Periplaneta americana glucan Pa1-B1, process for its preparation and uses thereof

Abstract

The application provides an American cockroach glucan Pa1-B1 and a preparation method and application thereof, and belongs to the technical field of biological medicines. The American cockroach glucan Pa1-B1 is a glucan with an alpha (1→4) linked main chain, and glucose on the side chain is alpha (1→6) connected to the main chain, and the weight average molecular weight is 36000 kDa to 42000 kDa. The American cockroach glucan Pa1-B1 is obtained by crushing whole American cockroaches, defatting with 95% ethanol, extracting under alkaline conditions by enzymatic hydrolysis, and then alcohol precipitation, ion exchange column chromatography and gel column chromatography separation and purification. The American cockroach glucan Pa1-B1 is a novel polysaccharide first discovered in American cockroaches, has the activity of enhancing immune regulation, and can effectively train the body immunity, significantly inhibit malignant tumor metastasis, prolong the survival period of tumor-bearing mice, and has long-term anti-tumor effect after training immunity. Therefore, the American cockroach glucan Pa1-B1 can be used for preparing an immune function enhancing, training immunity anti-tumor or anti-tumor metastasis drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a Periplaneta americana glucan Pa1-B1, a composition thereof, a preparation method and application thereof. BACKGROUND

[0002] Periplaneta americana belongs to the class of insects, order Blattaria, and family Blattidae, and is one of the most ancient and strongest insect species. As a traditional Chinese medicine, Periplaneta americana has the effects of activating blood and unblocking collaterals, promoting water and reducing swelling, and astringing and healing, and is used for treating trauma and chronic ulcer.

[0003] Chemical substances in Periplaneta americana include protein, nucleoside, peptide, sugar, fatty acid and other components. The chemical structure of Periplaneta americana polysaccharide has not been reported so far, and the biological activity is still unclear. The chemical structure and biological efficacy of Periplaneta americana polysaccharide need to be further studied.

[0004] Tumor is a major disease of human beings, which seriously threatens human life and health. There is still an urgent need for developing drugs for preventing and treating tumor diseases in clinic.

[0005] So far, there is no report on Periplaneta americana glucan Pa1-B1 and its activity in the prior art. SUMMARY

[0006] In view of the above problems in the prior art, the present application provides a Periplaneta americana glucan Pa1-B1, a preparation method, a pharmaceutical composition and application thereof. A novel Periplaneta americana glucan Pa1-B1 is extracted and purified from Periplaneta americana, which is reported for the first time. Pharmacological studies show that the Periplaneta americana glucan Pa1-B1 has significant immune-enhancing activity. Further studies have confirmed that the Periplaneta americana glucan Pa1-B1 can train immunity to inhibit tumor growth and significantly prolong the survival of tumor-bearing mice.

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

[0008] A Periplaneta americana glucan, which is a glucan with an alpha (1→4) linkage as a main chain and an alpha (1→6) linkage of glucose on the side chain to the main chain, and is denoted as Pa1-B1, and has the following general formula I:

[0009]

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

[0011] The weight average molecular weight of the Periplaneta americana glucan Pa1-B1 is about 36000kDa-42000kDa.

[0012] Further, the application also discloses a preparation method of the Periplaneta americana polysaccharide, and the method comprises the following steps:

[0013] The Periplaneta americana dry whole worm or fresh worm is crushed, defatted, and then enzymatically extracted to obtain a crude polysaccharide solution, and then alcohol precipitation is performed to obtain the Periplaneta americana glucan.

[0014] The defatting comprises extracting with ethanol to remove organic matters such as lipids contained therein; and the enzymatic extraction is to enzymatically extract the sample after defatting with alkaline protease, and then the supernatant is alcohol precipitated after centrifugation, and the precipitate is collected.

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

[0016] The preparation method of the Periplaneta americana glucan Pa1-B1, wherein in the defatting step, the concentration of ethanol is 95%, and the defatting time is 0.5h-3h.

[0017] The volume concentration of the alkaline protease is 1%-5%, the enzymatic extraction condition is pH=9, the enzymatic extraction temperature is 50-65 DEG C, and the enzymatic extraction time is 2h-24h.

[0018] The preparation method of the Periplaneta americana glucan Pa1-B1, wherein the alcohol precipitation comprises the following steps: alcohol precipitation is performed on the supernatant, the alcohol concentration is made to reach 35%-45%, and the precipitate is collected.

[0019] The Periplaneta americana glucan has the activity of significantly enhancing the immune activity of a body, training immune anti-tumor and / or anti-tumor metastasis.

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

[0021] The Periplaneta americana glucan Pa1-B1 is applied to preparation of an immunoregulatory active medicine, an immune anti-tumor medicine, a training immune anti-tumor medicine, an anti-tumor metastasis medicine and / or a food for improving immune function.

[0022] The pharmaceutical composition is applied to preparation of an immunoregulatory active medicine, an immune anti-tumor medicine, a training immune anti-tumor medicine and an anti-tumor metastasis medicine.

[0023] The preparation method of the pharmaceutical composition comprises the following steps:

[0024] Step one: after the whole body of Periplaneta americana is dried, crushed, defatted, the enzyme method is used for extraction, and the solution of the crude polysaccharide is obtained after centrifugation, and then alcohol precipitation is carried out to obtain the crude Periplaneta americana glucan, wherein the ethanol concentration in the defatting step is 95%, and the defatting time is 0.5h-3h; wherein the enzyme of the enzyme method is preferably alkaline protease, and the volume concentration is 1%-5%, the enzyme hydrolysis condition is pH=9, the enzyme hydrolysis temperature is 50-65 DEG C, and the enzyme hydrolysis time is 2h-24h;

[0025] Step two: the component with a weight average molecular weight of 36000kDa-42000kDa in the Periplaneta americana glucan is refined to obtain the Periplaneta americana glucan, and the refining step comprises one or more of alcohol precipitation method, anion exchange chromatography method and gel exclusion chromatography method; the alcohol precipitation method is that the supernatant is alcohol precipitated to make the alcohol concentration reach 35%-45%, and the precipitate is collected;

[0026] Step three: the Periplaneta americana glucan obtained in the above step is added with a pharmaceutically acceptable carrier.

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

[0028] The structural characteristics of the Periplaneta americana polysaccharide Pa1-B1 are that the main chain is an α(1→4) glycosidic bond connected glucan, the glucose on the side chain is connected to the main chain by α(1→6), and the weight average molecular weight is 36000kDa-42000kDa. The Periplaneta americana polysaccharide Pa1-B1 is reported for the first time, which fills the gap in the prior art.

[0029] The research of the present application proves that the Periplaneta americana 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 the secretion of cytokines by macrophages. Therefore, the Periplaneta americana glucan Pa1-B1 has good immune activity, can be used for preparing food for improving immune function, and can also be used for preparing therapeutic drugs for immunodeficiency diseases. After giving the mouse an effective dose of Periplaneta americana glucan Pa1-B1 to train the immune system, the tumor cell lung metastasis is significantly inhibited, the survival period of tumor-bearing mice is prolonged, and the anti-tumor metastasis effect is long-acting, which proves that the Periplaneta americana glucan Pa1-B1 has excellent training immune anti-tumor activity. Therefore, the present application discloses the use of Periplaneta americana glucan Pa1-B1 in the preparation of immune anti-tumor drugs for the first time. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments of the present application will be briefly introduced as follows.

[0031] Figure 1The structural formula of the American cockroach dextran Pa1-B1;

[0032] Figure 2 The molecular weight distribution curve of Pa1-B1, a dextran from the American cockroach;

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

[0034] Figure 4 The ultraviolet spectrum of Pa1-B1 glucan from the American cockroach;

[0035] Figure 5 Infrared spectrum of Pa1-B1 glucan from the American cockroach;

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

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

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

[0039] Figure 9 For American cockroach glucan Pa1-B1 1 H- 1 H TOCSY spectrum;

[0040] Figure 10 For American cockroach glucan Pa1-B1 1 H- 13 C HSQC spectrum;

[0041] Figure 11 For American cockroach glucan Pa1-B1 1 H- 13 C HMBC spectrum;

[0042] Figure 12 For American cockroach glucan Pa1-B1 1 H- 13 C HSQC-TOCSY spectrum;

[0043] Figure 13 For American cockroach glucan Pa1-B1 1 H- 1 H ROESY spectrum;

[0044] Figure 14 Effect of Periplaneta americana glucan Pa1-B1 on the viability of macrophage RAW264.7 (n=3, * means significant compared with the blank group, ** represents p<0.01);

[0045] Figure 15 Effect of Periplaneta americana glucan Pa1-B1 on the phagocytic ability of macrophage RAW264.7 (n=3, * means significant compared with the blank group, ** represents p<0.01);

[0046] Figure 16 Effect of Periplaneta americana glucan Pa1-B1 on the NO release of macrophage RAW264.7 (n=3, * means significant compared with the blank group, **** represents p<0.0001);

[0047] Figure 17 Effect of Periplaneta americana glucan Pa1-B1 on the secretion of IL-6 of macrophage RAW264.7 (n=3, * means significant compared with the blank group, **** represents p<0.0001);

[0048] Figure 18 Effect of Periplaneta americana glucan Pa1-B1 on the secretion of TNF-α of macrophage RAW264.7 (n=3, * means significant compared with the blank group, **** represents p<0.0001);

[0049] Figure 19 Periplaneta americana glucan Pa1-B1 inhibits lung metastasis of melanoma cells (n=5);

[0050] Figure 20 In vivo imaging evaluation of Periplaneta americana glucan Pa1-B1 inhibits lung metastasis of melanoma cells (n=5);

[0051] Figure 21 Periplaneta americana glucan Pa1-B1 group (2mg) lung tissue HE staining picture of tumor-bearing mice;

[0052] Figure 22 Periplaneta americana glucan Pa1-B1 significantly prolongs the survival of tumor-bearing mice (n=10);

[0053] Figure 23 Periplaneta americana glucan Pa1-B1 (2mg) plays a long-term training immune anti-tumor role. DETAILED DESCRIPTION

[0054] The present application will be further described in conjunction with the embodiments of the present application with reference to the accompanying drawings, but the present application is not limited by the embodiments. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without some of these specific details. In other instances, well-known features, structures or characteristics have not been described in detail in order to avoid obscuring the application. The features, structures or characteristics described hereinbelow can be combined in any suitable manner in one or more embodiments.

[0055] Unless otherwise specified, the conditions in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The instruments or reagents used are not specified by the manufacturers, and are all conventional products available on the market. The cockroach species used in the present application is Periplaneta americana, and all are from artificial breeding.

[0056] Periplaneta americana, also known as cockroach, is commonly known as "cockroach", and has other names such as "stone ginger", "slippery worm", "oil thief" and the like. Periplaneta americana was first recorded in "Shennong's Herbal Classic": "Periplaneta americana, bitter and cold, treats blood stasis, cold and heat, broken clusters, throat and pharynx, and cold and no children", and is listed as a medium. "Herbal Classic Supplement" records: "Cockroaches on the stove are not limited to a certain amount, and are crushed and applied, and the roots of the boils come out". Periplaneta americana is a dried whole worm for medicine, which is salty and cold, and enters the heart, liver, spleen, and kidney, and can invigorate the spleen, relieve malnutrition, promote blood circulation, remove water and relieve swelling, and astringe ulcers and promote tissue repair. In addition, Periplaneta americana is a traditional medicine of southwestern ethnic minorities, mainly used to treat external injuries and chronic ulcer wounds.

[0057] The chemical components of Periplaneta americana include polypeptides, nucleosides, polyols, steroids and terpenes, etc., and have the effects of anti-fibrosis, tissue repair, anti-inflammatory and swelling, myocardial protection, etc. However, polysaccharides in Periplaneta americana have not been reported, and their chemical structure has not been elucidated, and the biological activity of the polysaccharides is not clear. Based on this, the inventors have carried out systematic chemical and pharmacological activity research on Periplaneta americana polysaccharides, and for the first time reported a novel Periplaneta americana glucan Pa1-B1, and after in-depth research, it was surprisingly found that the glucan has significant immunomodulatory activity, and it was also found to have the pharmacological activity of training the body's immunity and having long-acting anti-tumor effect.

[0058] Periplaneta americana glucan Pa1-B1

[0059] The present application provides a Periplaneta americana glucan Pa1-B1 having the structure 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 Periplaneta americana glucan Pa1-B1 disclosed in the embodiment of the present application is composed of glucose, and the main chain is linked by alpha (1→4) glycosidic bond, and the side chain is linked by alpha (1→6) glycosidic bond.

[0063] The Periplaneta americana glucan disclosed in the embodiment of the present application is obtained by innovative extraction and purification of Periplaneta americana, and is novel in structure and is reported for the first time. Further activity research shows that the Periplaneta americana glucan Pa1-B1 significantly promotes the release of NO and the secretion of cytokines IL-6 and TNF-α of macrophage RAW264.7. It is shown that the Periplaneta americana glucan has significant immune regulation ability, and therefore, the present application further discloses a new use of the Periplaneta americana glucan in preparing a medicine and / or food for improving immunity.

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

[0065] In some embodiments, the dispersity coefficient of the Periplaneta americana glucan is 3.01.

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

[0067] According to the embodiment of the present application, the weight average molecular weight of the Periplaneta americana glucan is about 36000 kDa to 42000 kDa, the main chain is alpha (1→4) glycosidic bond, and the side chain is alpha (1→6) glycosidic bond.

[0068] Preparation method of Periplaneta americana glucan

[0069] The second aspect of the present application provides a preparation method of Periplaneta americana glucan Pa1-B1, comprising the following steps S100 to S200:

[0070] S100: extracting crude polysaccharide from Periplaneta americana;

[0071] S200: refining to obtain Periplaneta americana glucan Pa1-B1.

[0072] According to the embodiment of the present application, the Periplaneta americana glucan with novel structure and significant immune regulation activity can be obtained by the above preparation method, the weight average molecular weight of the Periplaneta americana glucan is about 36000 kDa to 42000 kDa, and the main chain is alpha (1→4) glycosidic bond and the side chain is alpha (1→6) glycosidic bond. Therefore, the preparation method provided in the second aspect of the present application can be used to prepare the Periplaneta americana glucan Pa1-B1 of the first aspect of the present application.

[0073] In the embodiments of the present application, the periplaneta americana body can be fresh insect body, or dried insect body or powder after crushing the dried insect body.

[0074] In some embodiments, the periplaneta americana body is powder after crushing the dried insect body.

[0075] In some embodiments, the extracting the crude polysaccharide from the periplaneta americana body comprises: crushing the periplaneta americana body, extracting by warm water, hot water and / or enzymolysis to obtain a solution of the crude polysaccharide.

[0076] Specifically, the extracting the crude polysaccharide from the periplaneta americana body comprises: taking the dried insect body powder, adding 95% ethanol at a material-liquid ratio of 1g:5mL, heating treatment at a water bath temperature of 90℃ for 3h, cooling to room temperature and filtering to obtain 95% ethanol extract, combining the extract after 3 times of extraction, concentrating and freeze-drying to obtain the defatted extract of periplaneta americana. Then, adding deionized water to the extract at a material-liquid ratio of 1g:5mL, extracting at a water bath temperature of 65℃ for 24h, extracting 3 times, centrifuging and taking the supernatant.

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

[0078] S210: enzymolysis extraction, alcohol precipitation, to obtain crude periplaneta americana glucan.

[0079] Specifically, the enzymolysis temperature is 50-65℃, the enzymolysis time is 2-24h, and the volume concentration of alkaline protease is 1%-5%, and the volume concentration of alkaline protease is 3%.

[0080] In the embodiments of the present application, the alcohol precipitation comprises: boiling the solution after enzymolysis to inactivate the enzyme, centrifuging, alcohol precipitation of the supernatant, and collecting the precipitate.

[0081] In the embodiments of the present application, the alcohol precipitation of the supernatant comprises the following steps:

[0082] The 95% ethanol is added to the inactivated enzyme solution under stirring until the final alcohol concentration is 35%-45%, centrifuged to obtain 35%-45% alcohol precipitate, and then re-dissolved in water and freeze-dried.

[0083] Specifically, the final alcohol concentration in step S211 is 40%, and the final alcohol concentration in step S211 is 40%.

[0084] The component with a weight average molecular weight of 36000kDa-42000kDa in the refined crude periplaneta americana glucan is refined to obtain periplaneta americana glucan Pa1-B1.

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

[0086] Specifically, crude American cockroach glucan can be reconstituted with deionized water, centrifuged to remove insoluble matter, and then purified by alcohol precipitation fractionation, gel size exclusion chromatography, dialysis or ultrafiltration. The fraction containing American cockroach glucan, the retentate or the dialysate is collected. If it contains salt, it needs to be desalted and then directly freeze-dried under vacuum or concentrated under reduced pressure followed by alcohol precipitation and vacuum drying to obtain purified American cockroach glucan.

[0087] Those skilled in the art will understand that, for size exclusion chromatography, the gel material is rationally selected based on the molecular weight of the dextran in the polysaccharide component of *Periplaneta americana*, such as the Sephadex series of dextran gels, the Sepharose series of agarose gels, the Bio-Gel P series of polyacrylamide gels, and gel packing materials formed by their cross-linking. Then, according to the actual performance of each packing material, the column is packed, the sample is loaded, and the eluent is eluted sequentially with salt-containing or salt-free elution solutions, and the fractions are collected. The elution peaks are combined, and the eluent may or may not be concentrated. It is then placed in a dialysis bag for dialysis or an ultrafiltration membrane for dialysis or ultrafiltration desalting. The desalted retentate is collected and then freeze-dried or vacuum-reduced to obtain purified *Periplaneta americana* dextran. Alternatively, when purifying the *Periplaneta americana* polysaccharide component using dialysis or ultrafiltration methods, an ultrafiltration membrane with an appropriate molecular weight is selected for ultrafiltration retentate treatment. For example, an aqueous solution of American cockroach polysaccharide component is first subjected to ultrafiltration through an ultrafiltration membrane with a molecular weight smaller than that of American cockroach dextran Pa1-B1. The permeate is collected to remove substances with low molecular weight. Then, it is subjected to dialysis or ultrafiltration through a dialysis bag or ultrafiltration membrane with a molecular weight larger than that of American cockroach dextran Pa1-B1. The retentate is collected, concentrated, and then freeze-dried or vacuum-reduced to obtain purified American cockroach dextran Pa1-B1.

[0088] Structural analysis of the *Periplaneta americana* glucan provided in this embodiment of the invention showed that it exhibited a well-symmetrical chromatographic peak on HPGPC gel chromatography, indicating high purity. Methylation analysis revealed that the *Periplaneta americana* glucan mainly contains α(1→4) and α(1→6) glycosidic bonds. One-dimensional and two-dimensional NMR spectral data clearly identified the NMR signals of the *Periplaneta americana* glucan.

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

[0090] Further, the weight average molecular weight of the Periplaneta americana glucan Pa1-B1 is about 41611 kDa, and the dispersion coefficient is 3.01.

[0091] Application of Periplaneta americana glucan Pa1-B1 in functional food and / or medicine

[0092] The embodiment of the third aspect of the application provides application of Periplaneta americana glucan in preparation of food and / or medicine for enhancing immune activity and / or training immune anti-tumor.

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

[0094] According to the embodiment of the application, the Periplaneta americana glucan Pa1-B1 is injected into the abdominal cavity of mice in advance, melanoma cells are inoculated into the tail vein after 7 days of training immunity, compared with a control group, the Periplaneta americana glucan Pa1-B1 can significantly inhibit tumor cell lung metastasis after training immunity, significantly prolong the survival period of tumor-bearing mice, and also has a long-acting anti-tumor metastasis effect, which proves that the Periplaneta americana glucan Pa1-B1 has significant training immunity anti-tumor activity. Therefore, the application further discloses application of the Periplaneta americana glucan Pa1-B1 in preparation of training immunity anti-tumor medicine.

[0095] Embodiment

[0096] The following specific embodiments are used to illustrate the application. It should be pointed out that the embodiments described below are exemplary and are used to explain the application, and cannot be understood as limiting the application. If the specific technology or condition is not indicated in the embodiments, the technology or condition described in the literature in the art or according to the product manual is used. If the specific condition is not indicated in the embodiments, the conventional condition or the condition recommended by the manufacturer is used, and if the instrument or reagent is not indicated by the manufacturer, it is a conventional product that can be obtained from the market.

[0097] Embodiment 1: Preparation of Periplaneta americana glucan

[0098] Extraction of Periplaneta americana crude polysaccharide

[0099] 1) After drying treatment of Periplaneta americana, the Periplaneta americana whole worm powder is obtained by crushing;

[0100] 2) Defatting: The dried powder of P. americana was weighed, and 95% ethanol was added according to the solid-liquid ratio of 1:5 (kg:L). The mixture was heated in a water bath at 90°C for 3 h, and then cooled to room temperature. The filtrate was obtained by filtration. The extraction was repeated 3 times, and the defatted filtrate was obtained by filtration. The ethanol was evaporated.

[0101] 3) Enzymatic extraction: 700 g of the defatted and dried filtrate was placed in a 5 L reactor, 3.5 L of deionized water and 21 g of alkaline protease (3%) were added, and the mixture was reacted at 65°C for 24 h in the reactor. The pH value of the reaction solution was monitored and maintained at 9 during the extraction process. The supernatant was collected by centrifugation (4000 rpm x 15 min), and the precipitate was extracted 3 times.

[0102] 4) Alcohol precipitation: The supernatant of step 3) was added with 95% ethanol to a final concentration of 40% under continuous stirring, and then placed at 4°C for 12 h. The precipitate was obtained by centrifugation (4000 rpm x 15 min).

[0103] 5) The precipitate was washed with anhydrous ethanol 3 times, and the alcohol was evaporated. The water was added to dissolve the precipitate, and then freeze-dried to obtain a crude polysaccharide sample of 22.61 g.

[0104] 6) Anion exchange column chromatography: After activation and column packing of DEAE-52 cellulose, the column was eluted with deionized water for 3 column volumes. The eluate was adjusted to pH 7, and then freeze-dried to obtain a crude P. americana polysaccharide.

[0105] 7) Gel column chromatography: Sephadex G100 gel was selected. About 200 mg of P. americana crude polysaccharide was dissolved in 10 mL of deionized water, centrifuged at 4000 rpm for 15 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was slowly and uniformly injected into the top of the Sephadex G100 gel column, and eluted with 0.1 M NaCl solution at a flow rate of 0.1 mL / min. Each tube was collected 2 mL, and the polysaccharide content was detected by the phenol-sulfuric acid method. The absorbance value was used as the vertical coordinate, and the tube number was used as the horizontal coordinate. The elution curve of the polysaccharide components was drawn. The eluate belonging to the same elution peak was combined, desalted with a 500 Da dialysis bag, and then freeze-dried and weighed. After Sephadex G100 purification, the components were analyzed by liquid chromatography, which was a single symmetrical peak. No obvious color change was observed by methylene blue detection, and purified P. americana glucan Pa1-B1 was obtained.

[0106] Example 2: Structure analysis of P. americana glucan.

[0107] The P. americana polysaccharide Pa1-B1 obtained in Example 1 was subjected to structure analysis, which specifically included:

[0108] (1) Molecular weight determination:

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

[0110] Detector: differential detector (RID) and diode array detector (DAD);

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

[0112] Determination process: accurately weigh 5 mg of Periplaneta americana glucan Pa1-B1 sample or known molecular weight dextran control into mobile phase to prepare a 5 mg / mL solution, pass through a 0.2 μm microporous filter, and then inject the filtrate into a high performance liquid chromatograph for analysis, and record the chromatogram. The data are processed by GPC software, a standard curve is drawn, the data are substituted into the equation, and the molecular weight is calculated.

[0113] Experimental results: the weight average molecular weight of Pa1-B1 is 41611 kDa, and the polydispersity coefficient is 3.01, and the specific molecular weight distribution is as shown in Figure 2 .

[0114] (2) Monosaccharide composition analysis: take 0.5 mL of the prepared sample solution, monosaccharide standard solution, and monosaccharide standard mixture solution (deionized water as blank control) into each COD test tube, add 0.5 mL of 4M trifluoroacetic acid (TFA) and vortex mix, and then seal and perform acid hydrolysis reaction at 110 °C for 4 h. Take out the hydrolysis solution, cool to room temperature, and evaporate to dryness on a rotary evaporator with a 50 °C water bath. Add 1 mL of methanol to promote deacidification, and repeat 2-3 times. Finally, dissolve the dried sample in 500 μL of deionized water, and take out 200 μL for use. Add 200 μL of 0.6M NaOH solution and 400 μL of 0.5M PMP-methanol solution to the 200 μL solution taken from the above COD tube, vortex mix, and then seal and perform derivatization reaction at 70 °C for 1 h. After the reaction is completed, cool to room temperature, slowly add 0.6M hydrochloric acid solution for neutralization, add chloroform for complete extraction for 3 times, filter the aqueous phase through a 0.22 μm filter membrane, and then take the filtrate as the liquid chromatography analysis sample solution.

[0115] Chromatographic conditions:

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

[0117] Column: Hadesil C18-BIO (5 μm, 4.6 x 250 mm); 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: 250 nm, detection time: 60 min.

[0120] As Figure 3 The results show that the polysaccharide of Periplaneta americana mainly contains glucose, which is a glucan.

[0121] (3) Methylation analysis:

[0122] Pre-treatment: weigh 3 mg of Pa1-B1 into a 10 mL screw tube. Add 2 mL of methanol and place in a vacuum desiccator for 2 times. Add 0.5 mL of dimethyl sulfoxide and ultrasonic treatment for 20-30 min.

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

[0124] Add 500 μL of DMSO / NaOH solution to the sample and ultrasonic treatment for 30 min.

[0125] Methylation: after adding 200 μL of CH3I to the sample and ultrasonic treatment for 10 min, add another 200 μL of CH3I and ultrasonic treatment for 10 min, and then add 400 μL of CH3I and 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, and combine the organic phase solution. 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 polysaccharide acid: dissolve the dried sample in 1 mL of deionized water, add 1 mL of 4M TFA solution to fully dissolve the methylation product, and perform acid hydrolysis in an oven at 121 °C for 2 h. After the reaction is completed, add 2 mL of methanol and dry by rotary evaporation at 50 °C, repeat 2 times.

[0127] Monosaccharide reduction: dissolve the dried sample in 1 mL of 2M NH4OH, and add 1 mL of freshly prepared 1M NaBD4. Ultrasonic treatment the sample for 1 min, and incubate at room temperature for 2.5 h. Slowly add 400 μL of acetic acid to destroy the excess reducing agent. Dissolve with 1 mL of 5% (v / v) acetic acid-methanol solution and dry by rotary evaporation 2 times. Dissolve with 1 mL of methanol and dry by rotary evaporation 2 times. Dry the dried sample in a vacuum desiccator.

[0128] Sugar alcohol acetylation: 1 mL of acetic anhydride and 1 mL of pyridine were added to the sample, vortexed to dissolve, and placed in a 100°C reaction for 1.5 h. 1 mL of water was added to terminate the reaction, and 3 mL of chloroform was added to extract 3 times. The organic phase layer was extracted with an equal volume of water 3 times, and the organic layer was spin-dried. 2 mL of methanol was added and spin-dried twice. 1 mL of chromatographic grade chloroform was added to dissolve, filtered with a 0.22 μm filter membrane, and then subjected to GC-MS analysis.

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

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

[0131] Table 1 GC-MS signal assignment of the methylation product of Periplaneta americana glucan Pa1-B1

[0132]

[0133]

[0134] The main ion peaks were assigned as shown in Table 1: 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 fragments 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 fragments in the sugar chain; the ion peak at 23.350 min was assigned to 2,3,4-tri-O-methyl-1,5,6-triacetyl-D-glucitol, indicating the presence of →6)-Glcp-(1→ structural fragments 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 →4,6)-Glcp-(1→ structural fragments in the sugar chain. It was inferred that the Periplaneta americana glucan contains α(1→4) glycosidic bonds and α(1→6) glycosidic bonds.

[0135] (4) UV spectrum analysis: 1 mg of Periplaneta americana polysaccharide Pa1-B1 was dissolved in water to 1 mg / mL, and the UV absorption spectrum of the sample was detected in the range of 190-800 nm.

[0136] The UV spectrum is shown in Figure 4 Figure 1, and the glucan Pa1-B1 has few impurities and high purity.

[0137] (5) Infrared spectrum analysis: 2 mg of Periplaneta americana glucan Pa1-B1 was vacuum dried at 40°C for 24 h, and then potassium bromide was pressed into a tablet. The sample was scanned by an infrared spectrometer in the range of 4000-400 cm -1 , and the spectrum was recorded.

[0138] The infrared spectrum of Pa1-B1 is shown in Figure 5 Figure 2, and the characteristic absorption peaks are as follows: 3417 cm -1 is caused by sugar ring alcohol hydroxyl O-H stretching vibration; 2929 cm -1 is a sugar ring methylene C-H absorption vibration peak; 1635 cm -1 is an aldehyde group stretching vibration peak; 1417-1200 cm -1 is a C-H variable angle vibration peak; 1200-1000 cm -1 is a sugar ring C-O-C stretching vibration; 927 and 848 cm -1 are C-H vibration characteristic peaks on the pyran ring; 700-500 cm -1 are a plurality of absorption bands of sugar ring stretching vibration.

[0139] (6) Nuclear magnetic resonance (NMR) spectrum analysis: about 10 mg of Periplaneta americana glucan Pa1-B1 sample was exchanged with D2O for 3 times, and was dissolved in D2O to 20 mg / mL. The sample was detected by an 800 MHz nuclear magnetic instrument to obtain 1 H-NMR / 13 C-NMR spectrum 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 nuclear magnetic results of Periplaneta americana polysaccharide Pa1-B1 are shown in Figure 6-13 Figures 3 and 4, and the chemical shift values of each C and H of each residue are assigned according to the nuclear magnetic spectrum,1 H, 13 C signal assignment is shown in Table 2.

[0141] Table 2 Signal assignment of Periplaneta americana glucan Pa1-B1 1 H and 13 C NMR signal assignment

[0142]

[0143] From 1 H- 13 C HSQC and 1 H- 13 C HMBC spectrum clearly showed the correlation signals between C-H on the sugar ring. Combined with the two-dimensional spectrum in 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 spectrum data confirmed that Pa1-B1 is a glucan with α(1→4) glycosidic bond as the main chain, and has α(1→6) glycosidic bond glucose side chain substitution.

[0144] Example 3: Periplaneta americana glucan Pa1-B1 enhances immune activity experiment.

[0145] The Periplaneta americana glucan Pa1-B1 prepared in the above examples was subjected to activity experiment, and the specific steps and results were as follows:

[0146] (1) The effect of Periplaneta americana glucan Pa1-B1 on RAW264.7 cell activity:

[0147] Take the logarithmic growth phase cells of RAW264.7, prepare a cell suspension with a cell density of 8×10 4 cells / mL, inoculate in a 96-well cell culture plate, 100 μL per well; after 24 h of culture, add Periplaneta americana glucan Pa1-B1 to make its final concentration 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively, and place in the incubator for 24 h; then add 10 μL CCK8 reagent to each well, and return to the incubator for 1-2 h; use the enzyme marker to measure the absorbance value at 450 nm, and calculate the effect of Periplaneta americana glucan Pa1-B1 on RAW264.7 cell activity at different concentrations.

[0148] As Figure 14It was found that the Periplaneta americana glucan Pa1-B1 had no cytotoxic effect on RAW264.7 cells at the concentration range of 100 μg / mL to 800 μg / mL.

[0149] (2) Effect of Periplaneta americana glucan Pa1-B1 on phagocytosis of RAW264.7 cells:

[0150] The logarithmic growth phase cells of RAW264.7 were taken to prepare a cell suspension with a cell density of 1×10 5 cells / mL, which was inoculated into 96-well cell culture plates at 100 μL per well. After 24 h of culture, Periplaneta americana glucan Pa1-B1 was added to make the final concentration 2 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL, respectively. After 24 h of culture in an incubator, the cell culture solution was removed, 150 μL of 0.075% neutral red solution was added to each well, and the culture was continued for 1 h. Then the neutral red solution was discarded, 200 μL of PBS was added to each well to wash the cells, and the operation was repeated for 3 times. Then 100 μL of ethanol: glacial acetic acid = 1:1 solution was added, and the mixture was allowed to stand at room temperature for 30 min to 1 h. The absorbance was measured at 540 nm using a microplate reader.

[0151] As shown in Table 1, Periplaneta americana glucan Pa1-B1 can improve the phagocytosis of RAW264.7 cells at the concentration of 50 μg / mL and 100 μg / mL. Figure 15

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

[0153] The logarithmic growth phase cells of RAW264.7 were taken to prepare a cell suspension with a cell density of 1×10 6 cells / mL, which was inoculated into 24-well cell culture plates at 100 μL per well, and the culture medium was supplemented to 600 μL. Different concentrations of polysaccharide sample Pa1-B1 were added to the experimental group, and LPS was used as the positive control group. The blank group was set, and the cell supernatant was collected after 24 h of culture under the original culture conditions as the experimental group sample and the control group sample. Different concentrations of polysaccharide sample and LPS control group were added to 96-well plates at 50 μL / well. Griess I and Griess II were added to each well at 50 μL each at room temperature, and the plate was shaken after 10 min of light protection. The absorbance was measured at 540 nm using a microplate reader.

[0154] As shown in Table 2, polysaccharide Pa1-B1 can promote the secretion of NO by macrophages at the concentration of 12.5-200 μg / mL. Figure 16

[0155] ​​(4) The influence of Periplaneta americana glucan Pa1-B1 on the secretion of IL-6 and TNF-α of macrophages:

[0156] Different concentrations of polysaccharide samples Pa1-B1 were taken, LPS was taken as a positive control group, a blank group was set, and the cell supernatant was collected after 24h of cell culture as experimental group samples and control group samples.

[0157] The operation of IL-6 and TNF-α content detection is as follows: 100ul of standard diluent and different concentrations of samples, and 50ul of 1 / 100 diluted detection antibody were added to each well of an enzyme-labeled plate, the plate was sealed, and incubated at room temperature for 2h with shaking at 300r / min, after incubation, the liquid was discarded and 300ul of washing solution was added for 6 times, after washing, the plate was dried on the water-absorbing paper, 100ul of 1 / 100 diluted horseradish peroxidase-labeled streptavidin was added to each well, the plate was sealed, and incubated at room temperature for 45min with shaking at 300r / min, and then the above step was washed, 100ul of color developing substrate TMB was added to each well, and the reaction was terminated by adding 100ul of stop solution after incubation at room temperature for 30min, and the OD450 was detected by using an enzyme-labeled instrument in a short time.

[0158] The influence of Periplaneta americana glucan Pa1-B1 on the secretion of IL-6 and TNF-α of macrophages RAW264.7 is shown in Figure 17 and Figure 18 respectively. The results show that Periplaneta americana glucan Pa1-B1 can significantly promote the secretion of cytokines IL-6 and TNF-α of macrophages at a concentration of 2-100ug / mL.

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

[0160] Example 4: Anti-tumor metastasis activity experiment of Periplaneta americana glucan Pa1-B1 training immunity.

[0161] The Periplaneta americana glucan Pa1-B1 prepared in the above example was taken to carry out an in vivo immune anti-tumor activity experiment, the animal experiment scheme was approved by the ethics committee of Kunming Institute of Botany, Chinese Academy of Sciences, and was strictly carried out in accordance with the relevant national animal experiment regulations, and the specific steps and results are as follows:

[0162] (1) Experimental process: C57BL / 6J mice were injected with 200ul of PBS or Periplaneta americana glucan Pa1-B1 (1mg or 2mg / 200ul of PBS) by single intraperitoneal injection, and were fed with normal feed and deionized water for 7 days.

[0163] C57BL / 6J mice syngeneic melanoma cells, B16-F10 and B16-F10 / luc with luciferase gene, respectively, were cultured in 10 cm culture dishes. The logarithmic growth phase melanoma cells were trypsinized for 1-2 min and then terminated with complete medium. After centrifugation (1000 rpm x 3 min), the supernatant was discarded, the cells were resuspended with 1x PBS and blown to a single cell suspension. The cell concentration was adjusted to 3x10 5 5 6 / mL-5x10 5

[0164] After 7 days of intraperitoneal administration, the mice were inoculated with 100 μL of tumor cells of adjusted concentration through the tail vein. After 14-18 days of tumor cell inoculation, the lung tissue was removed for photography or the mice were intraperitoneally injected with luciferase for small animal live imaging photography. The gray value of the tumor metastasis lesion was statistically analyzed by Image J to represent the degree of metastasis.

[0165] The lung tissue was paraffin-embedded and sectioned, and the tissue sections were HE stained to evaluate the immunological anti-tumor activity of Periplaneta americana dextran Pa1-B1.

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

[0167] (2) Experimental results: In the survival statistics of each experimental group, 7 mice in the Pa1-B1 (2 mg) group survived to the 50th day of tumor inoculation, and the lung tissue of two of them was observed to show no tumor lesions in the lungs, which were considered as Tumor-free mice. After 60 days from the administration treatment, 5 mice in the Pa1-B1 (2 mg) group were again inoculated with 3.5x10 5

[0168] As shown in Figure 19 and Figure 20 , after 7 days of training immunization with Periplaneta americana dextran Pa1-B1, the mice were inoculated with melanoma cells through the tail vein on the 18th day, and the lung tissue of the mice was taken and small animal live imaging was performed with luciferin to count the lung melanoma metastasis lesions. The experimental results showed that 1 mg of single dose of Periplaneta americana dextran had a tendency to inhibit lung metastasis of melanoma, and at a single dose of 2 mg, it could significantly inhibit lung metastasis of melanoma cells.

[0169] As​Figure 21 As shown in Figure 6, lung tissue HE staining showed that 2 mg single dose of P. americana glucan Pa1-B1 had significant inhibitory effect on tumor cell lung metastasis, indicating that P. americana glucan Pa1-B1 had significant trained immunity anti-tumor activity.

[0170] As shown in Figure 7, compared with the control group, P. americana glucan Pa1-B1 at 1 mg and 2 mg doses could significantly prolong the survival of tumor-bearing mice, and 7 mice in the 2 mg administration group were still alive on the 50th day after inoculation of tumor cells. Figure 22 As shown in Figure 8, 2 mice were found to have no melanoma metastasis in the lungs after dissection, and 7 surviving mice were considered to be tumor-free mice. Five of the mice were again inoculated with melanoma cells, and lung tissue was taken for observation after 18 days. The lung melanoma metastasis in 4 of the 5 mice was significantly less than that in the control group.

[0171] Figure 23 As shown in Figure 9, 2 mice were found to have no melanoma metastasis in the lungs after dissection, and 7 surviving mice were considered to be tumor-free mice. Five of the mice were again inoculated with melanoma cells, and lung tissue was taken for observation after 18 days. The lung melanoma metastasis in 4 of the 5 mice was significantly less than that in the control group.

[0172] Further, the bone marrow cells of the mice treated with P. americana glucan Pa1-B1 trained immunity and PBS control group for 7 days were transplanted into untrained immunity mice (recipients), and the recipients were inoculated with melanoma cells through the tail vein after 14 days. Lung tissue was taken for dissection after 18 days. It was found that the lung tumor metastasis of the recipient mice transplanted with the bone marrow cells of the P. americana glucan Pa1-B1 trained immunity mice was significantly less than that of the recipient mice transplanted with the bone marrow cells of the PBS treatment group mice.

[0173] The above results show that P. americana glucan Pa1-B1 has significant trained immunity anti-tumor activity, and therefore, the P. americana glucan Pa1-B1 has application value for preparing immune anti-tumor drugs and / or auxiliary anti-tumor drugs.

[0174] It should be noted that the example is taken as an example of P. americana glucan Pa1-B1 inhibiting lung metastasis of melanoma cells B16-F10 to prove the trained immunity anti-tumor activity of Pa1-B1, and it cannot be understood as a limitation of the immune anti-tumor activity of P. americana glucan Pa1-B1 in this example. The immune anti-tumor activity of P. americana glucan Pa1-B1 in this example includes but is not limited to the inhibitory activity of various types of tumor cells such as melanoma cells on the in vivo progression and metastasis of the tumor cells, and the prolongation of the survival period of tumor-bearing mice inoculated with different types of tumor cells. The trained immunity anti-tumor activity of P. americana glucan Pa1-B1 in this example is studied, which is administered before inoculation of tumor, and it cannot be understood as a limitation of the trained immunity anti-tumor effect of P. americana glucan Pa1-B1 after inoculation of tumor.

[0175] ​Example 5: Periplaneta americana glucan Pa1-B1 capsule.

[0176] Materials: Periplaneta americana glucan Pa1-B1 obtained by the method of Example 1, food or pharmaceutical grade starch.

[0177] Prescription:

[0178] Name of raw and auxiliary materials Dosage Periplaneta americana glucan Pa1-B1 500g Starch 300g Total 10000 pieces Name of raw and auxiliary materials

[0179] Preparation process: take the prescribed amount of Periplaneta americana glucan Pa1-B1 and starch, stir until mixed completely. Add an appropriate amount of talc, granulate by wet granulation with ethanol, sieve and dry, then fill into No. 2 capsule shells. Each capsule contains 50 mg of Periplaneta americana glucan Pa1-B1, and Periplaneta americana glucan Pa1-B1 capsules are prepared.

[0180] Example 6: Periplaneta americana glucan Pa1-B1 oral liquid.

[0181] Materials: Periplaneta americana glucan Pa1-B1 obtained by the method of Example 1, food or pharmaceutical grade flavoring agent is caramel flavor.

[0182] Prescription:

[0183] Dosage Periplaneta americana glucan Pa1-B1 Sucrose 100g Caramel flavor 0.6g Purified water 0.1g 2000 mL Total 500 pieces Name of raw and auxiliary materials Dosage

[0184] Preparation process: take the prescribed amount of Periplaneta americana glucan Pa1-B1, sucrose and caramel flavor, add purified water to completely dissolve, then filter through a 0.2 μm microporous filter. The filtrate is filled into bottles by an oral liquid filling machine at a volume of 4 mL per bottle, sealed and sterilized to obtain the product.

[0185] Example 7: Periplaneta americana glucan Pa1-B1 lyophilized powder for injection.

[0186] Materials: Periplaneta americana glucan Pa1-B1 obtained by the method of Example 1, water for injection.

[0187] Prescription:

[0188] Periplaneta americana glucan Pa1-B1 Water for injection 1000 mL 20g Total 1000 pieces ​ ​ ​

[0189] Take the prescribed amount of Periplaneta americana glucan Pa1-B1 and add water for injection, stir until dissolved completely, then sterilize by intermittent heat pressing. After the content is qualified, filter through a 0.22 μm microporous filter, then divide and fill into regulated vials at a volume of 0.5 mL per vial, half-tighten the stopper, place in a freeze-drying box, freeze-dry according to the set freeze-drying curve, tighten the stopper, take out of the box, crimp the cap, perform visual inspection, and package the finished product.

[0190] Freeze-drying process: Put the sample into the box, and keep the temperature of the baffle at -50°C for 5 h. Then, lower the temperature of the cold trap to -50°C, and start vacuumizing to 200 μbar. Start sublimation: keep the temperature at -15°C for 2 h, and then keep it at -5°C for 3 h. Keep the vacuum at 100-250 μbar. Then, dry the sample: keep the temperature at 5°C for 2 h, and then keep it at 15°C for 1 h. Keep the vacuum at 80-100 μbar. Finally, keep the temperature at 40°C for 0.5 h, and then keep it at 40°C for 4 h.

Claims

1. American cockroach glucan Pa1-B1, characterized in that, The American cockroach glucan is a glucan with a main chain linked by α(1→4) and glucose molecules on the side chains linked to the main chain by α(1→6), having the structure shown in general formula I below: ， In Equation I, n represents the number of repeating units and is a natural number, where 40000≦n≦47000.

2. The American cockroach dextran Pa1-B1 according to claim 1, characterized in that, The weight-average molecular weight of the American cockroach glucan is 36,000 kDa to 42,000 kDa.

3. The method for preparing the American cockroach dextran Pa1-B1 according to claim 1, characterized in that, The method includes the following steps: Step 1: After drying, pulverizing and defatting the whole American cockroach, extract it using an enzymatic method. After centrifugation, obtain a solution of crude polysaccharide, and then precipitate it with alcohol to obtain crude American cockroach glucan. Step 2: Refine the components of the American cockroach glucan with a weight average molecular weight of 36,000 kDa to 42,000 kDa to obtain American cockroach glucan. The purification steps include one or more of the following: alcohol precipitation, anion exchange chromatography, and gel size exclusion chromatography.

4. The method for preparing American cockroach dextran Pa1-B1 according to claim 3, characterized in that, In the defatting step, the ethanol concentration is 95%, and the defatting time is 0.5 h to 3 h; The enzyme used in the enzymatic method is an alkaline protease with a volume concentration of 1% to 5%. The enzymatic hydrolysis conditions are pH=9, the enzymatic hydrolysis temperature is 50℃ to 65℃, and the enzymatic hydrolysis time is 2 h to 24 h. The alcohol precipitation method is as follows: take the supernatant for alcohol precipitation, so that the alcohol concentration reaches 35% to 45%, and collect the precipitate.

5. A pharmaceutical composition comprising an effective amount of the American cockroach dextran Pa1-B of claim 1 and a pharmaceutically acceptable carrier.

6. The use of the American cockroach dextran Pa1-B as described in claim 1 or 2 in the preparation of foods that enhance immune function.

7. The use of the American cockroach dextran Pa1-B as described in claim 1 or 2 in the preparation of drugs that enhance immune function, train immune antitumor activity, provide immune antitumor activity, or inhibit tumor metastasis.

8. The use of the pharmaceutical composition of claim 5 in the preparation of a medicament for enhancing immune function, training immune antitumor activity, immune antitumor activity, or antitumor metastasis.

9. The method for preparing the pharmaceutical composition according to claim 5, characterized in that, The method includes the following steps: Step 1: After drying and pulverizing the whole American cockroach and defatting it, the solution of crude polysaccharide is obtained by enzymatic extraction and centrifugation. Then, it is precipitated with alcohol to obtain crude American cockroach dextran. In the defatting step, the ethanol concentration is 95% and the defatting time is 0.5 h to 3 h. The enzyme used in the enzymatic method is alkaline protease with a volume concentration of 1% to 5%. The enzymatic hydrolysis conditions are pH=9, the enzymatic hydrolysis temperature is 50℃ to 65℃, and the enzymatic hydrolysis time is 2 h to 24 h. Step 2: Purify the components of the American cockroach dextran with a weight average molecular weight of 36,000 kDa to 42,000 kDa to obtain American cockroach dextran. The purification step includes one or more of the following: alcohol precipitation, anion exchange chromatography, and gel size exclusion chromatography. The alcohol precipitation method is as follows: take the supernatant for alcohol precipitation to achieve an alcohol concentration of 35% to 45%, and collect the precipitate. Step 3: Take the American cockroach glucan obtained in the above steps and add it to a pharmaceutically acceptable carrier.

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