung tissue was dissected and it was found that the recipient mice transplanted with bone marrow cells of mice trained with American cockroach glucan Pa1-B1 had significantly fewer lung tumor metastases than the recipient mice transplanted with bone marrow cells of mice treated with PBS.

[0173] The above results indicate that the American cockroach glucan Pa1-B1 has significant training immune anti-tumor activity. Therefore, the American cockroach glucan Pa1-B1 has application value in preparing immune anti-tumor drugs and / or auxiliary anti-tumor drugs.

[0174] It should be noted that in this embodiment, the inhibition of lung metastasis of melanoma cells B16-F10 by American cockroach glucan Pa1-B1 is used as an example to demonstrate the trained immune anti-tumor activity of Pa1-B1, and it cannot be understood as a limitation on the immune anti-tumor activity of American cockroach glucan Pa1-B1 in this embodiment. The immune anti-tumor activity of American cockroach glucan Pa1-B1 in this embodiment includes but is not limited to the inhibitory activity on the progression and metastasis of various different types of tumor cells such as melanoma cells in vivo, and its extension of the survival of tumor-bearing mice inoculated with different types of tumor cells. In this embodiment, the trained immune anti-tumor activity of American cockroach glucan Pa1-B1 is fully studied, and the administration treatment is carried out before tumor inoculation, and it cannot be understood as a limitation on the ability of American cockroach glucan Pa1-B1 to play a trained immune anti-tumor role after tumor inoculation.

[0175] Example 5: American cockroach glucan Pa1-B1 capsules.

[0176] Materials: American cockroach glucan Pa1-B1 obtained in the same manner as in Example 1, and food or pharmaceutical grade starch.

[0177] prescription:

[0178] Name of raw materials Dosage American cockroach glucan Pa1-B1 500g starch 300g Made in total 10000 tablets

[0179] Preparation process: Weigh the prescribed amount of American cockroach glucan Pa1-B1 and starch, stir to mix thoroughly, add appropriate amount of talcum powder, wet granulate with ethanol, sieve and dry, and then put into No. 2 capsule shells, each capsule body is filled with 50 mg of American cockroach glucan Pa1-B1, and American cockroach glucan Pa1-B1 capsules are prepared.

[0180] Example 6: American cockroach glucan Pa1-B1 oral solution.

[0181] Materials: American cockroach glucan Pa1-B1 obtained by the same method as in Example 1; food or pharmaceutical grade flavoring agent is caramel essence.

[0182] prescription:

[0183] Name of raw materials Dosage American cockroach glucan Pa1-B1 100g sucrose 0.6g Caramel flavor 0.1g Purified water 2000mL Made in total 500 pieces

[0184] Preparation process: Weigh the prescribed amount of American cockroach glucan Pa1-B1, sucrose and caramel essence, add purified water to completely dissolve, filter through a 0.2 μm microporous filter membrane, and fill the filtrate into 4 mL per bottle using an oral liquid filling machine. Sterilize after sealing to obtain the product.

[0185] Example 7: American cockroach glucan Pa1-B1 freeze-dried powder injection.

[0186] Materials: American cockroach glucan Pa1-B1 obtained in the same manner as in Example 1, water for injection.

[0187] prescription:

[0188] Name of raw materials Dosage American cockroach glucan Pa1-B1 20g Water for injection 1000mL Made in total 1000 pieces

[0189] Weigh the prescribed amount of American cockroach glucan Pa1-B1, add injection water, stir to dissolve completely, sterilize by intermittent hot pressing. After the content is qualified, filter with a 0.22μm microporous filter membrane; pack into controlled vials, 0.5mL per bottle, half-stopper, place in a freeze drying box, freeze-dry according to the set freeze-drying curve, stopper, unbox, roll cap, visually inspect and package to obtain the finished product.

[0190] Freeze-drying process: put the sample into the box, lower the partition temperature to -50℃, keep it for 5 hours; lower the cold trap to -50℃, and start to evacuate to 200μbar. Start sublimation: uniformly increase the temperature to -15℃ in 2 hours, keep it for 3 hours; uniformly increase the temperature to -5℃ in 3 hours, keep it for 6 hours, and maintain the vacuum at 100-250μbar; then dry: increase the temperature to 5℃ in 2 hours, keep it for 2 hours, and maintain the vacuum at 150-200μbar; increase the temperature to 15℃ in 1 hour, keep it for 2 hours, and maintain the vacuum at 80-100μbar; increase the temperature to 40℃ in 0.5 hours, keep it for 4 hours, and evacuate to the lowest vacuum.

Claims

1. American cockroach glucan Pa1-B1, characterized in that The American cockroach glucan is a glucan whose main chain is α(1→4) connected, and the glucose on the side chain is connected to the main chain by α(1→6), and has a structure shown in the following general formula I: In Formula I, n represents the number of repeating units and is a natural number, 40000≦n≦47000.

2. The American cockroach glucan Pa1-B1 according to claim 1, characterized in that The weight average molecular weight of the American cockroach glucan is 36000 kDa to 42000 kDa.

3. The method for preparing the American cockroach glucan Pa1-B1 according to claim 1, characterized in that: The method comprises the following steps: Step 1: Dry and crush the whole American cockroach, defat it, extract it by enzyme method, obtain a crude polysaccharide solution after centrifugation, and then precipitate it with alcohol to obtain crude American cockroach glucan; Step 2: Refining the component with a weight average molecular weight of 36000 kDa to 42000 kDa in the American cockroach glucan to obtain American cockroach glucan, The refining step comprises: one or more of alcohol precipitation, anion exchange chromatography, and gel exclusion chromatography.

4. The method for preparing the American cockroach glucan Pa1-B1 according to claim 3, characterized in that: In the degreasing step, the ethanol concentration is 95%, and the degreasing time is 0.5h to 3h; The enzyme of the enzymatic method is preferably alkaline protease, with a volume concentration of 1% to 5%, enzymatic hydrolysis conditions of pH = 9, enzymatic hydrolysis temperature of 50°C to 65°C, and enzymatic hydrolysis time of 2h to 24h; The alcohol precipitation method comprises the following steps: taking the supernatant and subjecting it to alcohol precipitation, so that the alcohol concentration reaches 35% to 45%, and collecting the precipitate.

5. The American cockroach glucan Pa1-B1 according to claim 1, characterized in that The American cockroach glucan has the ability to significantly enhance the body's immune activity and has the activity of training immunity against tumors and / or against tumor metastasis.

6. A pharmaceutical composition comprising an effective amount of the American cockroach glucan Pa1-B according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the American cockroach glucan Pa1-B according to claim 1 or 2 in the preparation of food for improving immune function.

8. Use of the American cockroach glucan Pa1-B according to claim 1 or 2 in the preparation of drugs for enhancing immune function, training immune anti-tumor, immune anti-tumor or anti-tumor metastasis.

9. Use of the pharmaceutical composition according to claim 6 in the preparation of drugs for enhancing immune function, training immune anti-tumor, immune anti-tumor or anti-tumor metastasis.

10. The method for preparing the pharmaceutical composition according to claim 6, characterized in that: The method comprises the following steps: Step 1: Dry and crush the whole American cockroach and defatted it, then extract it by enzymatic method, obtain a crude polysaccharide solution after centrifugation, and then precipitate it with alcohol to obtain crude American cockroach glucan, wherein in the defatting step, the ethanol concentration is 95%, and the defatting time is 0.5h-3h; wherein the enzyme of the enzymatic method is preferably alkaline protease, and its volume concentration is 1%-5%, the enzymatic hydrolysis conditions are pH=9, the enzymatic hydrolysis temperature is 50°C-65°C, and the enzymatic hydrolysis time is 2h-24h; Step 2: purifying the component with a weight average molecular weight of 36000 kDa to 42000 kDa in the American cockroach glucan to obtain the American cockroach glucan, wherein the purification step comprises: one or more of an alcohol precipitation method, anion exchange chromatography, and gel exclusion chromatography; the alcohol precipitation method comprises: taking the supernatant and performing alcohol precipitation to make the alcohol concentration reach 35% to 45%, and collecting the precipitate; Step 3: Take the American cockroach glucan obtained in the above step and add a pharmaceutically acceptable carrier.

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