Method for extracting micromolecular water-soluble plant polysaccharide

Abstract

The invention relates to an extraction method of micromolecular water-soluble plant polysaccharide, and belongs to the technical field of plant active ingredient extraction. The method comprises the following steps: (1) crushing plant raw materials, adding absolute ethyl alcohol for soaking and degreasing treatment, and filtering to obtain filter residues; (2) adding 8-20 times of normal-temperature water into the filter residues, then adding trifluoroacetic acid which accounts for 1.5-5% of the mass of the mixture and hydrofluoric acid which accounts for 0.001-0.005% of the mass of the mixture, uniformly mixing, and only degrading polysaccharide components in the filter residues once to obtain a small-molecular water-soluble polysaccharide crude extract; (3) carrying out vacuum rotary evaporation to remove organic acid, adding absolute ethyl alcohol into the liquid subjected to rotary evaporation and concentration for alcohol precipitation, and collecting the precipitate; and (4) performing deproteinization, chromatographic purification and freeze-drying treatment to obtain the micromolecular water-soluble plant polysaccharide. According to the method, more micromolecular water-soluble plant polysaccharides can be rapidly and efficiently prepared, and in addition, the micromolecular content of the obtained micromolecular water-soluble plant polysaccharides is higher.

Description

Technical Field

[0001] This application relates to the field of plant active ingredient extraction technology, and in particular to a method for extracting small molecule water-soluble plant polysaccharides. Background Technology

[0002] Polysaccharides are one of the main active and indicator components of plants, widely distributed in various parts and organs of plants. Essentially, a polysaccharide is a long-chain molecule composed of multiple monosaccharide molecules linked by glycosidic bonds. Water-soluble plant polysaccharides refer to polysaccharides extracted from plants that can dissolve in water.

[0003] The bioactivity of polysaccharides largely determines their utilization value. The constituent components, structural mode, and spatial conformation of polysaccharides, their molecular weight and distribution range, and their water solubility are the main factors influencing their bioactivity. Numerous studies have shown that the molecular weight of active polysaccharides is a necessary condition for their biological activity. Larger molecular weights result in larger apparent molecular volumes, which hinders the polysaccharides from crossing multiple cell membrane barriers to enter the organism and exert their biological activities. Simultaneously, the larger molecular weight of polysaccharides is accompanied by poorer water solubility, which is another important condition for them to exert their biological activity.

[0004] For the extraction of water-soluble plant polysaccharides, the conventional technique in existing technologies involves extraction with water followed by precipitation with alcohols. For the extraction of small-molecule water-soluble plant polysaccharides, acid degradation is used to obtain polysaccharides with lower molecular weights. Furthermore, to obtain even lower molecular weight polysaccharides with specific molecular weights, the conventional technique involves further ultrafiltration fractionation of the solution obtained after preliminary acid degradation using ultrafiltration centrifuge tubes with specific molecular weights. While this method can obtain small-molecule polysaccharides with a certain molecular weight distribution, its disadvantages include sample loss due to polysaccharide adsorption in the ultrafiltration centrifuge tubes, and the formation of a gel layer on the membrane surface after polysaccharide concentration, which hinders further filtration of small molecules. This necessitates frequent pauses in centrifugation to clean the membrane, extending the operation time. Additionally, most ultrafiltration tubes are not recommended for reuse, as this degrades membrane performance (e.g., pore size deformation, increased adsorption), and high-throughput experiments are costly, with a single 30 kDa tube costing approximately 100-200 yuan.

[0005] The pursuit of efficiently and cost-effectively reducing the molecular weight of water-soluble plant polysaccharides has always been a goal of those skilled in the art. Summary of the Invention

[0006] To address or partially address the problems existing in related technologies, this application provides a method for extracting small molecule water-soluble plant polysaccharides, which can rapidly and efficiently prepare more small molecule water-soluble plant polysaccharides. Furthermore, the obtained small molecule water-soluble plant polysaccharides have a higher proportion of small molecules.

[0007] This application discloses a method for extracting small-molecule water-soluble plant polysaccharides, comprising the following steps: (1) After crushing the plant raw materials, add anhydrous ethanol for soaking and degreasing treatment, and filter to obtain filter residue; (2) Add 8-20 times the amount of water at room temperature to the filter residue, then add 1.5%-5% trifluoroacetic acid and 0.001-0.005% hydrofluoric acid, respectively, and mix well. The polysaccharide components in the filter residue are degraded only once to obtain a crude extract of small molecule water-soluble polysaccharides. (3) The organic acid was removed by vacuum rotary evaporation of the crude extract of small molecule water-soluble polysaccharide to obtain the concentrated liquid after rotary evaporation. Anhydrous ethanol was added to the concentrated liquid for alcohol precipitation and the precipitate was collected. (4) The obtained small molecule water-soluble plant polysaccharide is subjected to deproteinization, chromatography purification and freeze drying to obtain small molecule water-soluble plant polysaccharide.

[0008] Furthermore, the plant material is Panax notoginseng root, Dendrobium officinale, or Astragalus membranaceus.

[0009] Furthermore, the feature is that in step (2), the polysaccharide components in the filter residue are degraded once for 1-3 hours.

[0010] Further, in step (3), anhydrous ethanol is added until the final ethanol concentration is 40%-60% for alcohol precipitation.

[0011] Furthermore, in step (4), enzymatic hydrolysis combined with the Sevag method is used for deproteinization.

[0012] Furthermore, in step (4), DEAE-Sepharose Fast Flow anion exchange chromatography is used, with gradient elution using PBS at pH 7.6.

[0013] The beneficial effects of this application are: 1. In the process of dissolving polysaccharide-containing filter residue in water, this application innovatively adds a low concentration of hydrofluoric acid in addition to trifluoroacetic acid. The presence of a low concentration of hydrofluoric acid can promote the dissolution of polysaccharides and improve their water solubility, thereby resulting in a higher polysaccharide content in the final product, small-molecule water-soluble plant polysaccharides. On the other hand, in the process of degrading macromolecular polysaccharides into small-molecule polysaccharides by trifluoroacetic acid, the presence of a small amount of hydrofluoric acid also makes the acid hydrolysis process of macromolecular polysaccharides more efficient, and the reaction can be fully carried out at room temperature without the need for boiling water.

[0014] 2. Compared to existing technologies that only add trifluoroacetic acid to hydrolyze polysaccharides, the method of adding a lower concentration of hydrofluoric acid is more efficient. It achieves the hydrolysis of large-molecule polysaccharides into small-molecule polysaccharides in a single step, while also resulting in a narrower molecular weight distribution of the obtained small-molecule polysaccharides. The method in this application requires no ultrafiltration centrifuge tubes; it only requires a single step of acid hydrolysis to obtain small-molecule water-soluble plant polysaccharides, making it highly efficient, simple to operate, and low-cost.

[0015] 3. The introduction of a low concentration of fluoride ions in this application can, on the one hand, avoid uncontrollable fluorination modification of polysaccharides, and on the other hand, the fluoride ions can be fully removed by calcium salt precipitation. Detailed Implementation

[0016] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art. Example 1

[0017] This embodiment describes a method for extracting small-molecule water-soluble plant polysaccharides, comprising the following steps: (1) After the dried Panax notoginseng root is crushed, it is soaked in anhydrous ethanol for degreasing treatment, and then filtered to obtain the filter residue; (2) Add 10 times the amount of water at room temperature to the filter residue, then add 1.5% trifluoroacetic acid and 0.005% hydrofluoric acid by mass of the mixture, mix well, and degrade the polysaccharide components in the filter residue for 2 hours to obtain a crude extract of small molecule water-soluble polysaccharides. (3) The obtained crude extract of small molecule water-soluble polysaccharide was vacuum rotary evaporated to remove organic acids, and the concentrated liquid after rotary evaporation was obtained. Anhydrous ethanol was added to the concentrated liquid after rotary evaporation until the final ethanol concentration was 40% for alcohol precipitation, and the precipitate was collected. Fluoride ions were removed from the liquid after alcohol precipitation by adding calcium salt, a conventional defluorinating agent. The fluoride ion content in the defluorinated liquid obtained after calcium salt defluorination was found to be less than 1.0 mg / L. (4) The obtained small molecule water-soluble plant polysaccharide is subjected to enzymatic hydrolysis and Sevag method combined with deproteinization, chromatography purification and freeze drying to obtain small molecule water-soluble plant polysaccharide.

[0018] The specific procedure for deproteinization using a combination of enzymatic hydrolysis and the Sevag method is as follows: First, prepare 1 mg / ml streptoprotein and papain solutions separately, mix them in equal volumes, and set aside. Then, dissolve the small-molecule water-soluble crude plant polysaccharide in water to prepare a sample solution. Mix the above protease solution and sample solution at a volume ratio of 1:100, incubate at 37℃ for 2 hours, then add Sevag solution (chloroform:isoamyl alcohol = 4:1) at a volume ratio of 1:1, shake repeatedly, and let stand for 24 hours. Retain the aqueous phase, and deproteinize three times using the Sevag method. The aqueous phase is then precipitated again with ethanol, centrifuged after standing, washed with ethanol and acetone, and dried to obtain the deproteinized polysaccharide.

[0019] The specific chromatographic purification process is as follows: DEAE-Sepharose Fast Flow anion exchange chromatography was used, with gradient elution using PBS at pH 7.6. Example 2

[0020] This embodiment describes a method for extracting small-molecule water-soluble plant polysaccharides, comprising the following steps: (1) After the dried Panax notoginseng root is crushed, it is soaked in anhydrous ethanol for degreasing treatment, and then filtered to obtain the filter residue; (2) Add 15 times the amount of water at room temperature to the filter residue, then add 2% trifluoroacetic acid and 0.003% hydrofluoric acid by mass of the mixture, mix well, and degrade the polysaccharide components in the filter residue for 0.5 h to obtain a crude extract of small molecule water-soluble polysaccharides. (3) The organic acid was removed by vacuum rotary evaporation of the crude extract of small molecule water-soluble polysaccharide to obtain the concentrated liquid after rotary evaporation. Anhydrous ethanol was added to the concentrated liquid after rotary evaporation until the final ethanol concentration was 50% for alcohol precipitation and the precipitate was collected. (4) The obtained small molecule water-soluble plant polysaccharide is subjected to enzymatic hydrolysis and Sevag method combined with deproteinization, chromatography purification and freeze drying to obtain small molecule water-soluble plant polysaccharide. Example 3

[0021] This embodiment describes a method for extracting small-molecule water-soluble plant polysaccharides, comprising the following steps: (1) After the dried Panax notoginseng root is crushed, it is soaked in anhydrous ethanol for degreasing treatment, and then filtered to obtain the filter residue; (2) Add 10 times the amount of water at room temperature to the filter residue, then add 5% trifluoroacetic acid and 0.001% hydrofluoric acid by mass of the mixture, mix well, and degrade the polysaccharide components in the filter residue for 1 hour to obtain a crude extract of small molecule water-soluble polysaccharides. (3) The organic acid was removed by vacuum rotary evaporation of the crude extract of small molecule water-soluble polysaccharide to obtain the concentrated liquid after rotary evaporation. Anhydrous ethanol was added to the concentrated liquid after rotary evaporation until the final ethanol concentration was 60% for alcohol precipitation and the precipitate was collected. (4) The obtained small molecule water-soluble plant polysaccharide is subjected to enzymatic hydrolysis and Sevag method combined with deproteinization, chromatography purification and freeze drying to obtain small molecule water-soluble plant polysaccharide. Example 4

[0022] The extraction method in this embodiment differs from that in Example 3 in that, in step (2), 5% of the mass of the mixture is added with trifluoroacetic acid, but hydrofluoric acid is not added. All other steps are the same as in Example 1. Example 5

[0023] The extraction method in this embodiment differs from that in Example 1 in that water at 90°C is added in step (2), while the rest is the same as in Example 1. Example 6

[0024] The extraction method in this embodiment differs from that in Example 3 in that hydrofluoric acid of 0.006% by mass of the mixture is added in step (2), and trifluoroacetic acid is not added. All other steps are the same as in Example 3.

[0025] Experimental results determination 1. The polysaccharide content of the final products obtained in Examples 1-6 was determined. Specifically, the sulfuric acid-phenol method is used for determination, and the test method is as follows: ① Preparation of standard solution: Prepare a 0.1 mg / ml glucose standard solution by successively pipetting 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 mL into test tubes, adding ddH2O to each test tube to make up to 2 mL, and set aside for use.

[0026] ② Sample solution preparation: Accurately weigh the sample powder and add ddH2O to prepare a 0.1 mg / mL sample solution for testing. 5% phenol solution preparation: Accurately weigh 2.5 g of phenol solid powder, add 47.5 mL of ddH2O, dissolve at 75℃, and store in the dark.

[0027] ③ Reaction of standard solution and sample solution: 1 mL of the prepared standard solution and sample solution were sequentially added to test tubes, followed by 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution. The mixture was gently shaken, with ddH2O as a blank control. The absorbance was measured at 490 nm using an ELISA reader. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. The absorbance of the sample solution was substituted into the standard curve for calculation. The polysaccharide content determination results are shown in Table 1.

[0028] Table 1

[0029] As shown in Table 1, the extract obtained by the preparation method described in this invention has a higher polysaccharide content, significantly higher than the group that only underwent acid hydrolysis with trifluoroacetic acid. This result indicates that the increase in polysaccharide content is closely related to the introduction of hydrofluoric acid. Furthermore, the results of Example 6 also show that relying solely on the addition of hydrofluoric acid and preparing small molecule polysaccharides at room temperature resulted in a low polysaccharide content in the product, presumably related to insufficient dissolution of the polysaccharides.

[0030] 2. The molecular weight distribution of the final products obtained in Examples 1-5 was determined, and the results are shown below: Molecular weight distribution of the product obtained in Example 1

[0031] Molecular weight distribution of the product obtained in Example 2

[0032] Molecular weight distribution of the product obtained in Example 3

[0033] Molecular weight distribution of the product obtained in Example 4

[0034] As can be seen from the above determination of molecular weight distribution, the extract obtained by the preparation method of the present invention has a small molecule content (molecular weight less than 1115 Da) of more than 95%, which is significantly better than the prior art example that only uses trifluoroacetic acid for acid hydrolysis.

[0035] 3. Fluorine content detection Fluorine content was tested on the polysaccharide products obtained in Examples 1-4 and Example 6. It was found that the fluorine content in Examples 1-3, with the addition of hydrofluoric acid, was consistent with the result of Example 4 without the addition of hydrofluoric acid, both being below 1.0 mg / kg, meeting the requirements of the relevant regulations in the Limits for Contaminants in Food (GB2762-2005). However, in Example 6, due to the addition of a relatively high concentration of hydrofluoric acid, the fluorine content of the resulting polysaccharide product exceeded 1.0 mg / kg, which is presumably related to the fluorination modification of the polysaccharide with fluorine that occurred in this example. Considering the safety issues when the prepared small molecule polysaccharides are used as food or health products, the amount of hydrofluoric acid added in this application is determined to be 0.001-0.005%.

[0036] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for extracting a small water-soluble plant polysaccharide, characterized by, The method comprises the following steps: (1) crushing the plant raw material and then adding anhydrous ethanol to soak and defat the plant raw material, and filtering to obtain filter residue; (2) adding 8-20 times of water at room temperature to the filter residue, and then adding 1.5-5% of trifluoroacetic acid and 0.001-0.005% of hydrofluoric acid, and mixing, and only degrading the polysaccharide component in the filter residue once to obtain a small-molecule water-soluble polysaccharide crude extract; (3) removing the organic acid from the small-molecule water-soluble polysaccharide crude extract by vacuum rotary evaporation to obtain a rotary evaporation concentrated solution, and then adding anhydrous ethanol to the rotary evaporation concentrated solution to perform alcohol precipitation, and collecting the precipitate; (4) sequentially performing deproteinization, chromatography purification, and freeze-drying treatment on the obtained small-molecule water-soluble plant polysaccharide to obtain a small-molecule water-soluble plant polysaccharide.

2. The method of claim 1, wherein the small molecule water-soluble plant polysaccharide is extracted from the plant material by the following steps: The plant raw material is the root of Panax notoginseng, Dendrobium officinale, or Astragalus membranaceus. ​ 3. The method of claim 1, wherein the small molecule water-soluble plant polysaccharide is extracted by the following steps: The step (2) degrades the polysaccharide component in the filter residue for 1-3 hours. ​ 4. The method of claim 1, wherein the small molecule water-soluble plant polysaccharide is extracted from the plant material by the following steps: The step (3) adds anhydrous ethanol to an ethanol final concentration of 40%-60% to perform alcohol precipitation. ​ 5. The method of claim 1, wherein the small molecule water-soluble plant polysaccharide is extracted by the following steps of: The step (4) adopts enzymatic hydrolysis and the Sevag method to perform deproteinization. ​ 6. The method of claim 1, wherein the small molecule water-soluble plant polysaccharide is extracted from the plant material by the following steps: The step (4) adopts DEAE-Sepharose Fast Flow anion exchange chromatography and gradient elution with PBS at pH 7.

6. ​

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