Separation and purification method of polygonatum sibiricum oligosaccharide

Abstract

The invention relates to a method for separating and purifying polygonatum sibiricum oligosaccharide. According to the method, the polygonatum sibiricum polysaccharide and the filler or / and the filter aid are fully mixed and then separated and purified; wherein the filler is prepared from Sephadex G-15 (crosslinked glucose gel), DEAE-Sepharose CL-4B (agar gel) and Bio-Gel P-2 (polyacrylamide gel), the filter aid is prepared from kieselguhr, perlite, cellulose, activated carbon and red soil, and the method has the advantages that the Mwlt can be obviously improved; the yield of the 2KDa polygonatum sibiricum oligosaccharide is increased, and the effect of combined use of the filler and the filter aid is optimal. The addition of the filter aid can avoid the blockage of filler pores in the middle and later stages of elution, improve the elution performance of the filler on polysaccharide, and accelerate the elution and purification speed of the polygonatum kingianum oligosaccharide. Experiments show that the oligosaccharide can significantly enhance the proliferative activity and phagocytosis of macrophages, promote RAW264.7 cells to secrete TNF-alpha, IL-6, NO and other cell factors, and improve the cellular immunocompetence. The method has the characteristics of convenience, high efficiency and low cost, and has relatively high application value.

Description

Technical Field

[0001] The invention belongs to the field of separation and purification of traditional Chinese medicine polysaccharides, and particularly relates to separation and purification of polygonatum sibiricum oligosaccharides. Background Art

[0002] Polygonatum sibiricum is the dried rhizome of Polygonatum yunnanensis, Polygonatum sibiricum or Polygonatum multiflorum of the Liliaceae family. Depending on the different forms, it is usually called large Polygonatum sibiricum, Polygonatum jitou or Polygonatum jiangxing. It tastes sweet and is neutral in nature. It has the effect of replenishing qi and nourishing yin. Raw Polygonatum sibiricum contains a large amount of mucus and other ingredients, which may cause tongue numbness and throat discomfort, while processed Polygonatum sibiricum eliminates this numb tongue feeling. Polygonatum sibiricum is rich in Polygonatum sibiricum polysaccharides, flavonoids, triterpenoid saponins, alkaloids, anthraquinones, volatile oils, lignans and amino acids. Polygonatum sibiricum polysaccharides are one of the most important components of Polygonatum sibiricum and have significant biological activities, including immunomodulation, anti-tumor, anti-oxidation, anti-virus, myocardial protection and antihypertensive pharmacological functions.

[0003] Compared with high molecular weight polysaccharides, oligosaccharides have a small molecular weight, good water solubility, and are more easily absorbed by the human body. After adding wine and steaming at high temperature, the polysaccharides in polygonatum are hydrolyzed into oligosaccharides, and the oligosaccharide content increases. Pharmacological experiments show that polygonatum oligosaccharides with Mw < 2KDa have better immune effects and are its main immune active components. Therefore, the development of polygonatum oligosaccharides has broad application prospects.

[0004] Separation and purification are the key steps in the preparation of Polygonatum cyrtonema oligosaccharides. Currently, ion exchange chromatography based on the principle of ion exchange and gel chromatography separation based on the principle of molecular sieve are mostly used for separation and purification. However, these traditional separation and purification methods have slow separation speeds and low yields, which limits the application and development of Polygonatum cyrtonema oligosaccharides.

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

[0006] Therefore, the present invention develops an efficient and low-cost separation and purification technology to optimize the existing separation process, improve the separation efficiency of oligosaccharides, and improve the yield and purity of oligosaccharides to meet the urgent needs of scientific research and industrial applications. Summary of the invention

[0007] The purpose of the present invention is to provide a method for separating and purifying polygonatum oligosaccharides.

[0008] Specifically: a method for separating and purifying polygonatum oligosaccharides, using a column chromatography method, polygonatum polysaccharides are mixed with fillers and then purified, the fillers include Sephadex G-15, DEAE-Sepharose CL-4B, Bio-Gel P-2, preferably Sephadex G-15, and the mixed volume is 1%-5% of the column volume, preferably 3%.

[0009] At the same time, another separation and purification method of polygonatum oligosaccharide is provided: polygonatum polysaccharide is mixed with a filter aid and then purified, the filter aid comprises diatomaceous earth, perlite, cellulose, activated carbon, terracotta, preferably diatomaceous earth, and the mixed volume is 0.5%-3% column volume, preferably 2%.

[0010] As a further improvement, the present invention provides a method in which polygonatum polysaccharide is mixed with a filler, then mixed with a filter aid, and then purified.

[0011] As a further improvement, the present invention provides extraction and purification of polygonatum oligosaccharides comprising the following steps:

[0012] (1) Extraction: Take the slices of Polygonatum sibiricum and add 8 times the weight of water to the slices, heat and reflux at 100°C, extract twice, each time for 3 hours, and combine the water extracts;

[0013] (2) Alcohol precipitation: the aqueous extract in step (1) is concentrated, anhydrous ethanol is added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, filtered, and the precipitate is dissolved in an appropriate amount of water to make the concentration reach about 0.5 g / mL based on the crude drug amount, anhydrous ethanol is continued to be added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, and filtered to obtain the precipitate as crude polygonatum polysaccharide;

[0014] (3) Savage method for protein removal: add pure water to dissolve the crude polysaccharide from step (2), add the polysaccharide solution, chloroform and n-butanol into a separatory funnel in a volume ratio of 25:5:1, shake vigorously to mix evenly, and denature the protein into a gel-like state and precipitate. Let stand to allow the precipitate to separate into layers. The top aqueous solution is the polysaccharide. Centrifuge and take the polysaccharide aqueous solution. Repeat 5 times.

[0015] (4) Decolorization and impurity removal: A. Pretreatment of macroporous resin: After soaking the AB-8 macroporous resin in ultrapure water, the finely crushed resin particles are washed away with pure water, and then soaked and washed with anhydrous ethanol for multiple times until it is not turbid when added with ultrapure water, and then washed repeatedly with ultrapure water for standby use; B. Decolorization and impurity removal: The polysaccharide solution in step (3) is subjected to adsorption decolorization; the sample is loaded onto an AB-8 column, and 3 times the column volume of pure water is used for elution and decolorization, and the eluate is collected, concentrated under reduced pressure, and freeze-dried to obtain the refined polygonatum sibiricum polysaccharide;

[0016] (6) Purification: A 500 mg / mL polygonatum polysaccharide sample was mixed with 3% column volume of Sephadex G-15, loaded onto a pretreated Sephadex G-15 column, eluted with pure water, and the eluents at the same peak were collected.

[0017] The polygonatum sibiricum polysaccharide is selected from raw product and processed product, and the processed product is prepared by one of steaming, wine making, black bean making and honey roasting.

[0018] Beneficial Effects

[0019] In view of the shortcomings of the existing separation and purification methods of polygonatum oligosaccharides, the present invention adopts a method for separation and purification after polygonatum polysaccharides are fully mixed with fillers and / or filter aids; wherein the fillers include Sephadex G-15, DEAE-SepharoseCL-4B, Bio-Gel P-2, and the mixed volume is preferably 1%-5%, more preferably 3%, and the filter aids include diatomaceous earth, perlite, cellulose, activated carbon, and red clay, and the usage amount is preferably 0.5%-3% column volume, more preferably 2%. This method can significantly improve the yield of polygonatum oligosaccharides with Mw<2KDa, and the combined use of fillers and filter aids has the best effect. The addition of filter aids can avoid the clogging of filler pores in the middle and late stages of elution, improve the elution performance of fillers for polysaccharides, and accelerate the elution and purification speed of polygonatum oligosaccharides. Experiments show that the oligosaccharides can significantly enhance the proliferation activity and phagocytosis of macrophages, promote the secretion of cytokines such as TNF-α, IL-6 and NO by RAW264.7 cells, and improve cellular immune activity. This method is convenient, efficient, low-cost and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings:

[0021] Figure 1 The results of cell viability assay of different polysaccharide fractions of Polygonatum cyrtonema (n=3);

[0022] Figure 2 The results of the macrophage phagocytic activity assay of different polysaccharide fractions of Polygonatum sibiricum (n=3);

[0023] Figure 3 The results of determination of immune factor contents in different fractions of Polygonatum cyrtonema polysaccharide (n=3);

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

[0025] Figure 4This is the elution curve of Polygonatum cyrtonema polysaccharide directly loaded;

[0026] Figure 5 This is the elution curve of Polygonatum cyrtonema polysaccharide mixed with 3% column volume of Sephadex G-15 filler and loaded;

[0027] Figure 6 This is the elution curve of Polygonatum cyrtonema polysaccharide mixed with 2% diatomaceous earth and loaded;

[0028] Figure 7 This is the elution curve of Polygonatum cyrtonema polysaccharide mixed with 3% Sephadex G-15 column volume filler and 2% diatomaceous earth. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further explained below in conjunction with specific embodiments, which is not intended to limit the technical solution.

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

[0031] Embodiment 1: the processing of polygonatum

[0032] Steaming method: Wash 1 kg of Polygonatum odoratum, put it into a steamer and steam it until it turns brown and black, then take it out, cut it into thick slices and dry it. This method can enhance the effect of Polygonatum odoratum in replenishing qi and blood, nourishing yin and moistening the lungs.

[0033] Wine making method: Take 1kg of Polygonatum sibiricum, clean it, add yellow wine to moisten it for 10 hours, steam it for 12-16 hours, cut it into thick slices, mix it with the steaming liquid, dry it at 60℃ for 12 hours, and get the Polygonatum sibiricum slices. This method can increase the lung moistening effect of Polygonatum sibiricum.

[0034] Black bean preparation method: Cook 1 kg of Polygonatum odoratum with black beans, remove the beans and dry them in the sun, then steam them until they are moist and black inside and out, take them out, cut them into thick slices and dry them. This method can enhance the kidney-tonifying effect of Polygonatum odoratum.

[0035] Honey roasting method: After cleaning 1kg of polygonatum, mix it with honey and heat it until the surface of the polygonatum turns golden yellow, and use it after drying. This method can enhance the lung moistening effect of polygonatum.

[0036] Example 2: Extraction of Polygonatum sibiricum Polysaccharide and Separation and Purification of Polygonatum sibiricum Oligosaccharide

[0037] 1) Extraction: Take pieces of polygonatum, selected from 1 kg of raw polygonatum and 1 kg of processed polygonatum, wherein the processed product is prepared by the wine making method in the method of Example 1, add 8 times the weight of the pieces of water, heat under reflux at 100° C., extract twice, each time for 3 hours, and combine the water extracts;

[0038] 2) Alcohol precipitation: The aqueous extract in the concentration step 1) is concentrated, anhydrous ethanol is added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, filtered, and the precipitate is dissolved in an appropriate amount of water (so that the concentration reaches about 0.5 g / mL based on the crude drug amount), anhydrous ethanol is continued to be added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, and filtered to obtain the precipitate as the crude polysaccharide of polygonatum;

[0039] 3) Savage method for protein removal: add pure water to dissolve the crude polysaccharide of polygonatum sibiricum in step 2), add the polysaccharide solution, chloroform and n-butanol into a separatory funnel in a volume ratio of 25:5:1, shake vigorously to mix evenly, and the protein denatures into a gel and precipitates, let stand to allow the precipitate to separate, the top aqueous solution is the polysaccharide, centrifuge, take the polysaccharide aqueous solution, and repeat 5 times;

[0040] 4) Decolorization and impurity removal: A. Pretreatment of macroporous resin: After soaking AB-8 macroporous resin in ultrapure water, the fine resin particles are washed away with pure water, and then soaked and washed with anhydrous ethanol for multiple times until it is not turbid when added with ultrapure water, and then washed repeatedly with ultrapure water for standby use; B. Decolorization and impurity removal: The polysaccharide solution in step 3) is subjected to adsorption decolorization; the sample is loaded onto an AB-8 column, and 3 times the column volume of pure water is used for elution and decolorization, and the eluate is collected, concentrated under reduced pressure, and freeze-dried to obtain the refined polygonatum polysaccharide.

[0041] 5) Determination of polysaccharide content:

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

[0043] Take 8 clean stoppered test tubes, add 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL of glucose standard solution respectively, add water to 2 mL, then add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid to each tube, shake well and place for 5 minutes, heat in a 90°C water bath for 15 minutes, take out and quickly cool to room temperature, use tube 0 as blank to adjust to zero, and measure the absorbance at 490 nm. Draw a standard curve with glucose concentration X as the horizontal axis (μg / mL) and absorbance Y as the vertical axis.

[0044] ② Determination of the content of refined polygonatum polysaccharide: accurately dilute the refined polygonatum polysaccharide in step 4) by an appropriate multiple, aspirate 2 mL of the solution, operate according to the steps of the standard curve, measure its absorbance value, and substitute it into the standard curve to calculate the polysaccharide content.

[0045] 6) Purification: A polysaccharide sample prepared at a concentration of 500 mg / mL was directly loaded onto a pretreated Sephadex G-15 column, eluted with pure water, and the eluates at the same peak were collected.

[0046] Example 3 Comparison of the efficacy of different molecular weight polysaccharides of Polygonatum sibiricum

[0047] In order to investigate the efficacy of different molecular weight polysaccharides from Polygonatum sibiricum, wine-processed Polygonatum sibiricum was used as raw material, and polysaccharides with different molecular weights were extracted from it.

[0048] (I) The preparation method of different molecular weight polysaccharides of Polygonatum sibiricum is as follows: the eluates of each Polygonatum sibiricum polysaccharide obtained in Example 2 are concentrated and placed in dialysis bags with a cut-off relative molecular mass of 12KDa, 7KDa, and 2KDa, and dialyzed in purified water for 24 hours, and the dialysate outside the bag is collected. After each dialysate is concentrated under reduced pressure, it is freeze-dried to obtain different fractions of Polygonatum sibiricum polysaccharides.

[0049] (II) Efficacy evaluation: Using macrophage RAW264.7 as a cell model, the cell viability, phagocytic activity and immune factor content were examined. The specific methods are as follows:

[0050] 1) Culture and grouping of RAW264.7 macrophages: Mouse macrophage RAW264.7 cell line was revived with RPMI Medium 1640 culture medium containing 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO2. Cell growth was observed every day, and fresh culture medium was replaced in time, and logarithmic phase cells were taken for experiments. Mouse peritoneal macrophages were grouped into blank group, positive drug group (lipopolysaccharide group) and drug-treated group (Mw<2KDa Polygonatum sibiricum polysaccharide group, Mw2~7KDa Polygonatum sibiricum polysaccharide group, Mw7~12KDa Polygonatum sibiricum polysaccharide group, Mw>12KDa Polygonatum sibiricum polysaccharide group).

[0051] 2) Cell viability assay of Polygonatum sibiricum homogeneous polysaccharide: A cell suspension of macrophage RAW264.7 in the logarithmic growth phase was taken and 2×10 4 The cells were inoculated in a 96-well plate at a concentration of 100 μL / well. 100 μL of cell suspension was added to each well and cultured in a cell culture incubator. After changing the medium and washing the culture dish, non-adherent cells were removed to obtain purified adherent macrophages. Culture medium was added to the blank control group, 100 μL of culture medium and lipopolysaccharide were added to the positive control group, and 100 μL of culture medium and 250 μg / mL of purified polysaccharide solution of different fractions of Polygonatum sibiricum were added to the drug-treated group. Three replicate wells were set up in each group. After culturing in the incubator for 24 hours, 1 / 10 of the volume of CCK-8 solution of the cell culture medium was added to each well. The OD value was measured at 450 nm after 1 hour, and the cell viability was calculated according to the following formula: Cell viability % = OD experimental group / OD blank group*100%. Results are shown in Figure 1-3 .

[0052] Figure 1 The results of cell viability determination of different polysaccharide fractions of polygonatum sibiricum showed that the proliferation activity of RAW264.7 macrophages in the drug-treated group (different polysaccharide fractions of polygonatum sibiricum) was significantly enhanced compared with the blank control group (p < 0.001); the polygonatum sibiricum polysaccharide (Mw < 2KDa) had the best effect, and had significant differences in macrophage proliferation activity compared with the polygonatum sibiricum polysaccharide (Mw2-7KDa) part (p < 0.01), (Mw7-12KDa) part (p < 0.001), and (Mw>12KDa) part (p < 0.001). Comparing the results of cell viability determination of different molecular weight polysaccharides of polygonatum sibiricum, it can be seen that the pharmacological effect of polygonatum sibiricum oligosaccharide (Mw < 2KDa) is stronger.

[0053] 3) Determination of phagocytic activity of Polygonatum sibiricum homogeneous polysaccharide: RAW264.7 macrophages in the logarithmic growth phase were taken and 2×10 4 The concentration of cells / well was inoculated in a 96-well plate and cultured in a 37°C, 5% CO2 cell culture incubator. After 24 hours of intervention with 250μg / mL of each mass concentration of Polygonatum sibiricum homogeneous polysaccharide administration group, the supernatant was discarded, 100μL of neutral red solution was added, and the cells were placed in an incubator for further culture for 3 hours, and then the neutral red solution was discarded. The cells were rinsed with PBS buffer reagent 3 times, and 100μL / well of PBS was added each time. Subsequently, 100μL of glacial acetic acid-anhydrous ethanol (V:V=1:1) cell lysis solution was added to each well, and the cells were left to stand overnight at room temperature, and the OD value was measured at a wavelength of 490nm.

[0054] Figure 2 The results of the phagocytic activity of macrophages were detected by neutral red staining. In normal mouse macrophages, compared with the blank control group, the polysaccharide (Mw < 2KDa) part of polygonatum sibiricum significantly enhanced the phagocytic activity of RAW264.7 macrophages (p < 0.001); compared with other drug groups, the polysaccharide (Mw < 2KDa) part and the polysaccharide (Mw2-7KDa) part (p < 0.01), (Mw7-12KDa) part (p < 0.001), (Mw>12KDa) part (p < 0.001) had a very significant effect on enhancing the phagocytic activity of RAW264.7 macrophages, indicating that the oligosaccharide of Mw < 2KDa of polygonatum sibiricum can significantly increase the phagocytic activity of RAW264.7 macrophages under normal conditions, and the effect is stronger than that of other drug groups with other molecular weights.

[0055] 4) Determination of immune factor content: RAW264.7 macrophages in the logarithmic growth phase were taken and 2×10 5The concentration of cells / well was inoculated in a 96-well plate and cultured in a 37°C, 5% CO2 cell culture incubator. 100 μL complete culture medium was added to the blank control group, 100 μL LPS solution (2 μg / mL) was added to the positive control group, and the mass concentration of Polygonatum sibiricum uniform polysaccharide administration group was 250 μg / mL, and 3 replicates were set for each sample. Cultured in a cell culture incubator at 37°C for 24 hours, the supernatant was collected, and the supernatant was aspirated after centrifugation and stored at -20°C. TNF-α, IL-6 and NO in the cell supernatant were detected according to the instructions of the kit.

[0056] Figure 3 The results of the determination of immune factor content in different polysaccharide fractions of Polygonatum sibiricum showed that compared with the blank group, both the Polygonatum sibiricum polysaccharide (Mw < 2KDa) fraction (p < 0.001) and the Polygonatum sibiricum polysaccharide (Mw 2-7KDa) fraction could promote the release of IL-6, TNF-α and NO from RAW264.7 cells; compared with other drug administration groups, the Polygonatum sibiricum 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 in the secretion of cytokines, the phagocytic ability of macrophages is enhanced, and the body's disease resistance is enhanced accordingly, which indicates that Polygonatum sibiricum oligosaccharides with Mw < 2KDa can activate macrophages and thus exert an immune activity effect.

[0057] In summary, some oligosaccharides with Mw < 2KDa from Polygonatum sibiricum can significantly enhance the proliferation activity of macrophages, enhance the phagocytosis of macrophages, 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 Mw < 2 KDa

[0059] The 500 mg / mL polygonatum polysaccharide sample in Example 2 was separated and purified by the following methods:

[0060] 1) Direct loading method (traditional method): Load the sample directly onto the pretreated packing column, elute with pure water, and collect the eluate.

[0061] 2) Loading after mixing the polysaccharide and the filler (innovative method): After mixing the polygonatum polysaccharide with 1% to 5% of the column volume of the filler, load it into the pretreated filler column, elute with pure water, and collect the eluate; wherein the filler includes SephadexG-15, DEAE-Sepharose CL-4B, and Bio-Gel P-2. The eluates of each elution peak after purification are combined, concentrated, and placed in a dialysis bag with a retention relative molecular mass of 2KDa, and dialyzed in purified water for 24 hours. The dialysate outside the bag is collected, concentrated under reduced pressure, and freeze-dried to obtain a polygonatum polysaccharide fraction with Mw<2KDa. The glucose standard curve is redrawn according to the method in step 5) of Example 2, and the absorbance of polygonatum oligosaccharides is measured to calculate the oligosaccharide content. The calculation formula for the yield of Polygonatum sibiricum oligosaccharides with Mw<2KDa is: Yield (%) = (Polygonatum sibiricum oligosaccharide content*total mass of oligosaccharides) / (Polygonatum sibiricum refined polysaccharide content*total mass of purified polysaccharides loaded)*100%.

[0062] The yields of Polygonatum sibiricum oligosaccharides with Mw < 2KDa obtained by different separation and purification methods are shown in Table 1.

[0063] Table 1 Separation and purification yield of Polygonatum cyrtonema polysaccharide with different fillers (n=3)

[0064]

[0065] It can be seen that when using three different fillers, the oligosaccharide yields of the direct loading method are all lower than those of the loading method after the polysaccharide and filler are mixed. This shows that premixing of polysaccharides and fillers can increase the purification efficiency of Polygonatum sibiricum oligosaccharides with Mw < 2KDa.

[0066] Example 5: Effect of pretreatment of polysaccharides with different filter aids on the yield of oligosaccharides

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

[0068] 1) Direct loading method (traditional method): Load the sample directly onto a pretreated Sephadex G-15 column, elute with pure water, and collect the eluate.

[0069] 2) Mixing polysaccharide and filter aid before loading (innovative method): Mixing Polygonatum cyrtonema polysaccharide with 1% to 5% column volume of filter aid, loading the mixture into a pretreated Sephadex G-15 dextran gel column, eluting with pure water, and collecting the eluate; wherein the filter aid comprises diatomaceous earth, perlite, cellulose, activated carbon, and red clay. Other processing steps are the same as those in Example 4.

[0070] The yields of Polygonatum sibiricum oligosaccharides with Mw < 2KDa obtained by different separation and purification methods are shown in Table 2.

[0071] Table 2 Separation and purification yield of Polygonatum cyrtonema polysaccharide with different fillers (n=3)

[0072]

[0073] It can be seen that the oligosaccharide yield of the direct loading method is lower than that of the loading method after mixing the polysaccharide and the filter aid. It can be seen that pre-mixing the polysaccharide and the filter aid can increase the purification efficiency of the Polygonatum sibiricum oligosaccharide with Mw < 2KDa; among them, diatomaceous earth has the best effect.

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

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

[0076] 1) Direct loading method (traditional method): directly loading the sample onto the pretreated Sephadex G-15 gel column;

[0077] 2) Mixing the polysaccharide with the filler and then loading: Mixing the polygonatum polysaccharide with 3% column volume of the filler and then loading;

[0078] 3) Mixing the polysaccharide with the filter aid and then loading the sample: Mix the polygonatum polysaccharide with 2% column volume of the filter aid and then load the sample. In this embodiment, diatomaceous earth is used;

[0079] 4) Polysaccharide is mixed with filler and filter aid and then loaded (innovative method): Polygonatum sibiricum polysaccharide is mixed with 2% column volume of filter aid and then loaded. Other processing steps are the same as Example 4. The results are shown in Table 3.

[0080] Table 3 Effects of different improved methods on the yield of Polygonatum cyrtonema oligosaccharides (n=3)

[0081]

[0082] In this embodiment, Sephadex G-15 dextran gel is used as filler and diatomaceous earth is used as filter aid, but this is not a limitation of this solution. The results in Table 3 show that the purification efficiency of Mw<2KDa polygonatum oligosaccharide can be further improved by mixing the filler and filter aid with the polysaccharide and then loading the sample. Figure 4 This is the elution curve of Polygonatum cyrtonema polysaccharide directly loaded; Figure 5 This is the elution curve of Polygonatum cyrtonema polysaccharide mixed with 3% column volume of Sephadex G-15 filler and loaded; Figure 6 is the elution curve of Polygonatum cyrtonema polysaccharide mixed with 2% diatomaceous earth and loaded; Figure 4 , Figure 5 , Figure 6 Compared with the elution curve of Polygonatum cyrtonema polysaccharide, Figure 7The elution curve of polygonatum polysaccharide mixed with diatomaceous earth and Sephadex G-15 filler showed that the elution rate of polygonatum oligosaccharide was further accelerated and the yield was higher, and the two played a synergistic role.

Claims

1. A method for separating and purifying polygonatum oligosaccharides, characterized in that: The column chromatography method is adopted, the polygonatum polysaccharide is first mixed with the filler, loaded into the column, and then purified, eluted with pure water, and the eluate is collected; the filler is one or more of Sephadex G-15, DEAE-Sepharose CL-4B, Bio-Gel P-2.

2. The method for separating and purifying polygonatum oligosaccharides according to claim 1, characterized in that: The filler is Sephadex G-15.

3. The method for separating and purifying polygonatum oligosaccharides according to claim 1, characterized in that: The volume of the filler is 1-5% of the column volume.

4. The method for separating and purifying polygonatum oligosaccharides according to claim 3, characterized in that: The volume of the packing material was 3% of the column volume.

5. The method for separating and purifying polygonatum oligosaccharides as claimed in claim 1, characterized in that: In the preparation method of polygonatum polysaccharide, the polygonatum is selected from raw polygonatum or processed polygonatum, and the processing method of polygonatum is one of steaming method, wine making method, black bean making method and honey roasting method; the extraction and purification of polygonatum oligosaccharide includes the following steps: (1) Extraction: Take the slices of Polygonatum sibiricum and add 8 times the weight of water to the slices, heat and reflux at 100°C, extract twice, each time for 3 hours, and combine the water extracts; (2) Alcohol precipitation: the aqueous extract in step (1) is concentrated, anhydrous ethanol is added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, filtered, and the precipitate is dissolved in an appropriate amount of water to make the concentration reach about 0.5 g / mL based on the crude drug amount, anhydrous ethanol is continued to be added to make the alcohol content reach 80%, the extract is allowed to stand overnight, centrifuged, and filtered to obtain the precipitate as crude polygonatum polysaccharide; (3) Savage method for protein removal: add pure water to dissolve the crude polysaccharide from step (2), add the polysaccharide solution, chloroform and n-butanol into a separatory funnel in a volume ratio of 25:5:1, shake vigorously to mix evenly, and denature the protein into a gel-like state and precipitate. Let stand to allow the precipitate to separate into layers. The top aqueous solution is the polysaccharide. Centrifuge and take the polysaccharide aqueous solution. Repeat 5 times. (4) Decolorization and impurity removal: A. Pretreatment of macroporous resin: AB-8 macroporous resin is soaked in ultrapure water, and then washed with pure water to remove fine resin particles. The resin is then soaked and washed with anhydrous ethanol for multiple times until it is not turbid when ultrapure water is added. The resin is then repeatedly washed with ultrapure water for later use. B. Decolorization and impurity removal: Decolorize the polysaccharide solution in step (3) by adsorption; The sample was loaded onto an AB-8 column, and 3 times column volume of pure water was used for elution and decolorization. The eluate was collected, concentrated under reduced pressure, and freeze-dried to obtain the refined polygonatum sibiricum polysaccharide; (6) Purification: A 500 mg / mL polygonatum polysaccharide sample was mixed with 3% column volume of Sephadex G-15, loaded onto a pretreated Sephadex G-15 column, eluted with pure water, and the eluents at the same peak were collected.

6. The method for separating and purifying polygonatum oligosaccharides according to any one of claims 1 to 5, characterized in that: The polygonatum polysaccharide is mixed with a filler and then mixed with a filter aid, and then column loading, purification and elution are performed; the filter aid comprises one or more of diatomaceous earth, perlite, cellulose, activated carbon and red clay.

7. The method for separating and purifying polygonatum oligosaccharides according to claim 6, characterized in that: The filter aid is diatomaceous earth.

8. The method for separating and purifying polygonatum oligosaccharides as claimed in claim 6, characterized in that: The mixed volume of the filter aid is 0.5-3% column volume.

9. The method for separating and purifying polygonatum oligosaccharides according to claim 8, characterized in that: The mixed volume of the filter aid was 2% column volume.

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