A method for separating and purifying low oligosaccharides of rhizoma polygonati

Abstract

The application discloses a method for separating and purifying low-molecular-weight polysaccharides from Polygonatum sibiricum. The method comprises the following steps: mixing the polysaccharides with fillers and / or filter aids, and then separating and purifying the polysaccharides. The fillers comprise Sephadex G-15 (cross-linked glucose gel), DEAE-Sepharose CL-4B (agarose gel) and Bio-Gel P-2 (polyacrylamide gel), and the filter aids comprise diatomite, perlite, cellulose, activated carbon and red clay. The method can significantly improve the yield of the low-molecular-weight polysaccharides with a molecular weight of less than 2 KDa, and the effect of the combination of the fillers and the filter aids is the best. The addition of the filter aids can avoid the blockage of the pores of the fillers in the middle and late stages of elution, improve the elution performance of the fillers on the polysaccharides and accelerate the elution and purification speed of the low-molecular-weight polysaccharides. The experiments show that the low-molecular-weight polysaccharides can significantly enhance the proliferation activity and phagocytosis of macrophages, promote the secretion of cytokines such as TNF-alpha, IL-6 and NO by RAW264.7 cells and improve the immune activity of the cells. The method has the characteristics of convenience, high efficiency and low cost and has high application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of isolation and purification of traditional Chinese medicine polysaccharides, and particularly relates to a method for isolating and purifying low molecular weight polysaccharides from Polygonatum sibiricum. BACKGROUND

[0002] Polygonatum sibiricum is a dry rhizome of Polygonatum sibiricum, P. sibiricum or P. cyathopetalum. According to the different morphologies, it is usually referred to as large Polygonatum sibiricum, chicken head Polygonatum sibiricum or ginger-shaped Polygonatum sibiricum. It has a sweet taste and a flat nature, and has the effects of tonifying qi and nourishing yin. The raw product contains a large amount of mucilage and other components, which may cause tongue numbness and throat discomfort. However, the processed product eliminates this numb tongue feeling. Polygonatum sibiricum is rich in polysaccharides, flavonoids, triterpenoid saponins, alkaloids, anthraquinones, volatile oils, lignans and amino acids. Polygonatum sibiricum polysaccharide is one of the main components of Polygonatum sibiricum, and has significant biological activities, including immunomodulation, antitumor, antioxidant, antiviral, myocardial protection and antihypertensive pharmacological functions.

[0003] Compared with high molecular weight polysaccharides, low molecular weight polysaccharides have good water solubility and are more easily absorbed by the human body. After high-temperature steaming with wine, the polysaccharides in Polygonatum sibiricum are hydrolyzed into low molecular weight polysaccharides, and the content of low molecular weight polysaccharides is increased. Pharmacological experiments show that low molecular weight polysaccharides with a molecular weight of less than 2KDa have better immunological effects and are the main immunologically active components. Therefore, the development of low molecular weight polysaccharides from Polygonatum sibiricum has broad application prospects.

[0004] Isolation and purification is a key step in the preparation of low molecular weight polysaccharides from Polygonatum sibiricum. Currently, ion exchange chromatography based on ion exchange principle and gel chromatography separation based on molecular sieve principle are commonly used for isolation and purification. However, these traditional methods for isolation and purification have slow separation speed and low yield, which limits the application and development of low molecular weight polysaccharides from Polygonatum sibiricum.

[0005] Filter aids are usually some hard powdery or fibrous solids that can promote the formation of porous and stable filter cakes, thereby reducing the resistance of filter cakes or filter media, improving the filtration rate and the clarity of filtrate. Commonly used filter aids include diatomite, perlite and cellulose, etc. Filter aids and the like have a large specific surface area, can adsorb colloidal substances, and form a rigid and porous skeleton, reducing the compression between the fillers during elution, allowing the fillers to have more and larger pores, thereby reducing the specific resistance of the polysaccharide elution process, avoiding the blockage of the pores of the fillers in the middle and late stages of elution, increasing the elution rate and yield, and improving the elution performance of the fillers for polysaccharides.

[0006] Therefore, the present application develops a high-efficiency and low-cost separation and purification technology to optimize the existing separation process, improve the separation efficiency of low molecular weight polysaccharides, increase the yield and purity of low molecular weight polysaccharides, and meet the urgent needs of scientific research and industrial applications. SUMMARY

[0007] The present application aims to provide a method for separating and purifying low molecular weight polysaccharides from Rhizoma Polygonati.

[0008] Specifically, the present application provides a method for separating and purifying low molecular weight polysaccharides from Rhizoma Polygonati, which comprises the following steps: (1) mixing the polysaccharides with a filler; and (2) purifying the mixture.

[0009] The present application also provides another method for separating and purifying low molecular weight polysaccharides from Rhizoma Polygonati, which comprises the following steps: (1) mixing the polysaccharides with a filter aid; and (2) purifying the mixture.

[0010] As a further improvement, the present application provides a method for separating and purifying low molecular weight polysaccharides from Rhizoma Polygonati, which comprises the following steps: (1) mixing the polysaccharides with a filler; (2) mixing the mixture with a filter aid; and (3) purifying the mixture.

[0011] As a further improvement, the present application provides a method for separating and purifying low molecular weight polysaccharides from Rhizoma Polygonati, which comprises the following steps:

[0012] (1) extraction: adding Rhizoma Polygonati pieces into 8 times the weight of water at 100℃ and refluxing for 3 hours, repeating the extraction for 2 times, and combining the water extracts;

[0013] (2) alcohol precipitation: concentrating the water extract, adding anhydrous ethanol to reach an alcohol content of 80%, standing overnight, centrifuging, filtering, dissolving the precipitate in water to reach a concentration of about 0.5g / mL, and adding anhydrous ethanol to reach an alcohol content of 80%, standing overnight, centrifuging, and filtering to obtain the crude polysaccharides from Rhizoma Polygonati;

[0014] (3) Savage method for removing protein: dissolving the crude polysaccharides from Rhizoma Polygonati in water, adding the polysaccharide solution, chloroform, and n-butanol in a volume ratio of 25:5:1 into a separatory funnel, shaking vigorously to mix uniformly and denature the protein into a gel, standing to separate the layers, and obtaining the polysaccharide solution from the uppermost water layer, repeating the process for 5 times;

[0015] (4) decolorization and impurity removal: A. macroporous resin pretreatment: soaking AB-8 macroporous resin in ultrapure water, washing away the fine resin particles with ultrapure water, soaking and washing the resin with anhydrous ethanol for multiple times until the addition of ultrapure water does not cause turbidity, and repeatedly washing with ultrapure water; B. decolorization and impurity removal: adsorbing and decolorizing the polysaccharide solution from step (3), loading the solution onto an AB-8 column, eluting and decolorizing with 3 times the column volume of ultrapure water, collecting the eluate, concentrating under reduced pressure, and freeze-drying to obtain the refined polysaccharides from Rhizoma Polygonati;

[0016] (6) Purification: The sample of 500 mg / mL polygonatum sibiricum polysaccharide was mixed with 3% column volume of Sephadex G-15, and then was loaded into a pretreated Sephadex G-15 column, and was eluted with pure water, and the eluent of the same peak position was collected.

[0017] The polygonatum sibiricum polysaccharide is selected from crude products and processed products, and the processing method of the processed products is one of steaming, wine processing, black bean processing and honey processing.

[0018] Beneficial effects

[0019] In view of the deficiencies of the existing separation and purification method of polygonatum sibiricum oligosaccharide, the polygonatum sibiricum polysaccharide is mixed with a filler and / or a filter aid for separation and purification, wherein the filler comprises Sephadex G-15, DEAE-Sepharose CL-4B and Bio-Gel P-2, the mixed volume is preferably 1%-5%, more preferably 3%, the filter aid comprises diatomite, perlite, cellulose, activated carbon and red clay, and the usage amount is preferably 0.5%-3% column volume, more preferably 2%. The method can significantly improve the yield of Mw<2KDa polygonatum sibiricum oligosaccharide, and the effect of the combination of the filler and the filter aid is best. The addition of the filter aid can avoid the blockage of the pores of the filler in the later stage of elution, improve the elution performance of the filler for polysaccharide, and accelerate the elution and purification speed of the polygonatum sibiricum oligosaccharide. Experiments show that the oligosaccharide can significantly enhance the proliferation activity and phagocytosis of macrophages, promote the secretion of TNF-α, IL-6 and NO and other cytokines by RAW264.7 cells, and improve the immune activity of cells. The method has the characteristics of convenience, high efficiency and low cost, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0020] The application will be further described below with reference to the drawings:

[0021] Figure 1 Cell activity determination results of different polysaccharide fractions of polygonatum sibiricum (n=3);

[0022] Figure 2 Macrophage phagocytosis activity determination results of different polysaccharide fractions of polygonatum sibiricum (n=3);

[0023] Figure 3 Immune factor content determination results of different polysaccharide fractions of polygonatum sibiricum (n=3);

[0024] Figures 1-3 *, compared with the blank, *p≤0.05, **p≤0.01, ***p≤0.001; #, compared with the Mw<2KDa polygonatum sibiricum polysaccharide group, #p≤0.05, ##p≤0.01, ###p≤0.001.

[0025] Figure 4Elution curve for direct sample loading of polygonatum sibiricum polysaccharide;

[0026] Figure 5 Elution curve for sample loading of polygonatum sibiricum polysaccharide mixed with 3% column volume of Sephadex G-15 packing material;

[0027] Figure 6 Elution curve for sample loading of polygonatum sibiricum polysaccharide mixed with 2% diatomite;

[0028] Figure 7 Elution curve for sample loading of polygonatum sibiricum polysaccharide mixed with 3% column volume of Sephadex G-15 packing material and 2% diatomite. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further illustrated below in combination with specific examples, which are not limitations to the technical solutions.

[0030] The biological materials in the examples are as follows: polygonatum sibiricum medicinal materials are purchased from Qingyang County, Chizhou City, Anhui Province.

[0031] Example 1: Processing of polygonatum sibiricum

[0032] Steaming method: 1 kg of polygonatum sibiricum is washed and then placed in a steamer to steam until it is brown-black and moist, and then taken out and dried into thick slices. This method can enhance the effects of polygonatum sibiricum in tonifying qi and blood, nourishing yin and moistening the lungs.

[0033] Wine processing method: 1 kg of polygonatum sibiricum medicinal materials is cleaned and then soaked in yellow rice wine for 10 h, steamed for 12-16 h, cut into thick slices, mixed with the steaming liquid, and dried at 60°C for 12 h to obtain polygonatum sibiricum decoction pieces. This method can increase the effect of polygonatum sibiricum in moistening the lungs.

[0034] Black bean processing method: 1 kg of polygonatum sibiricum is cooked with black beans, the beans are removed, and then dried and steamed until the inside and outside are moist black, and then taken out and dried into thick slices. This method can enhance the effect of polygonatum sibiricum in tonifying the kidney.

[0035] Honey roasting method: 1 kg of polygonatum sibiricum is cleaned and then mixed with honey and heated until the surface of polygonatum sibiricum is golden yellow, and then dried for use. This method can enhance the effect of polygonatum sibiricum in moistening the lungs.

[0036] Example 2: Extraction of polygonatum sibiricum polysaccharide and separation and purification of polygonatum sibiricum oligosaccharide

[0037] 1) Extraction: polygonatum sibiricum decoction pieces are selected from 1 kg of raw polygonatum sibiricum and 1 kg of polygonatum sibiricum processed products, wherein the processing method of the processed products is the wine processing method in the method of example 1, 8 times the weight of water of the decoction pieces is added, heated to 100°C, and refluxed, extracted for 2 times, each for 3 h, and the water extracts are combined;

[0038] 2) Alcohol precipitation: Concentrate the water extract in step 1), add anhydrous ethanol to reach 80% alcohol content, stand overnight, centrifuge, filter, and dissolve the precipitate in water (to a concentration of about 0.5 g / mL based on crude drug weight), continue to add anhydrous ethanol to reach 80% alcohol content, stand overnight, centrifuge, and filter to obtain the crude polysaccharide from Polygonatum as a precipitate;

[0039] 3) Protein removal by Savage method: Dissolve the crude polysaccharide from Polygonatum in step 2) in pure water, add the polysaccharide solution, chloroform, and n-butanol in a volume ratio of 25:5:1 to a separatory funnel, shake vigorously to mix uniformly, and denature the protein into a gel that precipitates, stand to separate the layers, and the uppermost aqueous layer is the polysaccharide, centrifuge, and collect the polysaccharide aqueous solution, repeat 5 times;

[0040] 4) Decolorization and impurity removal: A. Macroporous resin pretreatment: Soak AB-8 macroporous resin in ultrapure water, wash away the fine resin particles with pure water, and wash with anhydrous ethanol multiple times until the addition of ultrapure water does not cause turbidity, and then repeatedly wash with ultrapure water for standby use; B. Decolorization and impurity removal: Adsorb and decolorize the polysaccharide solution in step 3), load it onto an AB-8 column, elute and decolorize with 3 column volumes of pure water, collect the eluate, concentrate under reduced pressure, and freeze-dry to obtain refined polysaccharide from Polygonatum.

[0041] 5) Polysaccharide content determination:

[0042] ① Preparation of glucose standard curve: Accurately weigh 100 mg of dried and constant-weight glucose, and dilute with distilled water to 100 mL to obtain a 1 mg / mL glucose solution. Shake well, accurately pipette 10 mL of the solution, dilute with distilled water to 100 mL, and obtain a 100 μg / mL glucose standard solution.

[0043] Take 8 clean stoppered test tubes, and add 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the glucose standard solution, respectively, and make up to 2 mL with water. Then add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid to each tube, shake well, and stand for 5 min. Place in a 90°C water bath for 15 min, remove quickly, cool to room temperature, and measure the absorbance at 490 nm with tube 0 as the blank. Plot the standard curve with glucose concentration X as the abscissa (μg / mL) and absorbance Y as the ordinate.

[0044] ② Determination of refined polysaccharide content from Polygonatum: Dilute the refined polysaccharide from Polygonatum in step 4) by the appropriate factor, pipette 2 mL of the solution, and follow the standard curve procedure to measure the absorbance value. Substitute into the standard curve to calculate the polysaccharide content.

[0045] 6) Purification: The sample of 500 mg / mL of polygonatum sibiricum polysaccharide was directly loaded on a pre-treated Sephadex G-15 column, and eluted with pure water, and the eluate of the same peak position was collected.

[0046] Example 3: Comparison of the pharmacological effects of different molecular weight polysaccharides of polygonatum sibiricum

[0047] To compare the pharmacological effects of different molecular weight polysaccharides of polygonatum sibiricum, wine-processed polygonatum sibiricum was used as the raw material, and polysaccharides of different molecular weights were extracted.

[0048] (I) The preparation method of polysaccharides of different molecular weights of polygonatum sibiricum is as follows: the eluate of each polysaccharide of polygonatum sibiricum obtained in Example 2 was concentrated and placed in a dialysis bag with a molecular weight cut-off of 12 KDa, 7 KDa and 2 KDa, respectively, and dialyzed in purified water for 24 h, and the dialysate outside the bag was collected. Each dialysate was concentrated under reduced pressure, and then freeze-dried to obtain polysaccharides of different fractions of polygonatum sibiricum.

[0049] (II) Evaluation of pharmacological effects: The macrophage cell RAW264.7 was used as the cell model to investigate the cell viability, phagocytic activity and immune factor content determination: the specific method is as follows:

[0050] 1) Culture and grouping of RAW264.7 macrophages: The mouse macrophage RAW264.7 cell strain was resuscitated with RPMI Medium 1640 culture solution containing 10% fetal bovine serum, and cultured in a 37℃, 5% CO2 incubator. The cell growth condition was observed every day, and fresh culture medium was replaced in time, and the logarithmic phase cells were used for experiment. The mouse peritoneal macrophages were divided into blank group, positive drug group (lipopolysaccharide group) and drug administration group (Mw<2KDa polysaccharide of polygonatum sibiricum group, Mw2-7KDa polysaccharide of polygonatum sibiricum group, Mw7-12KDa polysaccharide of polygonatum sibiricum group, Mw>12KDa polysaccharide of polygonatum sibiricum group).

[0051] 2) Cell viability determination of polysaccharides of polygonatum sibiricum: The cell suspension of logarithmic growth phase of macrophage RAW264.7 was inoculated in a 96-well plate at a concentration of 2×10 4 cells / well. 100 μL of cell suspension was added to each well, and it was placed in a cell culture incubator. After washing the culture dish, the non-adherent cells were removed to obtain purified adherent macrophages. The blank control group was added with culture solution, the positive control group was added with culture solution and lipopolysaccharide, each 100 μL, and the drug administration group was added with culture solution and polysaccharide solution of polygonatum sibiricum of different fractions at a concentration of 250 μg / mL, each 100 μL. Each group had 3 replicate wells. After being placed in the incubator for 24 h, 1 / 10 of the volume of CCK-8 solution was added to each well, and the OD value was determined at 450 nm after 1 h. The cell viability was calculated according to the following formula: cell viability % = OD experimental group / OD blank group * 100%. The results are shown in Figures 1-3 .

[0052] Figure 1 Cell viability assay results of different polysaccharide fractions of Polygonatum sibiricum, in normal mouse macrophages, the proliferation activity of RAW264.7 macrophages of the administration group (different polysaccharide fractions of Polygonatum sibiricum) was significantly enhanced compared with the blank control group (p<0.001); among them, the effect of the polysaccharide fraction (Mw<2KDa) was the best, and there was a significant difference in the proliferation activity of macrophages between the polysaccharide fraction (Mw<2KDa) and the polysaccharide fraction (Mw2-7KDa) (p<0.01), the polysaccharide fraction (Mw7-12KDa) (p<0.001), and the polysaccharide fraction (Mw>12KDa) (p<0.001). It can be seen from the cell viability assay results of different molecular weight polysaccharides of Polygonatum sibiricum that the polysaccharide fraction (Mw<2KDa) has a stronger pharmacodynamic effect.

[0053] 3) Cell phagocytosis activity assay of uniform polysaccharide of Polygonatum sibiricum: logarithmic growth period macrophage RAW264.7 was inoculated in a 96-well plate at a concentration of 2×10 4 μL / well of PBS. Then 100 μL of ice acetic acid-absolute ethanol (V:V=1:1) cell lysis solution was added to each well, and it was statically incubated at room temperature overnight. The OD value was measured at a wavelength of 490 nm.

[0054] Figure 2 The results of detecting the phagocytosis activity of macrophages by neutral red staining method. In normal mouse macrophages, the polysaccharide fraction (Mw<2KDa) of Polygonatum sibiricum had a very significant effect on enhancing the phagocytosis activity of RAW264.7 macrophages compared with the blank control group (p<0.001); compared with other administration groups, the polysaccharide fraction (Mw<2KDa) had a very significant effect on enhancing the phagocytosis activity of RAW264.7 macrophages compared with the polysaccharide fraction (Mw2-7KDa) (p<0.01), the polysaccharide fraction (Mw7-12KDa) (p<0.001), and the polysaccharide fraction (Mw>12KDa) (p<0.001), which indicated that the oligosaccharide fraction (Mw<2KDa) of Polygonatum sibiricum could significantly increase the phagocytosis activity of RAW264.7 macrophages under normal conditions, and the effect was stronger than that of other molecular weight administration groups.

[0055] 4) Immune factor content determination: logarithmic growth period macrophage RAW264.7 was inoculated in a 96-well plate at a concentration of 2×10 5Cells were seeded at a concentration of [number] cells / well in 96-well plates and cultured in a 37°C, 5% CO2 incubator. The blank control group received 100 μL of complete culture medium, the positive control group received 100 μL of LPS solution (2 μg / mL), and the groups treated with Polygonatum sibiricum polysaccharide had a concentration of 250 μg / mL. Each sample was tested in triplicate. Cells were incubated at 37°C for 24 h, and the supernatant was collected, centrifuged, and stored at -20°C. TNF-α, IL-6, and NO levels in the cell supernatant were detected according to the kit instructions.

[0056] Figure 3 The results show the content of immune factors in different polysaccharide fractions of Polygonatum odoratum. Compared with the control group, both the Polygonatum odoratum polysaccharide (Mw<2KDa) fraction (p<0.001) and the Polygonatum odoratum polysaccharide (Mw2-7KDa) fraction promoted the release of IL-6, TNF-α, and NO from RAW264.7 cells. Compared with other treatment groups, the Polygonatum odoratum polysaccharide (Mw<2KDa) fraction had a more significant effect on promoting the release of IL-6, TNF-α, and NO from RAW264.7 cells. With the increase of cytokine secretion, the phagocytic capacity of macrophages is enhanced, and the body's disease resistance is also enhanced. This indicates that Polygonatum odoratum oligosaccharides with Mw<2KDa can activate macrophages and thus exert an immune-active effect.

[0057] In summary, the oligosaccharide fraction of Polygonatum sibiricum with Mw < 2 kDa can significantly enhance the proliferative activity of macrophages, enhance their phagocytic function, promote the secretion of cytokines such as TNF-α, IL-6, and NO by RAW264.7 cells, and improve cellular immune activity.

[0058] Example 4: Purification of oligosaccharides from Polygonatum sibiricum with Mw < 2 kDa

[0059] The Polygonatum polysaccharide sample with a concentration of 500 mg / mL in Example 2 was separated and purified using the following methods:

[0060] 1) Direct sample loading method (traditional method): The sample is directly loaded into the pretreated packed column, eluted with pure water, and the eluent is collected.

[0061] 2) polysaccharide mixed with filler before loading (innovative method): polysaccharide was mixed with 1%~5% column volume of filler before loading into the pretreated filler column, and eluted with purified water, and the eluate was collected; wherein the filler comprises Sephadex G-15, DEAE-Sepharose CL-4B, Bio-Gel P-2. The eluate of each elution peak after purification was combined, concentrated and placed in a dialysis bag with a relative molecular mass of 2KDa, and dialyzed in purified water for 24h. The dialysate outside the bag was collected, concentrated under reduced pressure, and freeze-dried to obtain Mw<2KDa polysaccharide fraction. The glucose standard curve was redrawn according to the method in step 5) of Example 2, and the absorbance of rhizoma polygonati oligosaccharide was measured to calculate the oligosaccharide content. The yield of Mw<2KDa rhizoma polygonati oligosaccharide was calculated according to the formula: yield (%) = (rhizoma polygonati oligosaccharide content * total mass of oligosaccharide) / (rhizoma polygonati refined polysaccharide content * total mass of loaded refined polysaccharide) * 100%.

[0062] The yield of Mw<2KDa rhizoma polygonati oligosaccharide obtained after different separation and purification methods is shown in Table 1.

[0063] Table 1 Yield of rhizoma polygonati polysaccharide separated and purified by different fillers (n=3)

[0064]

[0065] It can be seen that the yield of oligosaccharide by direct loading method is less than that by polysaccharide mixed with filler before loading using three different fillers, and it can be seen that the pre-mixing of polysaccharide and filler can increase the purification efficiency of Mw<2KDa rhizoma polygonati oligosaccharide.

[0066] Example 5: Effect of different filter aids on the yield of oligosaccharide

[0067] The 500mg / mL rhizoma polygonati polysaccharide sample in Example 2 was separated and purified by the following methods:

[0068] 1) Direct loading method (traditional method): directly loaded into the pretreated Sephadex G-15 dextran gel column, eluted with purified water, and the eluate was collected.

[0069] 2) polysaccharide mixed with filter aid before loading (innovative method): polysaccharide was mixed with 1%~5% column volume of filter aid before loading into the pretreated Sephadex G-15 dextran gel column, and eluted with purified water, and the eluate was collected; wherein the filter aid comprises diatomite, perlite, cellulose, activated carbon, and red soil. Other processing steps are the same as in Example 4.

[0070] The yield of Mw<2KDa rhizoma polygonati oligosaccharide obtained after different separation and purification methods is shown in Table 2.

[0071] Table 2 The yield of purified polygonatum sibiricum polysaccharide with different fillers (n = 3)

[0072]

[0073] It can be seen that the yield of oligosaccharides by direct loading method is less than that by loading after mixing polysaccharides with filter aid. It can be seen that pre-mixing polysaccharides with filter aid can increase the purification efficiency of Mw<2KDa polygonatum sibiricum oligosaccharides. Diatomite has the best effect.

[0074] Example 6: Extraction and purification of polygonatum sibiricum oligosaccharides

[0075] The 500 mg / mL polygonatum sibiricum polysaccharide sample in Example 2 was separated and purified by the following methods:

[0076] 1) Direct loading method (traditional method): directly loaded on a pretreated Sephadex G-15 dextran gel column;

[0077] 2) Loading after mixing polysaccharides with fillers: mixing polygonatum sibiricum polysaccharides with 3% column volume of fillers and then loading;

[0078] 3) Loading after mixing polysaccharides with filter aid: mixing polygonatum sibiricum polysaccharides with 2% column volume of filter aid and then loading. Diatomite was used in this example;

[0079] 4) Loading after mixing polysaccharides with fillers and filter aid (innovative method): mixing polygonatum sibiricum polysaccharides with 2% column volume of filter aid and then loading. Other processing steps are the same as in Example 4. The results are shown in Table 3.

[0080] Table 3 Effect of different improved methods on the yield of polygonatum sibiricum oligosaccharides (n = 3)

[0081]

[0082] In this example, Sephadex G-15 dextran gel was used as the filler and diatomite was used as the filter aid, but this is not a limitation of the present solution. The results in Table 3 show that loading after mixing fillers and filter aid with polysaccharides can further improve the purification efficiency of Mw<2KDa polygonatum sibiricum oligosaccharides. Figure 4 Elution curve of polygonatum sibiricum polysaccharides directly loaded; Figure 5 Elution curve of polygonatum sibiricum polysaccharides loaded after mixing with 3% column volume of Sephadex G-15 fillers; Figure 6 Elution curve of polygonatum sibiricum polysaccharides loaded after mixing with 2% diatomite; and Figure 4 , Figure 5 , Figure 6 Compared with the elution curve of polygonatum sibiricum polysaccharides, Figure 7The elution curve of the mixture of polysaccharides from Polygonatum sibiricum, diatomite and Sephadex G-15 showed that the elution rate of oligosaccharides from Polygonatum sibiricum was further accelerated and the yield was higher.

Claims

1. A method for separating and purifying low molecular weight polysaccharides from Polygonatum sibiricum, the method comprising the following steps: The method comprises the following steps: mixing the polysaccharide with a filler, mixing the polysaccharide with a filter aid, loading the mixture into a column, and then purifying the polysaccharide by using pure water to elute the polysaccharide, and collecting the eluate; the filler is one or more of Sephadex G-15, DEAE-Sepharose CL-4B and Bio-Gel P-2; the column is loaded on a pretreated filler column; the volume of the filler is 1-5% of the column volume; and the mixed volume of the filter aid is 0.5-3% of the column volume. ​ 2. The method for separating and purifying low oligosaccharides of Polygonatum according to claim 1, characterized in that: The filler is Sephadex G-15.

3. The method for separating and purifying low molecular weight polysaccharides from Polygonatum according to claim 1, characterized in that: The volume of the filler is 3% of the column volume.

4. The method for isolating and purifying low molecular weight polysaccharides from Polygonatum according to claim 1, characterized in that: The preparation method of the polysaccharide comprises the following steps: selecting raw polygonatum or processed polygonatum as the polygonatum; and processing the polygonatum by one of the following methods: steaming, wine processing, black bean processing and honey processing; and extracting and purifying the polysaccharide, which comprises the following steps: (1) extraction: polygonatum slices are added into water with a weight of 8 times that of the polygonatum slices, and the mixture is heated to reflux at 100 DEG C for 3 hours, and the extraction is repeated twice, and the water extracts are combined; (2) alcohol precipitation: the water extracts are concentrated, and anhydrous ethanol is added to make the alcohol content reach 80%, and the mixture is left overnight, and then centrifuged and filtered, and the precipitate is dissolved in water to make the concentration reach 0.5 g / mL, and then anhydrous ethanol is added to make the alcohol content reach 80%, and the mixture is left overnight, and then centrifuged and filtered to obtain the crude polysaccharide of polygonatum; (3) Savage method for removing protein: the crude polysaccharide of polygonatum is dissolved in pure water, and the polysaccharide solution, chloroform and n-butanol are added into a separatory funnel in a volume ratio of 25:5:1, and the mixture is shaken vigorously to make the protein denatured into a gel, and the mixture is left to separate into layers, and the uppermost water solution is the polysaccharide, and the polysaccharide water solution is centrifuged and collected, and the process is repeated 5 times; (4) decolorization and impurity removal: A. macroporous resin pretreatment: AB-8 macroporous resin is soaked in ultrapure water, and the fine resin particles are washed away with pure water, and the resin is washed with anhydrous ethanol for multiple times until the water added is not turbid, and the resin is washed repeatedly with ultrapure water and reserved; B. decolorization and impurity removal: the polysaccharide solution in step (3) is subjected to adsorption decolorization; the polysaccharide solution is loaded on an AB-8 column, and 3 column volumes of pure water are used to elute and decolorize the polysaccharide, and the eluate is collected, and the polysaccharide is obtained by concentration under reduced pressure and freeze-drying; 5. The method for separating and purifying low molecular weight polysaccharides from Polygonatum according to any one of claims 1-4, characterized in that: (5) purification: a polysaccharide sample with a concentration of 500 mg / mL is mixed with 3% of the column volume of Sephadex G-15, and the mixture is loaded on a pretreated Sephadex G-15 column, and the polysaccharide is eluted with pure water, and the eluate at the same peak position is collected.

6. The method for separating and purifying the low oligosaccharides of Polygonatum according to claim 5, characterized in that: The filter aid comprises one or more of diatomite, perlite, cellulose and activated carbon.

7. The method for separating and purifying low oligosaccharides of Polygonatum according to claim 6, characterized in that: The filter aid is diatomite. The mixed volume of the filter aid is 2% of the column volume.

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