Extraction method of polysaccharide

Abstract

The invention discloses an extraction method of polygonatum polysaccharide, and belongs to the technical field of natural product extraction. The method comprises the steps of raw material pretreatment, squeezing extraction, standing centrifugation, macroporous resin adsorption separation, nanofiltration membrane grading purification, Sevage deproteinization, neutral alumina decoloration, freeze drying and the like. The method is mild in process condition and simple and convenient to operate, polysaccharide degradation can be effectively reduced through squeezing and low-temperature separation technologies, and the purity and yield of the polysaccharide are remarkably improved by combining resin separation and nanofiltration membrane technologies. Through detection, the purity of the obtained polysaccharide can reach 90% or above, the protein content is lower than 1%, and the method is suitable for polysaccharide preparation of plant rhizome raw materials and the fields of medicines, food or health care products.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and purification technology, specifically a method for extracting polysaccharides. Background Technology

[0002] Polysaccharides are natural macromolecules widely found in plants, fungi, and algae, possessing various biological activities such as immunomodulation, antioxidation, antitumor, and anti-fatigue. Current techniques for polysaccharide extraction typically employ hot water extraction or enzymatic methods, but these suffer from low extraction efficiency, poor purity, numerous impurities, and complex procedures. Therefore, there is an urgent need for a simple, efficient, and stable method to obtain high-purity polysaccharides. Summary of the Invention

[0003] The purpose of this invention is to provide a simple, high-purity, and low-protein polysaccharide extraction method, overcoming the shortcomings of unstable polysaccharide yield and incomplete impurity removal in the prior art.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for extracting polysaccharides, comprising the following steps: (1) Raw material pretreatment: Take 1 kg of fresh European Polygonatum rhizome, remove mud and fibrous roots, wash and cut into pieces of about 1 cm. Put the sample into a mechanical pressing device and press to obtain crude extract. Wash the residue three times with 300 mL of purified water until the residue is no longer slippery.

[0005] (2) Centrifugation: Combine the pressed liquids and let them stand at 0-10 ℃ for 12-24 hours. Set the centrifugation speed to 11,000 rpm and the time to 15 minutes to remove suspended impurities and obtain a clear supernatant.

[0006] (3) Macroporous resin adsorption separation: Weigh 200 g of AB-8 type macroporous adsorption resin, soak it in 95% ethanol for 24 hours, and then pack it into a column. Equilibrate the column bed with deionized water. Load the polysaccharide extract onto the column, and after static adsorption for 8 hours, elute with a gradient of deionized water and ethanol. The ethanol gradient concentration is 0-50%. Collect the water wash and low-concentration ethanol eluent, concentrate it, and use it for later use.

[0007] (4) Nanofiltration membrane fractionation purification: A polyamide nanofiltration membrane system with a membrane area of ​​0.3 m² and a molecular weight cutoff range of 300–10000 Da was used. The system operating pressure was controlled at 0.2–0.4 MPa and the temperature at 25 ℃. The eluent was fractionated according to molecular weight and concentrated to obtain a high-purity polysaccharide fraction.

[0008] (5) Sevage method for protein removal: Add Sevage reagent (chloroform: n-butanol = 4:1) to the obtained polysaccharide solution at a volume ratio of 3:1, shake to mix, let stand to separate the layers, and remove the lower organic phase. Repeat the operation twice to completely remove protein impurities.

[0009] (6) Decolorization and drying: Decolorization was performed using neutral alumina with a column particle size of 100 mesh and a flow rate controlled at 1-2 mL / min. The eluent was collected and freeze-dried to obtain a light white polysaccharide powder with a purity ≥90% and a protein content ≤1%.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The yield of European Polygonatum polysaccharides extracted by this method was 1.12%, with a polysaccharide purity of 93.5% and a protein content of 0.82%. The results indicate that the method described in this invention can achieve efficient extraction of high-purity polysaccharides under relatively mild conditions, and the resulting product has low impurity content, making it suitable for subsequent structural characterization and pharmacological studies. Attached Figure Description

[0011] Figure 1 shows the high-performance gel permeation chromatography (HPGPC) chromatogram of PMP. (a. crude polysaccharide; b. purified polysaccharide (PMP2)); Figure 2 The total ion chromatogram of PMP2 derivatives; Figure 3 The mass spectrum of the PMP2 derivative is shown in the (secondary) mass spectrum. Figure 4 The NMR spectra are as follows: (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum; (c) HSQC NMR spectrum; (d) COSY NMR spectrum; (e) HMBC NMR spectrum; (f) NOESY NMR spectrum. Figure 5 It is a PMP2 structure; Figure 6 The results of scanning electron microscopy (SEM) of PMP2 are shown. (a) × 400x; (b) × 2000x. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Example 1 The extraction of polysaccharides from European Polygonatum includes the following steps: (1) Raw material pretreatment: Take 1 kg of fresh European Polygonatum rhizome, remove mud and fibrous roots, wash and cut into pieces of about 1 cm. Put the sample into a mechanical pressing device and press to obtain crude extract. Wash the residue three times with 300 mL of purified water until the residue is no longer slippery.

[0014] (2) Centrifugation: Combine the pressed liquids and let them stand at 4-5 ℃ for 24 hours. The centrifugation speed is set to 11,000 rpm and the time is 15 minutes to remove suspended impurities and obtain a clear supernatant.

[0015] (3) Macroporous resin adsorption separation: Weigh 200 g of AB-8 type macroporous adsorption resin, soak it in 95% ethanol for 24 hours and then pack it into a column. Use deionized water and equilibrate the column bed. Load the polysaccharide extract onto the column. After static adsorption for 8 hours, elute with a gradient of deionized water and 30% ethanol. Collect the water wash and low concentration ethanol eluent, concentrate it and use it for later use.

[0016] (4) Nanofiltration membrane fractionation purification: A polyamide nanofiltration membrane system with a membrane area of ​​0.3 m² and a molecular weight cutoff range of 300–5000 Da was used. The system operating pressure was controlled at 0.3 MPa and the temperature at 25 ℃. The eluent was fractionated according to molecular weight and concentrated to obtain a high-purity polysaccharide fraction.

[0017] (5) Sevage method for protein removal: Add Sevage reagent (chloroform: n-butanol = 4:1) to the obtained polysaccharide solution at a volume ratio of 3:1, shake to mix, let stand to separate the layers, and remove the lower organic phase. Repeat the operation twice to completely remove protein impurities.

[0018] (6) Decolorization and drying: Neutral alumina was used for decolorization. The column particle size was 100 mesh, and the flow rate was controlled at 1-2 mL / min. The eluent was collected and freeze-dried to obtain a light white polysaccharide powder.

[0019] Example 2 The extraction of polysaccharides from European Polygonatum includes the following steps: (1) Raw material pretreatment: Take 500 g of fresh European Polygonatum rhizome, remove mud and fibrous roots, wash and cut into pieces of about 1 cm. Put the sample into a mechanical pressing device and press to obtain crude extract. Wash the residue three times with 150 mL of purified water until the residue is no longer slippery.

[0020] (2) Centrifugation: Combine the pressed liquids and let them stand at 4-5 ℃ for 24 hours. The centrifugation speed is set to 11,000 rpm and the time is 15 minutes to remove suspended impurities and obtain a clear supernatant.

[0021] (3) Macroporous resin adsorption separation: Weigh 100 g of AB-8 type macroporous adsorption resin, soak it in 95% ethanol for 24 hours and then pack it into a column. Use deionized water and equilibrate the column bed. Load the polysaccharide extract onto the column, and after static adsorption for 8 hours, elute with a gradient of deionized water and 30% ethanol. Collect the water wash and low concentration ethanol eluent, concentrate it and use it for later use.

[0022] (4) Nanofiltration membrane fractionation purification: A polyamide nanofiltration membrane system with a membrane area of ​​0.3 m² and a molecular weight cutoff range of 300–5000 Da was used. The system operating pressure was controlled at 0.3 MPa and the temperature at 25 ℃. The eluent was fractionated according to molecular weight and concentrated to obtain a high-purity polysaccharide fraction.

[0023] (5) Sevage method for protein removal: Add Sevage reagent (chloroform: n-butanol = 4:1) to the obtained polysaccharide solution at a volume ratio of 3:1, shake to mix, let stand to separate the layers, and remove the lower organic phase. Repeat the operation twice to completely remove protein impurities.

[0024] (6) Decolorization and drying: Neutral alumina was used for decolorization. The column particle size was 100 mesh, and the flow rate was controlled at 1-2 mL / min. The eluent was collected and freeze-dried to obtain a light white polysaccharide powder.

[0025] Example 3 The extraction of polysaccharides from European Polygonatum includes the following steps: (1) Raw material pretreatment: Take 2 kg of fresh European Polygonatum rhizome, remove mud and fibrous roots, wash and cut into pieces of about 1 cm. Put the sample into a mechanical pressing device and press to obtain crude extract. Wash the residue with 500 mL of purified water 4 times until the residue is no longer slippery.

[0026] (2) Centrifugation: Combine the pressed liquids and let them stand at 4-5 ℃ for 24 hours. The centrifugation speed is set to 11,000 rpm and the time is 15 minutes to remove suspended impurities and obtain a clear supernatant.

[0027] (3) Macroporous resin adsorption separation: Weigh 400 g of AB-8 type macroporous adsorption resin, soak it in 95% ethanol for 24 hours and then pack it into a column. Use deionized water and equilibrate the column bed. Load the polysaccharide extract onto the column, and after static adsorption for 8 hours, elute with a gradient of deionized water and 30% ethanol. Collect the water wash and low concentration ethanol eluent, concentrate it and use it for later use.

[0028] (4) Nanofiltration membrane fractionation purification: A polyamide nanofiltration membrane system with a membrane area of ​​0.3 m² and a molecular weight cutoff range of 300–5000 Da was used. The system operating pressure was controlled at 0.3 MPa and the temperature at 25 ℃. The eluent was fractionated according to molecular weight and concentrated to obtain a high-purity polysaccharide fraction.

[0029] (5) Sevage method for protein removal: Add Sevage reagent (chloroform: n-butanol = 4:1) to the obtained polysaccharide solution at a volume ratio of 3:1, shake to mix, let stand to separate the layers, and remove the lower organic phase. Repeat the operation twice to completely remove protein impurities.

[0030] (6) Decolorization and drying: Decolorization was performed using neutral alumina with a column particle size of 100 mesh and a flow rate controlled at 1-2 mL / min. The eluent was collected and freeze-dried to obtain a light white polysaccharide powder.

[0031] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0032] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting polysaccharides, characterized in that: Includes the following steps: (1) Raw material pretreatment; (2) Mechanical pressing extraction followed by low-temperature static centrifugation; (3) Separation and elution via macroporous adsorption resin; (4) Purification by nanofiltration membrane fractionation; (5) Use Sevage reagent to remove proteins; (6) Decolorization using a neutral alumina column; (7) High-purity polysaccharides were obtained by freeze drying.

2. The method for extracting polysaccharides according to claim 1, characterized in that: The temperature after pressing and extraction is 0–10 ℃, and the settling time is 12–24 hours.

3. The method for extracting polysaccharides according to claim 1, characterized in that: The macroporous adsorption resin is of type AB-8, and the ethanol gradient concentration is 0–50%.

4. The method for extracting polysaccharides according to claim 1, characterized in that: The nanofiltration membrane has a molecular weight cutoff of 300–10000 Da and a system pressure of 0.2–0.4 MPa.

5. The method for extracting polysaccharides according to claim 1, characterized in that: The volume ratio of chloroform to n-butanol in the Sevage reagent is 4:

1.

6. The method for extracting polysaccharides according to claim 1, characterized in that: The decolorization step uses neutral alumina, and the elution flow rate is 1–2 mL / min.

7. A method for extracting polysaccharides according to any one of claims 1-6, characterized in that: The obtained polysaccharide purity is ≥90%, and the protein content is ≤1%.

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