Periplaneta americana glucan PaN with enhanced immune function and preparation method and application thereof

Abstract

The application discloses an American cockroach glucan PaN, a pharmaceutical composition of the American cockroach glucan PaN, a preparation method of the American cockroach glucan PaN and application of the American cockroach glucan PaN in enhancing immune function of a body, and belongs to the technical field of biological medicines.The American cockroach glucan PaN is an alpha (1→6) linked glucan, and the weight average molecular weight is 3000 Da to 4000 Da.The preparation method comprises the following steps: crushing American cockroach whole worm, degreasing by using ethanol, extracting by using an enzymolysis method, and separating and purifying by using a fractional alcohol precipitation, an ion exchange column chromatography method and / or a gel column chromatography method.The American cockroach glucan PaN can enhance the phagocytosis of macrophage RAW264.7, and dose-dependently promotes the macrophage to release NO and to secrete cytokines (IL-6, TNF-alpha).Therefore, the American cockroach glucan PaN and the composition thereof can be used in preparation of an immune-regulating medicine or food.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and health technology, specifically relating to a cockroach glucan PaN and its pharmaceutically acceptable pharmaceutical composition, its preparation method and its application. Background Technology

[0002] The American cockroach (Periplaneta americana), belonging to the order Blattodea and family Blattodea, is a traditional Chinese medicine recorded in many classic texts. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) lists it as a medium-grade herb, describing it as "salty and cold in nature. It treats blood stasis, hard masses, chills and fever, breaks up accumulations, sore throat, and infertility due to internal cold." The *Bencao Gangmu* (Compendium of Materia Medica) records it as "a nocturnal insect, a type of cricket, consumed by the southwestern barbarians." The *Mingyi Bielu* (Records of Famous Physicians) also records the medicinal value of cockroaches. The *Chinese Materia Medica of Medicinal Animals* includes the American cockroach, using dried or fresh adult insects as medicine. It is ground into powder for internal or external use, and is believed to have effects such as promoting blood circulation, reducing swelling, promoting wound healing, and treating external injuries and chronic ulcers.

[0003] The chemical substances in the American cockroach mainly include proteins and amino acids, nucleosides, peptides, sugars, and fatty acids. Modern pharmacological studies have shown that the American cockroach possesses activities such as promoting tissue repair, antibacterial activity, and antioxidant activity, but its active ingredients are unclear. In particular, American cockroach polysaccharides have not yet been reported, and their biological activity remains uncertain. To date, there are no reports on American cockroach dextran PaN and its activity. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the existing technology by providing a *Periplaneta americana* dextran PaN, its pharmaceutical composition, its preparation method, and its application in the preparation of immunomodulatory drugs.

[0005] Based on this, the present invention isolates and purifies a novel American cockroach dextran, PaN, from the American cockroach. The structure and function of this American cockroach polysaccharide are reported for the first time. Pharmacological studies have revealed that the American cockroach dextran PaN has a significant physiological function of enhancing immune activity.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] This invention first provides a dextran from the American cockroach species, which has an α(1→6) linked dextran structure, abbreviated as PaN, and has the structural formula shown in General Formula I below:

[0008]

[0009]

[0010] In chemical formula I, n represents the number of repeating units and is a natural number, where 6 ≤ n ≤ 8.

[0011] The weight-average molecular weight (Mw) of the American cockroach glucan is 3000 Da to 4000 Da, and the polydispersity index (PDI) is 1.43.

[0012] This invention provides a method for preparing the American cockroach dextran PaN, comprising the following steps:

[0013] Step (1): Dry and pulverize the whole American cockroach, defatt it, and then extract it by enzymatic hydrolysis to obtain crude American cockroach glucan;

[0014] Step (I): The crude American cockroach glucan obtained in Step (I) is further separated and purified to obtain the component with a weight average molecular weight of 3000 Da to 4000 Da, thus obtaining American cockroach glucan PaN.

[0015] In the method for preparing the American cockroach glucan, the defatting step involves an ethanol concentration of 95% and a defatting time of 0.5 h to 3 h.

[0016] The method for preparing American cockroach glucan includes the following: the amount of alkaline protease used is 1% to 5% of the weight of American cockroach; the enzymatic hydrolysis conditions are pH=9; the enzymatic hydrolysis temperature is 50 to 65℃; and the enzymatic hydrolysis time is 9 to 24 hours.

[0017] The method for preparing American cockroach glucan, wherein the fractional alcohol precipitation includes: taking the supernatant for fractional alcohol precipitation, so that the alcohol concentration reaches 35%-45% and 55%-65% respectively, and collecting the 55%-65% alcohol precipitate.

[0018] The method for preparing American cockroach glucan, wherein the purification method includes one or more of the following: fractional alcohol precipitation, anion exchange chromatography, gel size exclusion chromatography, dialysis, and ultrafiltration.

[0019] The American cockroach glucan has a significant physiological function of enhancing immune activity.

[0020] The present invention also provides a pharmaceutical composition comprising an effective amount of the aforementioned American cockroach dextran PaN and a pharmaceutically acceptable carrier.

[0021] The application of the aforementioned American cockroach glucan in the preparation of drugs that enhance immune function, and / or in the preparation of drugs for treating immunodeficiency diseases.

[0022] The application of the aforementioned American cockroach dextran Pa1-B in the preparation of foods that enhance immune function.

[0023] The use of the pharmaceutical composition in the preparation of a drug for enhancing immune function, and / or in the preparation of a drug for treating immunodeficiency diseases.

[0024] The method for preparing the pharmaceutical composition includes the following steps:

[0025] (1) The whole American cockroach is dried and pulverized, defatted and then enzymatically extracted to obtain crude American cockroach dextran. The defatting includes defatting with ethanol for 0.5 h to 3 h. The enzymatic hydrolysis includes hydrolysis with alkaline protease, the amount of alkaline protease is 1% to 5% of the mass of American cockroach, the hydrolysis conditions are pH=9, the hydrolysis temperature is 50℃ to 65℃, and the hydrolysis time is 2 h to 24 h.

[0026] (2) Purify the components of American cockroach dextran with a weight average molecular weight of 3000 Da to 4000 Da to obtain American cockroach dextran. The purification includes one or more of the following: fractional alcohol precipitation, anion exchange chromatography, and gel size exclusion chromatography. The fractional alcohol precipitation includes taking the supernatant for fractional alcohol precipitation, so that the alcohol concentrations reach 35% to 45% and 55% to 65% respectively, and collecting 55% to 65% precipitate.

[0027] (3) Take the American cockroach glucan obtained in the above steps and add it to a pharmaceutically acceptable carrier.

[0028] This invention reveals that the novel American cockroach dextran PaN significantly enhances the phagocytic capacity of macrophages and promotes the release of TNF-α and IL-6 cytokines. Enhanced macrophage phagocytosis effectively engulfs and digests pathogens such as bacteria and viruses, preventing their spread and infection within the body. The upregulation of TNF-α and IL-6 plays a crucial role in inflammatory and immune responses; they promote the recruitment and activation of inflammatory cells, induce the production of pro-inflammatory cytokines, regulate immune cell function, and participate in multiple mechanisms of the inflammatory cascade, coordinating the body's defense against infection and injury. Therefore, American cockroach dextran PaN has significant application value in enhancing the body's immune function.

[0029] Compared with the prior art, the present invention has the following technical effects:

[0030] The structure and function of the American cockroach polysaccharide PaN of this invention are reported for the first time, filling a gap in the prior art. The American cockroach polysaccharide PaN is a dextran linked by α(1→6) glycosidic bonds, with a weight-average molecular weight of 3000 Da to 4000 Da. The American cockroach dextran PaN can significantly enhance the phagocytic capacity of macrophages, promote the release of NO from RAW264.7 macrophages, significantly 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 dextran PaN of this invention has an immune-enhancing effect and can be used to prepare drugs for treating immunodeficiency diseases, as well as foods that improve immune function. Attached Figure Description

[0031] Figure 1 The structural formula of PaN, a dextran from the American cockroach;

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

[0033] Figure 3 HPLC chromatogram of standard monosaccharide mixtures of American cockroach dextran PaN and its PMP derivatives;

[0034] Figure 4 The ultraviolet spectrum of PaN, a dextran from the American cockroach;

[0035] Figure 5 Infrared spectrum of PaN, a dextran from the American cockroach;

[0036] Figure 6 For American cockroach dextran PaN 1 H-NMR spectrum;

[0037] Figure 7 For American cockroach dextran PaN 13 C-NMR spectrum;

[0038] Figure 8 For American cockroach dextran PaN 1 H- 1 H COSY spectrum;

[0039] Figure 9 For American cockroach dextran PaN 1 H- 1 H TOCSY spectrum;

[0040] Figure 10 For American cockroach dextran PaN 1 H- 13 C HSQC spectrum;

[0041] Figure 11For American cockroach dextran PaN 1 H- 13 C HMBC spectrum;

[0042] Figure 12 For American cockroach dextran PaN 1 H- 13 C HSQC-TOCSY spectrum;

[0043] Figure 13 For American cockroach dextran PaN 1 H- 1 H ROESY spectrum;

[0044] Figure 14 The effect of PaN, a dextran from the American cockroach, on the activity of RAW264.7 macrophages (n=3, * indicates significant difference compared with the control group, ** indicates p<0.01);

[0045] Figure 15 The effect of PaN, a dextran from the American cockroach, on the phagocytic capacity of RAW264.7 macrophages (n=3, * indicates significant difference compared to the control group, ** indicates p<0.01);

[0046] Figure 16 The effect of PaN, a dextran from the American cockroach, on NO release from RAW264.7 macrophages (n=3, * indicates significant difference compared to the control group, **** represents p<0.0001);

[0047] Figure 17 The effect of PaN, a dextran from the American cockroach, on IL-6 secretion by RAW264.7 macrophages (n=3, * indicates significant difference compared to the control group, **** represents p<0.0001);

[0048] Figure 18 The effect of American cockroach glucan PaN on TNF-α secretion by RAW264.7 macrophages (n=3, * indicates significant difference compared with the control group, **** represents p<0.0001). Detailed Implementation

[0049] The following description, in conjunction with the accompanying drawings, uses specific embodiments to further illustrate the substantive content of the present invention. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Instruments or reagents used, unless the manufacturer is specified, are all commercially available conventional products. The features, structures, or characteristics described below can be combined in any suitable manner in one or more embodiments.

[0050] [Example 1] Preparation of American cockroach glucan

[0051] 1.1 Extraction of crude polysaccharides from American cockroaches

[0052] The American cockroach (Periplaneta americana) used in this invention is commercially available.

[0053] Pulverization: After drying the whole American cockroach, it is pulverized to obtain American cockroach powder.

[0054] Degreasing: Weigh dry American cockroach powder, add 95% ethanol at a material-to-liquid ratio of 1g:5mL, reflux extract in a 90℃ water bath for 3h, after extraction, cool to room temperature and filter to obtain defatted residue.

[0055] Enzymatic hydrolysis: Weigh 700g of defatted American cockroach residue and place it in a 5L reactor. Add 3.5L of deionized water and 21g of alkaline protease (3% of the American cockroach residue). React in the reactor at 65℃ for 24h. Adjust the pH value to 9 with low concentration NaOH. Centrifuge (4000rpm×15min) and collect the supernatant.

[0056] Fractional alcohol precipitation: The supernatant was added to 95% ethanol to a final concentration of 40% under continuous stirring, allowed to stand at 4°C for 12 h, and centrifuged (4000 rpm × 15 min) to obtain the supernatant; 95% ethanol was added to the 40% alcohol precipitation supernatant to a final concentration of 60%, allowed to stand at 4°C for 12 h, and centrifuged (4000 rpm × 15 min) to obtain the precipitate; the precipitate was washed three times with anhydrous ethanol, the ethanol was evaporated, the precipitate was reconstituted in water, and then freeze-dried to obtain 10.03 g of crude polysaccharide sample.

[0057] 1.2 Purification of American cockroach dextran

[0058] Anion exchange column chromatography: After activation and packing of DEAE-52cellulose, deionized water was used for elution, with an elution volume of 3 column volumes. The pH of the eluent was adjusted to 7, and the eluent was concentrated and lyophilized to obtain crude American cockroach glucan.

[0059] Gel column chromatography: Sephadex G100 gel was used. 200 mg of crude polysaccharide from American cockroaches was weighed, dissolved in 10 mL of deionized water, and centrifuged (4000 rpm × 15 min). The supernatant was filtered through a 0.45 μm filter membrane and then slowly and evenly injected into the gel column. Elution was performed with 0.1 M NaCl solution at a flow rate of 0.1 mL / min, and 2 mL was collected from each tube. The polysaccharide content was detected by the phenol-sulfuric acid method. The elution curve was plotted with the absorbance value as the ordinate and the number of tubes as the abscissa, and the eluents belonging to the same elution peak were combined. The sample solution was desalted using a 500 Da dialysis bag, and then concentrated and lyophilized.

[0060] Purity analysis: The polysaccharide obtained by freeze-drying was subjected to high performance liquid chromatography (HPLC) analysis using methylene blue detection. Based on the single peak shape of the HPLC and the absence of color change in methylene blue, the purified American cockroach dextran PaN was finally obtained. The results showed that its purity was greater than 95%.

[0061] Chromatographic conditions: Agilent Technologies 1260 series high performance liquid chromatograph;

[0062] Detectors: Differential Identifier (RID) and Diode Array Detector (DAD);

[0063] Chromatographic conditions: OHpak SB-804HQ (8.0×300mm); injection volume: 30μL; column temperature: 35℃; mobile phase: 0.1M sodium chloride solution; flow rate: 0.5mL / min; detection time: 40min.

[0064] [Example 2] Structural analysis of American cockroach glucan

[0065] 2.1 Determination of molecular weight and its distribution

[0066] Weigh approximately 5 mg of PaN sample and add it to the mobile phase to prepare a 5 mg / mL solution. Filter the solution through a 0.22 μm microporous membrane and perform liquid chromatography analysis on the filtrate. Weigh a dextran reference standard with a known molecular weight and prepare a 5 mg / mL solution. Perform chromatographic conditions as described above and record the chromatogram. Process the data using GPC software, plot a standard curve, and substitute the data into the equation to calculate the molecular weight.

[0067] The weight-average molecular weight of PaN was determined to be 3373 Da, and the polydispersity index was 1.43. The specific molecular weight distribution is as follows: Figure 2 As shown.

[0068] 2.2 Monosaccharide Composition

[0069] Take 0.5 mL each of the prepared PaN sample solution, monosaccharide standard solution, and monosaccharide standard mixed solution and place them in their respective COD tubes. Add 0.5 mL of 4M trifluoroacetic acid (TFA) and vortex to mix. Under sealed conditions, perform acid hydrolysis in an oven at 110°C for 4 hours. Cool to room temperature and evaporate to dryness using a rotary evaporator at 50°C. Add 1 mL of methanol to remove acid, repeating twice. After dissolving in 500 μL of deionized water, take 200 μL for later 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 COD tube. Vortex to mix and under sealed conditions, perform derivatization reaction in an oven at 70°C for 1 hour. Cool to room temperature and slowly add 0.6M hydrochloric acid solution for neutralization. Extract thoroughly three times with chloroform. Filter the aqueous phase through a 0.22 μm filter membrane. The filtrate is the test solution for liquid chromatography analysis.

[0070] Chromatographic conditions: Agilent Technologies 1260 series high performance liquid chromatograph;

[0071] Column: Hadesil C18-BIO (5μm, 100A, 4.6×250nm), column temperature: 25℃;

[0072] Mobile phase: phosphate buffer (pH=6.8)-acetonitrile (83:17), flow rate 1 mL / min; injection volume 20 μL;

[0073] Detection: DAD detector, detection wavelength: 250nm, detection time: 60min.

[0074] like Figure 3 The results showed that the monosaccharide composition of PaN contained only glucose, indicating that the polysaccharide was a glucan.

[0075] 2.3 Methylation Analysis

[0076] Pretreatment: Weigh 3 mg of PaN, put it into a 10 mL spiral tube, add 2 mL of methanol, and dry it in a vacuum drying oven. Repeat twice. Add 0.5 mL of dimethyl sulfoxide and sonicate for 20–30 min. Weigh 1200 mg of NaOH, add 10 mL of DMSO to dissolve it, and obtain a 120 mg / mL sodium hydroxide DMSO solution. Add 500 μL of the prepared DMSO / NaOH solution to the sample and sonicate for 30 min.

[0077] Methylation: Add 200 μL of CH3I to the sample and sonicate for 10 min, then add another 200 μL of CH3I and sonicate for 10 min, followed by 400 μL of CH3I and sonicate for 30 min. Stop the reaction by adding 1 mL of water, then extract the sample with 3 mL of chloroform. Extract three times and combine the organic phase solutions. Wash the chloroform phase three times with an equal volume of water, and dry the organic layer by rotary evaporation at 45 °C.

[0078] Acid hydrolysis: Dissolve the dried sample in 1 mL of deionized water, then add 1 mL of 4 M TFA solution to dissolve the methylation product. Hydrolyze the sample in an oven at 121 °C for 2 h, then add 2 mL of methanol and dry by rotary evaporation at 50 °C. Repeat twice.

[0079] Reduction reaction: Dissolve the dried sample in 1 mL of 2M NH4OH and add 1 mL of freshly prepared 1M NaBD4. Sonicate the sample for 1 min and incubate at room temperature for 2.5 h. Slowly add 400 μL of acetic acid to destroy excess reducing agent. Reduce the sample to dryness by rotary evaporation, dissolve in 1 mL of 5% (v / v) acetic acid-methanol solution and reduce to dryness twice. Dissolve in 1 mL of methanol and reduce to dryness, repeating twice. Dry the reduced sample in a vacuum oven.

[0080] Sugar alcohol acetylation: Add 1 mL of acetic anhydride and 1 mL of pyridine to the sample, vortex to dissolve, and react at 100 °C for 1.5 h. Terminate the reaction by adding 1 mL of water, extract three times with 3 mL of chloroform, and extract the organic phase three times with an equal volume of water. Dry the organic layer by rotary evaporation, then add 2 mL of methanol and dry twice. Dissolve in 1 mL of chromatographic grade chloroform, filter through a 0.22 μm filter membrane, and perform GC-MS analysis.

[0081] GC detection conditions: Column: DB-5MS quartz capillary column (30m×0.25mm×0.25μm); Column temperature program: Initial temperature 80℃, hold for 11 min, program temperature increase at 10℃ / min to 195℃, hold for 1 min; continue at 2℃ / min to 210℃, hold for 2 min; finally, increase at 10℃ / min to 250℃, hold for 5 min; Column flow rate: 1.5mL / min; Injector temperature: 270℃; Splitless mode: Splitless; Carrier gas: High-purity helium, injection volume: 1μL.

[0082] MS detection conditions: Ionization mode, EI; Electron energy: 70 eV; Transfer line temperature, 290℃; Ion source temperature: 230℃; Quadrupole temperature, 150℃; Mass range, 50–600; Inspection time, 60 min.

[0083] Table 1. GC-MS signal assignments of PaN methylated products of glucan from American cockroaches.

[0084]

[0085] As shown in Table 1, the ion peaks at 20.117 min are 87, 102.1, 118, 129, 145, 162, and 205.1, which are assigned to 2,3,4,6-tetra-O-methyl-1,5-diacetyl-D-glucanol, located at the end of the sugar chain, indicating the presence of the Glcp-(1→)-G ...

[0086] 2.4 UV spectral analysis

[0087] Dissolve American cockroach glucan PaN in water to a concentration of 1 mg / mL, and detect the ultraviolet absorption spectrum of the sample in the range of 190–800 nm.

[0088] Ultraviolet spectrum such as Figure 4 As shown, the American cockroach glucan PaN is free of impurities such as protein and nucleic acid, and has high purity.

[0089] 2.5 Infrared Spectroscopy Analysis

[0090] Take 2 mg of American cockroach dextran (PaN), vacuum dry at 40℃ for 24 h, then compress with potassium bromide into tablets, and spectroscopy the sample at 4000–400 cm⁻¹. -1 Scan and record the spectrum.

[0091] The infrared spectrum of PaN is as follows: Figure 5 As shown, its characteristic absorption peak is at 3409 cm⁻¹. -1 This is due to the stretching vibration of the hydroxyl group (OH) in the sugar ring; 2929 cm -1 The peak represents the stretching vibration of the methylene group of the sugar ring (CH); 1637 cm⁻¹ -1 The peak for aldehyde stretching vibration is 1417–1200 cm⁻¹. -1 The peak is a CH-angle vibration peak; 1200–1000 cm⁻¹ -1 For the COC stretching vibration on the sugar ring; 916 and 862 cm⁻¹ -1 The characteristic peak of the CH vibration on the pyranose ring; 700–500 cm⁻¹ -1 The multiple absorption bands are due to the stretching vibration of the sugar ring.

[0092] 2.6 Nuclear Magnetic Resonance Spectroscopy Analysis

[0093] Approximately 10 mg of American cockroach glucan (PaN) was dissolved to a concentration of 20 mg / mL after three D2O exchanges and detected using an 800 MHz NMR spectrometer. 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 C HSQC-TCOSY 1 H- 1 H ROESY map.

[0094] The 1D / 2D NMR results of PaN, a dextran from the American cockroach, are as follows: Figures 6 to 13 As shown, the chemical shifts of each C and H residue were assigned, and the results are shown in Table 2.

[0095] Table 2. American cockroach glucan PaN 1 H and 13 Signal attribution of C NMR

[0096]

[0097] Chemical shifts of PaN can be assigned and their linkage mode inferred through 1D and 2D NMR analysis. 1 Preliminary H NMR analysis indicated that the molecule has only one terminal hydrogen atom with a chemical shift greater than 4.90 ppm, suggesting that the sugar is in the α configuration. 1 H- 1 The H COSY and TOCSY spectra can be individually assigned to their hydrogen signals; combined with 13 C NMR and 1 H- 13 C HSQC determines its carbon signal; through 1 H- 13 CHMBC and 1 H- 1 H ROESY spectroscopy can determine its linkage mode, with correlation signals between positions 1 and 6, indicating a (1→6) linkage. Furthermore, compared to the chemical shift of the glucose standard, the carbon chemical shift at position 6 shifts to a lower field, indicating substitution at that position. Combined with the methylation results, this confirms that the American cockroach polysaccharide PaN is a structurally regular α(1→6) glucan.

[0098] [Example 3] Bioactivity experiment of PaN, a glucan from the American cockroach.

[0099] 3.1 Detection of the cytotoxic activity of American cockroach dextran PaN against RAW264.7 cells

[0100] RAW264.7 cells in the logarithmic growth phase were used to prepare cells with a density of 8 × 10⁻⁶ cells / year. 4 Cell suspensions of 100 μL / well were seeded into 96-well cell culture plates. After culturing for 24 h, American cockroach dextran (PaN) was added to final concentrations of 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL. After culturing for 24 h, 10 μL of CCK8 reagent was added to each well, and the plates were returned to the incubator for 1–2 h. The absorbance was measured at 450 nm using a microplate reader to calculate the effect of different concentrations of American cockroach dextran (PaN) on the viability of RAW264.7 cells.

[0101] like Figure 14 Data showed that the American cockroach dextran PaN had no cytotoxic effect on RAW264.7 cells in the concentration range of 100 μg / mL to 800 μg / mL.

[0102] 3.2 Effect of American cockroach dextran PaN on phagocytic capacity of RAW264.7 cells

[0103] RAW264.7 cells in the logarithmic growth phase were diluted to a cell density of 1×10⁻⁶. 5 Cell suspensions of 100 μL per well were seeded into 96-well cell culture plates. After culturing for 24 h, American cockroach dextran (PaN) was added to achieve final concentrations of 2 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. After culturing for 24 h, the cell culture medium was removed, and 150 μL of 0.075% neutral red solution was added to each well. After culturing for another 1 h, the neutral red solution was discarded, and 200 μL of PBS was added to each well to wash the cells. This process was repeated 3 times. 100 μL of ethanol-acetic acid solution was added, and the cells were incubated at room temperature for 30 min to 1 h. The absorbance was measured at 540 nm using a microplate reader.

[0104] The measurement results are as follows Figure 15 As shown, at a concentration of 100 μg / mL, the American cockroach dextran PaN significantly enhanced the phagocytic ability of RAW264.7 cells.

[0105] 3.3 Effect of American cockroach dextran PaN on NO release from RAW264.7 cells

[0106] RAW264.7 cells in the logarithmic growth phase were used to prepare cells with a density of 1×10⁻⁶. 6Cell suspensions of 100 μL / well were seeded into 24-well cell culture plates, and culture medium was added to a final volume of 600 μL. Different concentrations of polysaccharide sample PaN were added to the experimental groups. LPS was used as a positive control, and a blank control group was included. After culturing under the original conditions for 24 h, cell supernatant was collected as samples for the experimental and control groups. Different concentrations of polysaccharide sample and LPS control were added to 96-well plates at 50 μL / well. Griess I and Griess II were added sequentially at room temperature. After incubation in the dark for 10 min, the plates were shaken, and absorbance was measured at 540 nm using a microplate reader.

[0107] The measurement results are as follows Figure 16 As shown, the polysaccharide PaN at concentrations of 2–100 μg / mL promotes the secretion of NO by macrophages.

[0108] 3.4 Effects of American cockroach dextran PaN on macrophage secretion of IL-6 and TNF-α

[0109] Different concentrations of polysaccharide samples PaN were collected, with LPS as the positive control group and a blank group. After culturing the cells for 24 hours, the cell supernatant was collected as the experimental group sample and the control group sample.

[0110] The detection procedure for IL-6 and TNF-α levels was performed according to the kit instructions. The steps were as follows: Add 100 μL each of standard dilution buffer, samples of different concentrations, and positive control LPS to the microplate. Add 50 μL of 1 / 100 diluted detection antibody to each well. Seal the plate and incubate at room temperature with shaking at 300 rpm for 2 hours. After incubation, discard the liquid and add 300 μL of washing buffer 6 times. After washing, blot dry on absorbent paper. Add 100 μL of 1 / 100 diluted horseradish peroxidase-labeled streptavidin to each well. Seal the plate and incubate at room temperature with shaking at 300 rpm for 45 minutes. Wash as before, add 100 μL of chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 30 minutes. Add 100 μL of stop solution to terminate the reaction. Detect OD450 using a microplate reader within a short time.

[0111] The effects of American cockroach glucan PaN on the secretion of cytokines IL-6 and TNF-α by RAW264.7 macrophages are as follows: Figure 17 and Figure 18 As shown in the figure. The results indicate that the American cockroach dextran PaN at concentrations of 2–100 μg / mL significantly and in a dose-dependent manner promotes the secretion of cytokines IL-6 and TNF-α by macrophages.

[0112] The above results indicate that the American cockroach dextran PaN has no cytotoxic effect on macrophages, but it promotes the release of NO from macrophages and stimulates the secretion of cytokines IL-6 and TNF-α. Therefore, the American cockroach dextran PaN can safely and effectively activate the immune response of macrophages and has a significant physiological function of enhancing immune activity.

[0113] [Example 4] American cockroach glucan PaN droplets

[0114] 4.1 Materials

[0115] The same method as in Example 1 was used to obtain American cockroach dextran PaN and polyethylene glycol.

[0116] 4.2 Prescription:

[0117] Raw material names Dosage American cockroach glucan PaN 100g polyethylene glycol 6000g Made into 1000 pills

[0118] 4.3 Preparation process

[0119] Weigh out the prescribed amount of American cockroach dextran (PaN), add an appropriate amount of anhydrous ethanol, dissolve by gentle heating, then add to the prescribed amount of molten polyethylene glycol. Stir and mix thoroughly until the ethanol evaporates completely. Let stand in a 60°C water bath for 30 minutes to remove air bubbles. Transfer the bubble-free molten mixture into a storage container. Under conditions of 80-85°C, control the dripping rate and add the mixture drop by drop into the condensate. After complete condensation, pour off the condensate and collect the pellets. The temperature and density of the condensate should be appropriate to ensure the pellets solidify fully and do not stick together. Drain and remove the condensate from the pellets with filter paper, then dry under reduced pressure. Remove the completely condensed pellets from the condensate and wash with a suitable solvent to remove surface condensate. Dry the washed pellets in a low-temperature oven. Select pellets of uniform size and round shape from the prepared pellets, and remove any substandard pellets. Coat the pellets to increase their stability.

[0120] [Example 5] American cockroach glucan PaN capsules

[0121] 5.1 Materials

[0122] The same method as in Example 1 yields American cockroach dextran PaN, which is a food or pharmaceutical grade starch.

[0123] 5.2 Prescription:

[0124] Raw material names Dosage American cockroach glucan PaN 500g starch 300g Made into 10,000 pills

[0125] 5.3 Preparation process

[0126] Weigh out the prescribed amounts of American cockroach glucan (PaN) and starch, and stir until fully mixed. Add an appropriate amount of talc, granulate using ethanol wet granulation, sieve and dry, then fill into No. 2 capsule shells, each capsule containing 50 mg of American cockroach glucan (PaN), to obtain capsules.

[0127] [Example 6] American cockroach glucan PaN oral solution

[0128] 6.1 Materials

[0129] The American cockroach dextran PaN obtained by the same method as in Example 1, and the food or pharmaceutical grade flavoring agent is caramel flavoring.

[0130] 6.2 Prescription

[0131] Raw material names Dosage American cockroach glucan PaN 100g sucrose 0.6g Caramel flavoring 0.1g Purified water 2000mL Made into 500 pieces

[0132] 6.3 Preparation process

[0133] Weigh out the prescribed amounts of American cockroach glucan PaN, sucrose, and caramel flavoring, dissolve them completely in purified water, filter through a 0.2μm microporous membrane, and fill each bottle with 4mL of the filtrate using an oral liquid filling machine. After sealing, sterilize to obtain the final product.

[0134] [Example 7] American cockroach dextran PaN freeze-dried powder injection

[0135] 7.1 Materials

[0136] The same method as in Example 1 was used to obtain American cockroach dextran Pa1-B1, and water for injection.

[0137] 7.2 Prescription:

[0138] Raw material names Dosage American cockroach glucan PaN 20g Water for Injection 1000mL Made into 1000

[0139] 7.3 Preparation process

[0140] Weigh the prescribed amount of American cockroach dextran (PaN) and add water for injection to the final volume. Stir until completely dissolved and sterilize using intermittent autoclaving. After the content meets the requirements, filter through a 0.22 μm microporous membrane. Dispense into controlled vials, 0.5 mL per vial, partially stopper, place in a freeze-drying oven, freeze-dry according to the set freeze-drying curve, stopper, remove from the oven, cap, visually inspect for compliance, and package to obtain the finished product.

[0141] Freeze-drying process: Place the sample in the chamber, lower the partition temperature to -50℃, and hold for 5 hours; lower the cold trap to -50℃ and begin evacuation to 200 μbar. Sublimation begins: Heat to -15℃ at a constant rate for 2 hours and hold for 3 hours; heat to -5℃ at a constant rate for 3 hours and hold for 6 hours, maintaining a vacuum of 100–250 μbar; Drying continues: heat to 5℃ for 2 hours and hold for 2 hours, maintaining a vacuum of 150–200 μbar; heat to 15℃ for 1 hour and hold for 2 hours, maintaining a vacuum of 80–100 μbar; heat to 40℃ for 0.5 hours and hold for 4 hours, then evacuate to the lowest possible vacuum.

Claims

1. A Periplaneta americana glucan PaN, characterized in that, The Periplaneta americana glucan PaN is an α(1→6) linked glucan, and has the structure shown in general formula I: ， I In chemical formula I: n represents the number of repeating units, and is a natural number, 6≦n≦8.

2. The Periplaneta americana glucan PaN according to claim 1, characterized in that, The weight average molecular weight of the Periplaneta americana glucan is 3000 Da ~ 4000 Da.

3. The method for preparing Periplaneta americana polysaccharide PaN according to claim 1, characterized in that, The method comprises the following steps: (1) drying and crushing the whole Periplaneta americana, defatting, and then enzymatic extraction to obtain crude Periplaneta americana glucan; (2) refining the component with a weight average molecular weight of 3000 Da ~ 4000 Da in the Periplaneta americana glucan to obtain Periplaneta americana glucan, The refining step comprises one or more of fractional alcohol precipitation method, anion exchange chromatography method, and gel exclusion chromatography method.

4. The preparation method of the Periplaneta americana glucan PaN according to claim 3, characterized in that: The defatting comprises defatting with ethanol, and the defatting time is 0.5 h ~ 3 h; The enzymatic extraction comprises enzymatic extraction with alkaline protease, and the amount of the alkaline protease is 1% ~ 5% of the mass of the Periplaneta americana, the enzymatic extraction condition is pH = 9, the enzymatic extraction temperature is 50℃ ~ 65℃, and the enzymatic extraction time is 2 h ~ 24 h; The fractional alcohol precipitation method comprises taking the supernatant for fractional alcohol precipitation, so that the alcohol concentration reaches 35% ~ 45% and 55% ~ 65% respectively, and the 55% ~ 65% precipitate is collected.

5. A pharmaceutical composition comprising an effective amount of the Periplaneta americana glucan PaN according to claim 1 and a pharmaceutically acceptable carrier.

6. Use of the Periplaneta americana glucan according to claim 1 or 2 in the preparation of a drug for enhancing immune function, or / and in the preparation of a drug for treating immune deficiency diseases.

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

8. Use of the pharmaceutical composition according to claim 5 in the preparation of a drug for enhancing immune function, or / and in the preparation of a drug for treating immune deficiency diseases.

9. A method of preparing the pharmaceutical composition of claim 5, characterized by, The method comprises the following steps: (1) drying and crushing the whole Periplaneta americana, defatting, and then enzymatic extraction to obtain crude Periplaneta americana glucan, the defatting comprises defatting with ethanol, and the defatting time is 0.5 h ~ 3 h; the enzymatic extraction comprises enzymatic extraction with alkaline protease, and the amount of the alkaline protease is 1% ~ 5% of the mass of the Periplaneta americana, the enzymatic extraction condition is pH = 9, the enzymatic extraction temperature is 50℃ ~ 65℃, and the enzymatic extraction time is 2 h ~ 24 h; (2) refining the component with a weight average molecular weight of 3000 Da ~ 4000 Da in the Periplaneta americana glucan to obtain Periplaneta americana glucan, the refining comprises one or more of fractional alcohol precipitation method, anion exchange chromatography method, and gel exclusion chromatography method; wherein the fractional alcohol precipitation method comprises taking the supernatant for fractional alcohol precipitation, so that the alcohol concentration reaches 35% ~ 45% and 55% ~ 65% respectively, and the 55% ~ 65% precipitate is collected; (3) taking the Periplaneta americana glucan obtained in the above step, and adding a pharmaceutically acceptable carrier.

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