Lentinus edodes residue polysaccharide-chitosan oligosaccharide nano-composite as well as preparation method and application thereof

Abstract

The invention provides a shiitake mushroom dreg polysaccharide-chitosan oligosaccharide nano-composite as well as a preparation method and application thereof, and belongs to the technical field of biological nano-materials. The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nano-composite is prepared by dropwise adding a shiitake mushroom residue polysaccharide solution into a chitosan oligosaccharide solution. The shiitake mushroom dreg polysaccharide-chitosan oligosaccharide nano-composite prepared by dropwise adding the shiitake mushroom dreg polysaccharide solution into the chitosan oligosaccharide solution has good stability and good biological safety; meanwhile, the shiitake mushroom dreg polysaccharide-chitosan oligosaccharide nano-composite can play a better role in inhibiting inflammation and can be applied to medicines. The preparation method provided by the invention is simple, rapid, convenient to operate and convenient for practical popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials technology, specifically relating to a polysaccharide-chitosan oligosaccharide nanocomposite from shiitake mushroom residue, its preparation method, and its application. Background Technology

[0002] Natural polysaccharides and oligosaccharides possess high biocompatibility, biodegradability, and good controlled-release properties, making them commonly used functional carriers in drug delivery systems and providing a feasible pathway for precise drug delivery and controlled release. Chitosan oligosaccharides are the main degradation products of chitosan or chitin after chemical or enzymatic hydrolysis and deacetylation. Compared to chitosan, chitosan oligosaccharides have lower molecular weight, lower viscosity, and complete water solubility. Due to their abundant functional groups, excellent biocompatibility, and biodegradability, chitosan oligosaccharides are widely used in the preparation of drug delivery systems.

[0003] Shiitake mushroom residue polysaccharide is a low-molecular-weight polysaccharide extracted and purified from shiitake mushroom residue. It possesses anti-inflammatory and antioxidant bioactivities and may be a potential component for functional food or pharmaceutical development. Currently, the resource utilization of shiitake mushroom residue has received widespread attention, and the isolation of shiitake mushroom residue polysaccharide from it has also attracted scholarly interest, leading to a gradual increase in research on this topic. However, most studies are not in-depth and have a narrow scope of application. Therefore, to further expand the application research scope of shiitake mushroom residue polysaccharide, there is an urgent need in this field to obtain a nanocomposite with good stability, high bioactivity, and a simple preparation method for in-depth development and utilization. Summary of the Invention

[0004] The purpose of this invention is to provide a shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, which is prepared by dropwise addition of a shiitake mushroom residue polysaccharide solution to a chitosan oligosaccharide solution; the concentration of the chitosan oligosaccharide solution is 0.5-1.5 mg / mL, and the concentration of the shiitake mushroom residue polysaccharide solution is 0.5-2.5 mg / mL.

[0007] The present invention also provides a method for preparing the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, the method comprising adding a shiitake mushroom residue polysaccharide solution dropwise to a chitosan oligosaccharide solution at a rate of 2 mL / min, stirring for 30 min after the addition is complete, and then freeze-drying to obtain the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite.

[0008] Preferably, the volume ratio of the shiitake mushroom residue polysaccharide solution to the chitosan oligosaccharide solution is (1-8):(1-2).

[0009] Preferably, the addition is carried out at a stirring speed of 500 rpm / min; the solvents for preparing the chitosan oligosaccharide solution and the shiitake mushroom residue polysaccharide solution are both distilled water; the pH of the chitosan oligosaccharide solution is 5.0.

[0010] Preferably, the preparation of the shiitake mushroom residue polysaccharide solution includes:

[0011] Add the shiitake mushroom residue to sodium dihydrogen phosphate-acetic acid buffer at a mass-volume ratio of 1:20 (g / mL), mix well, sterilize, and then perform enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, inactivate the enzyme, centrifuge, and take the first supernatant to obtain the shiitake mushroom residue enzymatic hydrolysate.

[0012] Anhydrous ethanol was added to the enzymatic hydrolysate of shiitake mushroom residue until the volume fraction of ethanol reached 80%. After ethanol precipitation and standing for 12 hours, the precipitate was collected by centrifugation.

[0013] The precipitate was dissolved in water, and the second supernatant was collected by centrifugation. The second supernatant was dialyzed in distilled water for 72 hours using a dialysis bag of size 5000 Daltons. The second supernatant after dialyzing was freeze-dried to obtain shiitake mushroom residue polysaccharide.

[0014] Preferably, the sodium dihydrogen phosphate-acetic acid buffer solution has a sodium dihydrogen phosphate concentration of 0.2 mol / L, an acetic acid concentration of 0.05 mol / L, and a pH of 5.0; the weight-to-volume ratio of the precipitate to water is 1:20 (mg / mL).

[0015] Preferably, the enzyme used in the enzymatic hydrolysis is a complex enzyme; the complex enzyme is cellulase, glucanase and xylanase, and the amount of enzyme added is calculated based on the amount of shiitake mushroom residue powder added, which is 1000U / g, 800U / g and 900U / g respectively.

[0016] Preferably, the enzymatic hydrolysis temperature is 50°C and the enzymatic hydrolysis time is 4 hours.

[0017] The present invention also provides the application of the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite or the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared by the preparation method in the preparation of anti-inflammatory products.

[0018] Preferably, the product includes pharmaceuticals.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite of the present invention exhibits good stability, low toxicity to RAW264.7 macrophages, and good biocompatibility. Simultaneously, the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite demonstrates superior anti-inflammatory effects, inhibiting NO release and the release of inflammatory factors such as TNF-α, IL-6, and IL-1β, making it suitable for pharmaceutical applications. The preparation process of the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite provided by this invention is simple, rapid, and convenient, exhibiting good stability and high bioactivity, and can be further developed and utilized as a drug delivery system. Attached Figure Description

[0021] Figure 1 The image shows a transmission electron microscope (TEM) image of LEP-COS NPs9 from Example 1, where 200 nm is the numerical value of the scale bar. LEP-COS NPs9 is the mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 prepared in Example 4.

[0022] Figure 2 The images show the FT-IR spectra of LEP, COS, and LEP-COS NPs9 in Example 1, where LEP is shiitake mushroom residue polysaccharide, COS is chitosan oligosaccharide, and LEP-COS NPs9 is the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 prepared in Example 10.

[0023] Figure 3 The effects of LEP, COS, and LEP-COS NPs9 on the cell viability of RAW 264.7 macrophages in Experiment Example 2 are shown. LEP is shiitake mushroom residue polysaccharide; COS is chitosan oligosaccharide; and LEP-COS NPs9 is the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 prepared in Example 10.

[0024] Figure 4 The effects of LEP, COS, and LEP-COS NPs9 on NO release from LPS-stimulated RAW 264.7 macrophages in Experiment Example 2 were investigated. LEP is shiitake mushroom residue polysaccharide; COS is chitosan oligosaccharide; and LEP-COS NPs9 is the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 prepared in Example 10. Data are presented as mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences at the P < 0.01 level.

[0025] Figure 5The results of the effects of LEP, COS, and LEP-COS NPs9 on the release of TNF-α (A), IL-1β (B), and IL-6 (C) from LPS-stimulated RAW 264.7 macrophages in Experiment Example 2 are presented. LEP is lentinan from shiitake mushroom residue; COS is chitosan oligosaccharide; and LEP-COS NPs9 is a lentinan-chitosan oligosaccharide nanocomposite. Data are presented as mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences at the P < 0.01 level. Detailed Implementation

[0026] This invention provides a shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, which is prepared by adding a shiitake mushroom residue polysaccharide solution dropwise to a chitosan oligosaccharide solution.

[0027] The present invention also provides a method for preparing the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, the method comprising slowly adding a shiitake mushroom residue polysaccharide solution to a chitosan oligosaccharide solution at a rate of 2 mL / min, stirring for 30 min after the addition is complete, and then freeze-drying to obtain the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite.

[0028] In this invention, during the preparation of the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, the mass ratio of the shiitake mushroom residue polysaccharide solution to the chitosan oligosaccharide solution after mixing is preferably (2-4):1, more preferably (3-4):1, and most preferably 4:1. The concentration of the chitosan oligosaccharide solution is preferably 0.5-1.5 mg / mL, more preferably 0.8-1.2 mg / mL, and most preferably 1 mg / mL; the concentration of the shiitake mushroom residue polysaccharide solution is 0.5-2.5 mg / mL, more preferably 0.5-2.0 mg / mL, and most preferably 0.5 mg / mL. The shiitake mushroom residue polysaccharide solution is added dropwise to the chitosan oligosaccharide solution while stirring at 500 rpm / min.

[0029] In this invention, the chitosan oligosaccharide is preferably Mw ≤ 3000 Da; the shiitake mushroom residue polysaccharide is preferably Mw = 78 kDa. The solvent used to prepare both the chitosan oligosaccharide solution and the shiitake mushroom residue polysaccharide solution is distilled water; the pH of the chitosan oligosaccharide solution is 5.0.

[0030] In this invention, the preparation of the shiitake mushroom residue polysaccharide solution includes:

[0031] Add the shiitake mushroom residue powder to sodium dihydrogen phosphate-acetic acid buffer at a mass-volume ratio of 1:20 (g / mL), mix well, sterilize in an autoclave, cool, add a compound enzyme for enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis reaction is completed, centrifuge and take the supernatant to obtain shiitake mushroom residue enzymatic hydrolysate.

[0032] Add anhydrous ethanol to the enzymatic hydrolysate of shiitake mushroom residue until the ethanol content reaches 80% by volume. Let it stand at room temperature for 12 hours, centrifuge to remove the supernatant precipitated by alcohol, and collect the precipitate.

[0033] The precipitate was dissolved in water until fully dissolved, with a weight-to-volume ratio of precipitate to water of 1:20 (mg / mL). The supernatant was collected by centrifugation and dialyzed with distilled water for 72 hours at room temperature using a 5000 Dalton dialysis bag. The dialysate was then freeze-dried to obtain shiitake mushroom residue polysaccharide.

[0034] In this invention, when preparing the shiitake mushroom residue polysaccharide solution, the shiitake mushroom residue powder is prepared by drying and pulverizing the waste shiitake mushroom logs after harvesting, and passing them through a 100-mesh sieve to obtain the shiitake mushroom residue powder. The sodium dihydrogen phosphate-acetic acid buffer solution has a sodium dihydrogen phosphate concentration of 0.2 mol / L, an acetic acid concentration of 0.05 mol / L, and a pH of 5.0. During high-pressure sterilization, the temperature is set at 120℃, the pressure at 0.25 MPa, and the sterilization time at 30 min. After the high-pressure sterilization reaction, the mixture is removed and cooled to room temperature. During enzymatic hydrolysis, the complex enzyme consists of cellulase, glucanase, and xylanase (with enzyme dosages of 1000 U / g, 800 U / g, and 900 U / g, respectively). Taking 1000 U / g cellulase as an example, U / g means that each gram of shiitake mushroom residue powder contains 1000 U of cellulase. Enzymatic hydrolysis was performed in a shaker at 150 r / min and 50℃ for 4 h. After the enzymatic hydrolysis reaction was completed, the enzyme was inactivated by boiling water bath for 5 min, and then centrifuged at 9500 r / min for 20 min. The supernatant was collected to obtain the enzymatic hydrolysate of shiitake mushroom residue.

[0035] The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared by the above-described method of this invention has a particle size of less than 270 nm, enhanced stability, and uniform nanoparticles. The preparation process is simple, quick, and convenient, facilitating practical application.

[0036] This invention also provides the application of the aforementioned nanocomposite or the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared by the aforementioned preparation method in the preparation of products with anti-inflammatory effects. The products include pharmaceuticals.

[0037] The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 prepared in this invention showed better inhibition of the release of TNF-α, IL-6 and IL-1β from LPS-stimulated macrophages RAW264.7 compared to shiitake mushroom residue polysaccharide, and had significant anti-inflammatory activity.

[0038] The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared by this invention significantly inhibited the release of NO from LPS-stimulated RAW264.7 macrophages within a concentration range of 5–50 μg / mL.

[0039] The shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared in this invention can significantly inhibit the release of TNF-α from LPS-stimulated RAW264.7 macrophages at a concentration of 5–50 μg / mL; and can significantly inhibit the release of IL-1β and IL-6 from LPS-stimulated RAW264.7 macrophages at a concentration of 25–50 μg / mL.

[0040] In the following examples, the chitosan oligosaccharide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S31060-100g; Mw≤3000Da.

[0041] In the following examples, the cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10041-25g; the dextranase was purchased from Qingdao Keyuan Biotechnology Co., Ltd., catalog number D11806-25g; and the xylanase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S59712-100g.

[0042] In the following examples, the RAW264.7 cells were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The colorless RPMI1640 cell culture medium was purchased from Gibco, USA. The Alamar Blue reagent was purchased from Sigma, USA.

[0043] In the following examples, LPS represents lipopolysaccharide, purchased from Merck, Germany, catalog number P4516-1G. The Griess reagent was purchased from Beyotime Biotechnology Co., Ltd., catalog number S0021S. The ELISA kits were purchased from Sizhengbai Biotechnology Co., Ltd., catalog numbers CME0006, CME0005, and CME0004, which, in sequence, detect IL-6, IL-1β, and TNF-α, respectively.

[0044] In the following embodiments, the freeze-drying instrument used is an Alpha 2-4LD plus freeze dryer (Martin Christ Freeze Dryers, Germany), with parameters set to a temperature of -50±5℃ and a vacuum of approximately 1mbar.

[0045] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0047] Example 1

[0048] Preparation method of polysaccharide from shiitake mushroom residue

[0049] (1) Preparation of shiitake mushroom residue powder: The waste shiitake mushroom logs after harvesting are dried, crushed, and passed through a 100-mesh sieve to obtain shiitake mushroom residue powder.

[0050] (2) High-pressure complex enzymatic extraction of polysaccharides from shiitake mushroom residue:

[0051] Weigh 100.0g of shiitake mushroom residue powder into an Erlenmeyer flask, add 2L of sodium dihydrogen phosphate-acetic acid buffer (sodium dihydrogen phosphate concentration is 0.2mol / L, acetic acid concentration is 0.05mol / L, pH=5.0); shake well and place in an autoclave, set the temperature to 120℃, pressure to 0.25MPa, sterilize for 30min. After the reaction is complete, remove and cool to room temperature. Add a complex enzyme for enzymatic hydrolysis, wherein the complex enzyme is cellulase, glucanase and xylanase (the amount of enzyme added is calculated based on the amount of shiitake mushroom residue powder added, which is 1000U / g, 800U / g and 900U / g respectively). Hydrolyze at 150r / min and 50℃ for 4h under constant temperature shaking conditions. After the enzymatic hydrolysis reaction is complete, boil in water for 5min to inactivate the enzyme, then centrifuge at 9500r / min for 20min, and take the supernatant as the shiitake mushroom residue enzymatic hydrolysate.

[0052] (3) Ethanol precipitation of polysaccharides from shiitake mushroom residue: slowly add anhydrous ethanol to the above enzymatic hydrolysate while stirring until the ethanol content reaches 80% (volume fraction), let stand at room temperature for 12 hours, centrifuge to remove the supernatant of ethanol precipitation, and collect the precipitate.

[0053] (4) Dialysis with a dialysis bag: The above precipitate was fully dissolved in water, with a weight-to-volume ratio of precipitate to water of 1:20 (mg / mL). The supernatant was collected by centrifugation at 9500 rpm for 20 min. Dialysis was performed with distilled water in a 5000 Dalton dialysis bag at room temperature for 72 hours. The dialysate was freeze-dried to obtain a gray shiitake mushroom residue polysaccharide sample, which is the shiitake mushroom residue polysaccharide.

[0054] High-performance liquid chromatography (HPLC) gel size exclusion chromatography (GSC): Separation was performed using HPLC gel size exclusion chromatography (Waters 2695 pump separation system) with TSK-GEL series G3000PWXL and G4000PWXL columns (7.8 mm × 300 mm, TOSOH, Japan). Analysis was performed using an eight-angle laser light scattering detector (MALLS, Wyatt) and a Waters 2414 differential detector (RI). The results showed that the weight-average molecular weight of the polysaccharide from the shiitake mushroom residue prepared in Example 1 was approximately 78 kDa.

[0055] Example 2

[0056] Preparation of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0057] (1) Dissolve 0.2g of chitosan oligosaccharide (COS, Mw≤3000Da) in 200mL of distilled water, adjust the initial pH of the chitosan oligosaccharide solution to 5.0 with acetic acid, and prepare a solution of 1mg / mL; dissolve 0.4g of shiitake mushroom residue polysaccharide (LEP, Mw=78kDa) in 200mL of distilled water, and then dilute with distilled water to prepare a solution of 2mg / mL.

[0058] (2) At room temperature, with a magnetic stirrer at a stirring speed of 500 rpm / min, 60 mL of shiitake mushroom residue polysaccharide solution (2 mg / mL) was slowly added dropwise to 60 mL of chitosan oligosaccharide solution (1 mg / mL) at a speed of 2 mL / min using a microfluidic syringe. After the addition was complete, the mixture was stirred for 30 min and then freeze-dried to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 1 (LEP-COS NPs1).

[0059] Example 3

[0060] Preparation of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0061] The difference from Example 2 is that 60 mL of shiitake mushroom residue polysaccharide solution (2 mg / mL) was slowly added dropwise to 40 mL of chitosan oligosaccharide solution (1 mg / mL) to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 2 (LEP-COS NPs2).

[0062] Example 4

[0063] The difference from Example 2 is that 60 mL of shiitake mushroom residue polysaccharide solution (2 mg / mL) was slowly added dropwise to 30 mL of chitosan oligosaccharide solution (1 mg / mL) to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 3 (LEP-COS NPs3).

[0064] Example 5

[0065] Preparation of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0066] (1) Dissolve 0.2g of chitosan oligosaccharide (COS, Mw≤3000Da) in 200mL of distilled water, adjust the initial pH of the chitosan oligosaccharide solution to 5.0 with acetic acid, and prepare a solution of 1mg / mL; dissolve 0.2g of shiitake mushroom residue polysaccharide (LEP, Mw=78kDa) in 200mL of distilled water, and then dilute with distilled water to prepare a solution of 1mg / mL.

[0067] (2) At room temperature, with a magnetic stirrer at a stirring speed of 500 rpm / min, 120 mL of mushroom residue polysaccharide solution (1 mg / mL) was slowly added dropwise to 60 mL of chitosan oligosaccharide solution (1 mg / mL) at a speed of 2 mL / min using a microfluidic syringe. After the addition was complete, the mixture was stirred for 30 min and then freeze-dried to obtain mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 4 (LEP-COS NPs4).

[0068] Example 6

[0069] The difference from Example 5 is that 120 mL of shiitake mushroom residue polysaccharide solution (1 mg / mL) was slowly added dropwise to 40 mL of chitosan oligosaccharide solution (1 mg / mL) to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 5 (LEP-COS NPs5).

[0070] Example 7

[0071] The difference from Example 5 is that 120 mL of shiitake mushroom residue polysaccharide solution (1 mg / mL) was slowly added dropwise to 30 mL of chitosan oligosaccharide solution (1 mg / mL) to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 6 (LEP-COS NPs6).

[0072] Example 8

[0073] Preparation of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0074] (1) Dissolve 0.2g of chitosan oligosaccharide (COS, Mw≤3000Da) in 200mL of distilled water, adjust the initial pH of the chitosan oligosaccharide solution to 5.0 with acetic acid, and prepare a solution of 1mg / mL; dissolve 0.1g of shiitake mushroom residue polysaccharide (LEP, Mw=78kDa) in 200mL of distilled water, and then dilute with distilled water to prepare a solution of 0.5mg / mL.

[0075] (2) At room temperature, with a magnetic stirrer at a stirring speed of 500 rpm / min, 120 mL of shiitake mushroom residue polysaccharide solution (0.5 mg / mL) was slowly added dropwise to 30 mL of chitosan oligosaccharide solution (1 mg / mL) at a speed of 2 mL / min using a microfluidic syringe. After the addition was complete, the mixture was stirred for 30 min and then freeze-dried to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 7 (LEP-COS NPs7).

[0076] Example 9

[0077] The difference from Example 8 is that 120 mL of shiitake mushroom residue polysaccharide solution (0.5 mg / mL) was slowly added dropwise to 20 mL of chitosan oligosaccharide solution (1 mg / mL) to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 8 (LEP-COS NPs8).

[0078] Example 10

[0079] The difference from Example 8 is that 120 mL of shiitake mushroom residue polysaccharide solution (0.5 mg / mL) was slowly added dropwise to 15 mL of chitosan oligosaccharide solution (1 mg / mL), and the mixture was stirred for 30 min after the addition was complete. Then, the mixture was freeze-dried to obtain shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COS NPs9).

[0080] Test Example 1

[0081] Characterization of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0082] (1) Determination of particle size, zeta potential and polymer polydispersity coefficient of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite (LEP-COS NPs)

[0083] The particle size, zeta potential, and polymer polydispersity index of the different shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposites prepared in Examples 2-7 were determined using a laser particle size analyzer (Zetasizer NanoZS90, Malvern, UK).

[0084] The results are shown in Table 1. The lower the concentration of shiitake mushroom residue polysaccharide, the smaller the particle size of the formed nanocomposite, the larger the absolute value of the Zeta potential, the better the stability, the smaller the PDI value, and the more uniform the nanocomposite. At the same concentration of shiitake mushroom residue polysaccharide, the larger the mass ratio of shiitake mushroom residue polysaccharide to chitosan oligosaccharide, the smaller the particle size of the formed nanocomposite, the larger the absolute value of the Zeta potential, the enhanced stability, the smaller the PDI value, and the more uniform the nanocomposite.

[0085] Table 1. Particle size, zeta potential, and polymer polydispersity index (PDI) of different LEP-COS NPs

[0086]

[0087]

[0088] Note: LEP is shiitake mushroom residue polysaccharide; COS is chitosan oligosaccharide; LEP-COS NPs is shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite; m:m is the mass ratio.

[0089] Based on the analysis of Table 1, it can be seen that the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COS NPs9) prepared in Example 10 has the smallest particle size. Therefore, the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COS NPs9) prepared in Example 10 was selected for structural characterization and in vitro bioactivity experiments.

[0090] (2) Morphology of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite

[0091] The surface morphology of the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COS NPs9) prepared in Example 10 was observed using a bio-type field emission transmission electron microscope (Talos F200C G2). Figure 1 As shown, the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COS NPs9) is basically spherical or subspherical and relatively uniform in size.

[0092] (3) Fourier transform infrared spectroscopy analysis

[0093] 2.0 mg of the freeze-dried shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 (LEP-COSNPs9) prepared in Example 10, along with purchased chitosan solid samples and the shiitake mushroom residue polysaccharide prepared in Example 1, were placed at designated positions on an infrared spectrometer for scanning. The spectral acquisition range was 500–4000 cm⁻¹. -1 The resolution is set to 4cm. -1 .

[0094] like Figure 2 As shown, it is located between 3200 and 3550 cm. -1 The broad characteristic peak at 2936.64 cm⁻¹ is attributed to the stretching vibration of OH or NH. This peak is relatively broad because the absorption bands of the OH and NH stretching vibrations overlap. -1 The peak at 1563.98 cm is an absorption peak due to the stretching vibration of the CH bond. -1 and 1070.291cm -1 The peak at this point is an absorption peak due to the CH bending vibration. Compared with shiitake mushroom residue polysaccharide and chitosan oligosaccharide, the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite has a peak at 3277.82 cm⁻¹. -1 The blue shift at the location is 2936.64cm. -1 The redshift at the peak indicates that the polysaccharides in shiitake mushroom residue and the chitosan oligosaccharides are linked by hydrogen bonds. Therefore, the hydrogen bonds between the shiitake mushroom residue polysaccharides and the chitosan oligosaccharides may be one of the main forces promoting the formation of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposites.

[0095] Test Example 2

[0096] Evaluation of the in vitro anti-inflammatory activity of LEP-COS NPs9

[0097] (1) Cytotoxicity against RAW264.7 macrophages

[0098] The cytotoxicity of the shiitake mushroom residue polysaccharide prepared in Example 1, the purchased chitosan oligosaccharide solid sample, and the LEP-COS NPs9 prepared in Example 10 against RAW264.7 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was determined using the Alamar blue method. RAW264.7 cell suspensions were inoculated into 96-well plates at a cell count of 5 × 10⁶ cells / mL. 5 200 μL of each sample was incubated at 37°C in a 5% CO2 incubator for 24 h. The colorless RPMI1640 cell culture medium (purchased from Gibco, USA) was removed from the wells, and replaced with 200 μL of fresh colorless RPMI1640 culture medium containing different concentrations of shiitake mushroom residue polysaccharide prepared in Example 1, purchased chitosan oligosaccharide solid samples, and LEP-COS NPs9 samples (final concentrations set at 5, 10, 15, and 25 μg / mL). After incubation at 37°C for 48 h, 20 μL of Alamar Blue reagent (purchased from Sigma, USA) was added to each well for further incubation. The effect of the selected concentration range of samples on the viability of RAW264.7 cells was evaluated using the Alamar Blue method, with each sample tested in triplicate.

[0099]

[0100] like Figure 3 As shown, at concentrations of 2.5, 5, 10, 25, and 50 μg / mL, the macrophage RAW264.7 cells treated with the shiitake mushroom residue polysaccharide (LEP) prepared in Example 1, the purchased chitosan oligosaccharide solid sample (COS), and the LEP-COS NPs9 prepared in Example 10 had the lowest cell viability (90.3%). The results indicate that the shiitake mushroom residue polysaccharide prepared in Example 1, the purchased chitosan oligosaccharide solid sample, and the LEP-COS NPs9 prepared in Example 10 showed no cytotoxicity at concentrations ranging from 2.5 to 50 μg / mL.

[0101] (2) Effects on NO release from LPS-stimulated RAW264.7 macrophages

[0102] RAW264.7 cells were diluted with colorless RPMI 1640 medium to a concentration of 5 × 10⁶ cells / mL. 5A suspension of cells was added to 96-well plates, 160 μL per well. Cells were cultured at 37°C until fully adherent. The colorless RPMI1640 medium was then removed from the wells, and replaced with 200 μL of fresh colorless RPMI1640 medium containing different concentrations of lentinan (LEP) prepared in Example 1, purchased chitosan oligosaccharide solid sample (COS), and LEP-COS NPs9 sample prepared in Example 10 (final concentrations of 5, 10, 15, and 25 μg / mL). RAW264.7 cells were stimulated with 1 μg / mL LPS (lipopolysaccharide), and a blank control group (PBS) was set up. After culturing at 37°C for 48 h, 100 μL of the supernatant was added to 50 μL of Griess reagent (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0021S). The reaction was allowed to proceed for 10 min, and the absorbance at 543 nm was measured to determine the NO release.

[0103] like Figure 4 As shown, within the concentration range of 2.5–50 μg / mL, shiitake mushroom residue polysaccharide, chitosan oligosaccharide, and shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomplex 9 all inhibited the release of NO from LPS-stimulated RAW264.7 macrophages in a concentration-dependent manner. Chitosan oligosaccharide showed weaker activity in inhibiting the release of NO from LPS-stimulated RAW264.7 macrophages within this concentration range. Within the concentration range of 5–50 μg / mL, the activity of shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomplex 9 in inhibiting the release of NO from LPS-stimulated RAW264.7 macrophages was significantly higher than that of shiitake mushroom residue polysaccharide.

[0104] (3) Effects on the release of inflammatory factors TNF-α, IL-1β and IL-6 from LPS-stimulated macrophages RAW264.7

[0105] RAW264.7 cells were diluted with colorless RPMI 1640 medium to a concentration of 5 × 10⁶ cells / mL. 5 A suspension of cells was added to each well of a 96-well plate at 160 μL. Cells were cultured at 37°C until fully adherent. The colorless RPMI1640 medium was then removed from the wells, and replaced with 200 μL of fresh colorless RPMI1640 medium containing different concentrations of shiitake mushroom residue polysaccharide prepared in Example 1, purchased chitosan oligosaccharide solid sample, and LEP-COS NPs9 sample prepared in Example 10 (final concentrations set at 5, 10, 15, and 25 μg / mL). RAW264.7 cells were stimulated with 1 μg / mL LPS (lipopolysaccharide), and a blank control group (PBS group) was set up. After culturing at 37°C for 48 h, the supernatant was collected, centrifuged at 12000g for 10 min, and the levels of inflammatory factors IL-6, IL-1β, and TNF-α were strictly measured according to the ELISA kit requirements.

[0106] like Figure 5 As shown, at concentrations of 2.5–50 μg / mL, lentinan from shiitake mushroom residue, chitosan oligosaccharide, and lentinan-chitosan oligosaccharide nanocomplex 9 all inhibited the release of IL-6, IL-1β, and TNF-α from LPS-stimulated RAW264.7 macrophages in a concentration-dependent manner. Chitosan oligosaccharide showed weaker activity in inhibiting the release of IL-6, IL-1β, and TNF-α from LPS-stimulated RAW264.7 macrophages. At concentrations of 5–50 μg / mL, the lentinan-chitosan oligosaccharide nanocomplex 9 showed significantly higher activity in inhibiting the release of TNF-α from LPS-stimulated RAW264.7 macrophages than lentinan. At concentrations of 25–50 μg / mL, the lentinan-chitosan oligosaccharide nanocomplex 9 showed significantly higher activity in inhibiting the release of IL-1β and IL-6 from LPS-stimulated RAW264.7 macrophages than lentinan. The results showed that, without causing cytotoxicity, the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite 9 exhibited a good effect in inhibiting the release of TNF-α, IL-6 and IL-1β from LPS-stimulated macrophages RAW264.7, and had significant anti-inflammatory activity.

[0107] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite, characterized in that, The solution was prepared by adding a polysaccharide solution from shiitake mushroom residue to a chitosan oligosaccharide solution; the concentration of the chitosan oligosaccharide solution was 0.5–1.5 mg / mL, and the concentration of the shiitake mushroom residue polysaccharide solution was 0.5–2.5 mg / mL.

2. The method for preparing the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite according to claim 1, characterized in that, The preparation method includes adding a shiitake mushroom residue polysaccharide solution to a chitosan oligosaccharide solution at a rate of 2 mL / min, stirring for 30 min after the addition is complete, and then freeze-drying to obtain a shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite.

3. The preparation method according to claim 2, characterized in that, The volume ratio of the mushroom residue polysaccharide solution to the chitosan oligosaccharide solution is (1-8):(1-2).

4. The preparation method according to claim 2, characterized in that, The addition was carried out with a stirring speed of 500 rpm / min; the solvents for preparing the chitosan oligosaccharide solution and the shiitake mushroom residue polysaccharide solution were both distilled water; the pH of the chitosan oligosaccharide solution was 5.

0.

5. The preparation method according to claim 2, characterized in that, The preparation of the shiitake mushroom residue polysaccharide solution includes: Add the shiitake mushroom residue to sodium dihydrogen phosphate-acetic acid buffer at a mass-volume ratio of 1:20 (g / mL), mix well, sterilize, and then perform enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, inactivate the enzyme, centrifuge, and take the first supernatant to obtain the shiitake mushroom residue enzymatic hydrolysate. Anhydrous ethanol was added to the enzymatic hydrolysate of shiitake mushroom residue until the volume fraction of ethanol reached 80%. After ethanol precipitation and standing for 12 hours, the precipitate was collected by centrifugation. The precipitate was dissolved in water, and the second supernatant was collected by centrifugation. The second supernatant was dialyzed in distilled water for 72 hours using a dialysis bag of size 5000 Daltons. The second supernatant after dialyzing was freeze-dried to obtain shiitake mushroom residue polysaccharide.

6. The preparation method according to claim 5, characterized in that, The sodium dihydrogen phosphate-acetic acid buffer solution has a sodium dihydrogen phosphate concentration of 0.2 mol / L, an acetic acid concentration of 0.05 mol / L, and a pH of 5.0; the weight-to-volume ratio of the precipitate to water is 1:20 (mg / mL).

7. The preparation method according to claim 5, characterized in that, The enzymes used in the enzymatic hydrolysis are complex enzymes; the complex enzymes are cellulase, glucanase and xylanase, and the enzyme dosages, calculated based on the added shiitake mushroom residue powder, are 1000U / g, 800U / g and 900U / g, respectively.

8. The preparation method according to claim 5, characterized in that, The enzymatic hydrolysis temperature is 50℃, and the enzymatic hydrolysis time is 4 hours.

9. The application of the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite as described in claim 1 or the shiitake mushroom residue polysaccharide-chitosan oligosaccharide nanocomposite prepared by any of the preparation methods described in claims 2 to 8 in the preparation of anti-inflammatory products.

10. The application according to claim 9, characterized in that, The products include pharmaceuticals.

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