CN116178580B - Wine-processed Polygonatum sibiricum polysaccharide extract and preparation method thereof - Google Patents
Wine-processed Polygonatum sibiricum polysaccharide extract and preparation method thereof Download PDFInfo
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- CN116178580B CN116178580B CN202310138851.1A CN202310138851A CN116178580B CN 116178580 B CN116178580 B CN 116178580B CN 202310138851 A CN202310138851 A CN 202310138851A CN 116178580 B CN116178580 B CN 116178580B
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- polysaccharide
- rhizoma polygonati
- pspw
- supernatant
- water
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention provides a wine-processed rhizoma polygonati polysaccharide extract and a preparation method thereof, and the wine-processed rhizoma polygonati polysaccharide extract with different molecular weights and polarities can be separated and purified by adopting fractional alcohol precipitation, so that a 70% rhizoma polygonati water-soluble polysaccharide alcohol precipitation component is obtained, wherein monosaccharide in the component has simple composition, high sugar content, low protein content, high galactose proportion and strong immunocompetence. The preparation method has the advantages of simple operation and low cost, is an effective method for separating and purifying the polygonatum polysaccharide, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of chemical extraction and separation of traditional Chinese medicines, and particularly relates to a wine-processed polygonatum sibiricum polysaccharide extract and a preparation method thereof.
Background
Rhizoma Polygonati is dried rhizome of Polygonatum sibiricum of Polygonatum of Liliaceae Polygonatum sibiricum Red. Mainly produced in Shaanxi, hubei, henan and Zhejiang, etc., is a famous "Qin medicine" in Shaanxi province. Rhizoma Polygonati was first recorded in Ming Yi Bie Ji, which is compiled from the pottery Honggang in the North and south dynasties, and takes rhizoma Polygonati as the prime name. The "Ben Cao gang mu" records that rhizoma Polygonati "is effective in tonifying deficiency, replenishing essence, caring skin, and prolonging life. "rhizoma Polygonati is one of the Chinese medicines used as both medicine and food, has long history of medicine and definite curative effect, has the effects of invigorating spleen and replenishing qi, nourishing kidney and moistening lung, and is a good medicine for invigorating spleen and replenishing qi in the past. In recent years, rhizoma Polygonati has a high nutritive value and is widely used for development of nourishing foods such as beverages, yogurt, wine, vinegar, etc. Research of related literature shows that rhizoma Polygonati mainly contains polysaccharide, saponin, anthraquinone, flavone, alkaloid, amino acid, etc. Wherein, the rhizoma polygonati polysaccharide is taken as a main active ingredient and has the functions of biological activity such as immunoregulation, antioxidation, blood sugar reduction, anti-tumor, anti-aging and the like.
Rhizoma Polygonati is used for raw materials with throat irritation side effect, and is processed before clinical application, and common processing method of rhizoma Polygonati mainly comprises wine preparation method, honey preparation method and method of co-processing with other adjuvants, wherein wine preparation method is the most common method. In recent years, research on polysaccharide components in rhizoma polygonati by students at home and abroad is mainly focused on: extracting, separating, purifying, and pharmaceutically active. The extraction method of the rhizoma polygonati polysaccharide mainly comprises the following steps: water extraction and alcohol precipitation, stepwise alcohol precipitation, alkali extraction, ultrasonic assisted enzyme method, microwave assisted extraction and the like. The common separation and purification methods of polysaccharide mainly comprise a column chromatography method, a membrane filtration method, a step alcohol precipitation method, a quaternary ammonium salt precipitation method and the like, wherein the step alcohol precipitation method is an effective polysaccharide separation and purification method developed in recent years, and the method has the advantages of simple operation, low cost, less polysaccharide loss in the separation process and the like, and is widely used.
The patent document CN 113501890A (published day: 2021-10-15) discloses an extraction method of polygonatum cyrtonema polysaccharide and application of polygonatum cyrtonema polysaccharide, wherein the polygonatum cyrtonema polysaccharide with bidirectional moisture retention is obtained through extraction by a thermal extraction-grading alcohol precipitation method. Patent document CN 100554326C (publication date: 2006-07-05) discloses the use of an immune polysaccharide of Polygonatum sibiricum and a composition thereof, and the publication states that: the polysaccharide is purified by a DEAE-cellulose column to obtain 4 different Polygonatum sibiricum polysaccharide components, each of which can promote the proliferation of T, B lymphocytes. Patent document CN 113121715A (publication day: 2021-07-16) discloses a method for separating and extracting rhizoma Polygonati polysaccharide having intestinal mucosa immunocompetence, which comprises extracting rhizoma Polygonati medicinal particles, precipitating the extractive solution with ethanol for 2 times, and grading components having intestinal mucosa regulating activity. The above reports on the separation and activity of polysaccharide in rhizoma Polygonati crude products are not related to the separation, physicochemical properties and immunological activity of polysaccharide in rhizoma Polygonati wine products of Qin medicine.
According to the invention, the in-vivo and in-vitro immunoregulatory activity of the water-soluble polysaccharide PSPW of the rhizoma polygonati wine is obviously enhanced after the rhizoma polygonati wine is prepared through earlier-stage researches. Since PSPW is not a single polysaccharide component, it is difficult to determine the optimal polysaccharide active component, which makes it difficult to study the basis and mechanism of its immunomodulating substance. Therefore, the crude polysaccharide of Polygonatum sibiricum Red needs to be further separated and purified to find the optimal active polysaccharide component so as to provide a reference basis for developing Polygonatum sibiricum red polysaccharide functional food.
Disclosure of Invention
The invention aims to provide a wine-processed rhizoma polygonati polysaccharide extract and a preparation method thereof, and the wine-processed rhizoma polygonati polysaccharide extract and the preparation method thereof can separate and purify wine-processed rhizoma polygonati polysaccharide with different molecular weights and polarities by adopting fractional alcohol precipitation, so as to obtain a 70% rhizoma polygonati water-soluble polysaccharide alcohol precipitation component, wherein monosaccharide in the component has simple composition, high galactose proportion and strong immunocompetence. The preparation method has the advantages of simple operation and low cost, is an effective method for separating and purifying the polygonatum polysaccharide, and has wide application prospect.
The technical scheme of the wine-processed rhizoma polygonati polysaccharide extract is as follows:
the Polygonatum sibiricum polysaccharide extract is characterized by comprising the following raw material medicines in percentage by mole: 50-85% of galactose, 0-40% of galacturonic acid, 2-40% of mannose, 2-15% of rhamnose, 1-8% of glucose and 0-5% of arabinose.
Preferably, the rhizoma polygonati polysaccharide extract further comprises the following raw material medicines in percentage by mole: 0 to 0.1 percent of fucose, 0 to 0.1 percent of xylose, 0 to 0.1 percent of glucuronic acid.
Preferably, the Polygonatum sibiricum polysaccharide extract comprises the following raw material medicines in percentage by mole: galactose 52.75%, galacturonic acid 30.15%, mannose 4.48%, rhamnose 7.37%, glucose 2.33%, arabinose 2.92%.
Preferably, the Polygonatum sibiricum polysaccharide extract comprises the following raw material medicines in percentage by mole: 82.25% of galactose, 0.02% of galacturonic acid, 2.22% of mannose, 11.01% of rhamnose, 1.57% of glucose and 2.95% of arabinose.
As a further preferred aspect of the invention, the Polygonatum sibiricum polysaccharide extract comprises the following raw material medicaments in percentage by mole: 79.3% of galactose, 0.05% of galacturonic acid, 15.33% of mannose, 2.92% of rhamnose, 2.45% of glucose and 0.01% of arabinose.
Preferably, the Polygonatum sibiricum polysaccharide extract comprises the following raw material medicines in percentage by mole: 56.43% galactose, 0.03% galacturonic acid, 36.28% mannose, 0.01% rhamnose, 7.29% glucose and 0.02% arabinose.
Preferably, the preparation method of the polygonatum polysaccharide extract comprises the following steps:
preparation of water-soluble polysaccharide of rhizoma polygonati prepared by wine
Taking rhizoma polygonati raw products, adding yellow wine according to a volume ratio of 5:1, stirring uniformly, sealing and moistening for 10-12 h, steaming for 4h at 120 ℃ in an autoclave, steaming to the inside and outside for moistening black, taking out, slicing, drying in a baking oven at 60 ℃ to obtain rhizoma polygonati wine prepared decoction pieces, crushing, sieving with a 100-mesh sieve to obtain rhizoma polygonati wine prepared decoction piece powder, adding 10 times of distilled water, extracting at 80 ℃ for 2h each time, filtering the extracting solution, combining 2 times of extracting solutions, recovering filtrate, adding 4 times of absolute ethyl alcohol to ensure that the alcohol concentration reaches 80%, precipitating with alcohol for 12h at 4 ℃, filtering, recovering alcohol, adding distilled water for redissolution, deproteinizing, centrifuging, discarding precipitate, placing supernatant in a 3000Da dialysis bag for running water dialysis, concentrating the dialyzate under reduced pressure, and freeze-drying to obtain the rhizoma polygonati water-soluble polysaccharide prepared by wine;
separation and purification of polygonatum sibiricum polysaccharide: weighing the water-soluble polysaccharide of rhizoma polygonati prepared by using the wine, adding double distilled water for dissolving to enable the mass concentration to be 0.5-2%, slowly adding a certain volume of ethanol solution to enable the mass fraction of the ethanol in the solution to be 30%, standing for 8-16 h at the temperature of 2-6 ℃, centrifuging, respectively collecting sediment and supernatant I, adding double distilled water for dissolving the sediment, discarding the sediment, and freeze-drying the supernatant to obtain a 30% water-soluble polysaccharide alcohol sediment component PSPW-1;
taking supernatant I in the step, continuously adding ethanol until the mass fraction is 50%, standing for 8-16 h at 2-6 ℃, centrifuging, respectively collecting precipitate and supernatant II, performing related operation on the precipitate as in the step, and freeze-drying to obtain 50% of rhizoma polygonati water-soluble polysaccharide alcohol precipitation component PSPW-2;
taking the supernatant II in the step, adding ethanol to the supernatant II until the mass fraction is 60%, performing related operations on precipitation as the step, and freeze-drying to obtain supernatant III and 70% of rhizoma polygonati water-soluble polysaccharide alcohol precipitation component PSPW-3;
and fifthly, taking the supernatant III in the step, adding ethanol to the mass fraction of 80%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain an 80% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-4.
Preferably, in the step, the centrifugal speed is 8000-12000 r/min and the centrifugal time is 8-12 min.
Preferably, the freeze-drying conditions in the step (a) are as follows: the sample solution is placed at the temperature of minus 75 ℃ to minus 85 ℃ for 2 to 4 hours, the freezing temperature of a freeze dryer is minus 50 ℃ to minus 60 ℃, the temperature of a frozen shelf in a cold trap chamber is minus 20 ℃ to minus 30 ℃, the sublimation drying temperature is 45 ℃ to 55 ℃, the analysis drying temperature is 30 ℃ to 40 ℃, and the analysis time is 7 to 9 hours.
Preferably, the freeze-drying conditions in the step (a) are as follows: the sample solution is placed at-80 ℃ for 3 hours of pre-freezing, the freezing temperature of a freeze dryer is-55 ℃, the temperature of a frozen shelf in a cold trap chamber is-25 ℃, the sublimation drying temperature is 50 ℃, the analysis drying temperature is 35 ℃, and the analysis time is 8 hours.
Preferably, the Polygonatum sibiricum polysaccharide extract is applied to preparation of the Polygonatum sibiricum Red polysaccharide extract which plays an immunoregulatory role and is used as a functional food additive.
In order to highlight the innovativeness of the technical scheme of the invention, part of experiments screened in the experimental process are provided as follows.
According to the technical scheme, a step-by-step alcohol precipitation method is adopted, and according to the principle of similar compatibility, the polysaccharide with different polarities is precipitated step by adopting ethanol with different concentrations. The research adopts a step-by-step alcohol precipitation method to separate and purify the wine-made rhizoma polygonati water-soluble crude polysaccharide PSPW, obtains the wine-made rhizoma polygonati polysaccharide component with all molecular weight sections, and researches the physicochemical properties and in vitro immunocompetence of 4 alcohol precipitation components PSPW-1, PSPW-2, PSPW-3 and PSPW-4.
The results show that the physicochemical properties of the alcohol precipitation components are obviously different: the components are increased and then decreased, wherein PSPW-2 is the highest and PSPW-1 is the lowest. The monosaccharide composition results show that as the mass fraction of the ethanol increases, the monosaccharide composition types of each alcohol precipitation component gradually decrease, and PSPW-1 mainly contains 6 types of monosaccharides (rhamnose, arabinose, mannose, glucose, galactose and galacturonic acid), and PSPW-1 contains a high proportion of galacturonic acid (30.15%) as acidic heteropolysaccharide; PSPW-4 mainly contains 3 kinds of monosaccharides (mannose, glucose and galactose), and 4 alcohol precipitation components mainly contain galactose (> 52.75%) in terms of monosaccharide composition ratio. The infrared detection results show that the structural characteristics of all the components are the polysaccharide connected by the beta-glycosidic bond. The in vitro immunocompetence results of the components show that the components can promote RAW264.7 cell activity, phagocytose neutral red ability, secrete NO ability and secrete acid phosphatase activity, and the PSPW-3 immunocompetence is strongest.
The invention has the beneficial effects that:
the method comprises the steps of separating and purifying water-soluble polysaccharide of rhizoma polygonati in wine production by adopting a step-by-step alcohol precipitation method, and sequentially obtaining 0-30%, 30-50%, 50-70% and 70-80% polysaccharide components which are named as PSPW-1, PSPW-2, PSPW-3 and PSPW-4 along with the increase of the concentration of ethanol used in the step-by-step alcohol precipitation. The results show that the yield of each component is as follows: PSPW-2 (1.48%) > PSPW-3 (1.03%) > PSPW-4 (0.71%) > PSPW-1 (0.49%). From the sugar content and the protein content results, the rhizoma polygonati water-soluble polysaccharide content PSPW-3 (57.82 +/-0.16%) > PSPW-4 (57.44 +/-1.23%) > PSPW-2 (45.89 +/-0.97%) > PSPW-1 (43.21 +/-0.38%); rhizoma Polygonati protein content PSPW-1 (34.33+ -1.72%) > PSPW-4 (33.82 + -0.54%) > PSPW-2 (24.71+ -0.26%) > PSPW-3 (20.08+ -0.72%).
And the physicochemical properties of the alcohol precipitation components are researched by adopting methods such as Gas Chromatography (GC), high Performance Gel Permeation Chromatography (HPGPC), fourier transform infrared spectroscopy (FT-IR), and the like, and the physicochemical property results show that the 4 alcohol precipitation components (the molecular weight is 2.26 multiplied by 10 respectively 4 、2.24×10 4 、1.15×10 4 6.58×10 3 u) are glycoprotein complexes; the monosaccharide composition types and proportions of the alcohol precipitation components are obviously different, and the alcohol precipitation components all contain galactose with high proportion.
The influence of each alcohol precipitation component on the immune activity of RAW264.7 cells is evaluated by adopting an MTT method, a neutral red phagocytosis experiment and a Griess method, and the immune activity research shows that each component can promote the activity of RAW264.7 cells, the neutral red phagocytosis capability, the NO secretion capability and the acid phosphatase secretion activity, wherein the PSPW-3 has the strongest immune activity.
Separating and purifying active polysaccharide after the wine preparation of the rhizoma polygonati of the Qin medicine to obtain a polysaccharide component with optimal immunoregulatory activity; separating and purifying the water-soluble polysaccharide of the rhizoma polygonati prepared by wine by adopting a fractional alcohol precipitation method to obtain polysaccharide fractions with all molecular weight sections, and extracting all galacturonic acid in 30% of the fractions, so that the interference of uronic acid on the activity of other components is avoided, and the separation method is simple and convenient and easy to operate; the wine-processed Polygonatum sibiricum polysaccharide is separated by fractional alcohol precipitation, and 70% of the obtained components have simple monosaccharide composition, high sugar content, low protein content, high galactose ratio and strong immunocompetence. The invention can also provide technical reference and reference for separation and purification of polysaccharide, physical and chemical properties and activity research in rhizoma polygonati and other medicinal and edible traditional Chinese medicinal materials, and the preparation method of the water-soluble polysaccharide of rhizoma polygonati processed by wine has the advantages of simple operation and low cost, and has obvious industrial application prospect in development and application of rhizoma polygonati polysaccharide health-care food.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention.
FIG. 1-shows a gas chromatogram of monosaccharide composition of each of PSPW-1, PSPW-2, PSPW-3, and PSPW-4, wherein PSPW-1 is 30% rhizoma Polygonati water-soluble polysaccharide alcohol precipitation component, PSPW-2 is 50% rhizoma Polygonati water-soluble polysaccharide alcohol precipitation component, PSPW-3 is 70% rhizoma Polygonati water-soluble polysaccharide alcohol precipitation component, and PSPW-4 is 80% rhizoma Polygonati water-soluble polysaccharide component prepared by alcoholic liquor. Wherein Rha in the chromatogram is rhamnose, fuc is fucose, ara is arabinose, xyl is xylose, man is mannose, glc is glucose, gal is galactose, glcA is glucuronic acid, galA is galacturonic acid);
FIG. 2-HPGPC (high Performance gel permeation chromatography) diagram of various alcohol precipitation components of PSPW;
FIG. 3-IR spectrum of PSPW-1-4;
FIG. 4-PSPW-1-4 scanning electron microscope result diagram;
FIG. 5-PSPW and alcohol precipitation fractions were used for regulating immune function of RAW264.7 cells. (a) cell viability; (b) neutral red phagocytosis index; (c) an acid phosphatase activity index; (d) NO secretion amount
(n is more than or equal to 5; in contrast to the blank set of the samples, a P<0.05， b P<0.01; in contrast to the group of PSPWs, c P<0.05， d P<0.01)。
Detailed Description
The invention will be further described by way of exemplary embodiments in order to provide a more thorough understanding of the practice of the invention. Unless defined otherwise, technical or scientific terms used in the specification and claims of the present application should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present invention pertains.
Example 1: the invention relates to a preparation method of a Polygonatum sibiricum polysaccharide extract
1.1 materials and reagents
RAW264.7 cell, basic medical institute cell of Chinese medical science academy of sciencesA resource center; cation exchange resin (Dowex 50×8, 400 mesh), rhamnose, xylose, fucose, mannose, glucose, galactose, glucuronic acid, galacturonic acid standard and dextran series standard (0.05X10) 3 ～1.50×10 3 u), sigma-Aldrich, USA; CCK-8 kit, shanghai Tao Su Biochemical technology Co., ltd; IL-1 beta, TNF-alpha and IFN-gamma ELISA kits, shanghai Xinle Biotech Co., ltd; ethanol, pyridine, dichloromethane, etc. (analytical grade), national medicine group chemical reagent company, inc.
1.2 instruments and apparatus
Shimadzu 2010A Shimadzu high performance liquid chromatograph, shimadzu corporation; GC-2010 shimadzu gas chromatograph, shimadzu corporation, japan; a TENSOR27 fourier transform infrared spectrometer, bruk, germany; scanning electron microscope, ZEISS company, germany; SBS-IOOA numerical control automatic part collector, shanghai Xie analytical instruments works Co., ltd; DZF-6020 vacuum drying oven, shangshengxue instruments, inc.; R-1001-VN rotary evaporator, zhengzhou great wall family industry and trade company; HS840 ultra clean bench, tianjin Test instruments Co., ltd; a Herocell 180 carbon dioxide incubator, shanghai comfort biotechnology limited; DW-86L288 ultra-low temperature refrigerator, qingdao sea Co., ltd; RVC 2-18CD freeze dryer, CHRIST company, germany.
1.3 method
1.3.1PSPW extraction and fractional alcohol precipitation separation and purification
1.3.1.1PSPW (rhizoma Polygonati water-soluble polysaccharide) extraction and preparation method thereof
Preparation of water-soluble polysaccharide PSPW from rhizoma polygonati prepared by wine
Rhizoma polygonati wine product: taking a proper amount of rhizoma polygonati raw product, placing the rhizoma polygonati raw product into a proper container, adding yellow wine according to a volume ratio of 5:1, stirring uniformly, moistening for 10-12 h, placing the rhizoma polygonati raw product into a high-pressure steamer, steaming at 120 ℃ for 4h, steaming to moisten black inside and outside, taking out, slicing, and drying in a baking oven at 60 ℃ to obtain rhizoma polygonati wine prepared decoction pieces. Pulverizing the rhizoma Polygonati wine decoction pieces, and sieving with 100 mesh sieve to obtain rhizoma Polygonati wine decoction piece powder. Weighing 500g of rhizoma polygonati wine product powder, adding 10 times of distilled water into a stainless steel pot, extracting for 2 times with hot water at 80 ℃ under stirring, filtering the extracting solution for 2 hours each time, mixing the 2 times of extracting solutions, recovering the filtrate to small volume, adding 4 times of absolute ethyl alcohol (the concentration of the ethyl alcohol reaches 80%) respectively according to the volume ratio, and precipitating with alcohol at 4 ℃ for 12 hours; filtering, recovering ethanol, dissolving the precipitate with distilled water, deproteinizing by Savage method (dichloromethane: n-butanol=4:1), centrifuging at 10000rpm for 10min, discarding the precipitate, repeating for 5-6 times until no precipitate, dialyzing the supernatant with flowing water in 3000Da dialysis bag for 3 days, concentrating the dialysate under reduced pressure, and lyophilizing to obtain rhizoma Polygonati wine product crude polysaccharide PSPW (Polygonatum sibiricum wine-processdpolysacchades);
1.3.1.2 separation and purification of PSPW by fractional alcohol precipitation
Weighing 500mg of PSPW (rhizoma Polygonati water-soluble polysaccharide prepared by wine), and adding a proper amount of double distilled water for dissolution to make the mass concentration be 1%: (1) Slowly adding a certain volume of ethanol solution to make the mass fraction of ethanol in the solution reach 30%, standing at 4deg.C for 12h, centrifuging for 10min, collecting precipitate and supernatant I respectively, dissolving the precipitate with appropriate amount of double distilled water, centrifuging for 10min, discarding the precipitate, and lyophilizing the supernatant to obtain 30% ethanol precipitation component PSPW-1. (2) Continuously and slowly adding ethanol into the supernatant I until the mass fraction is 50%, standing for 12h at 4 ℃, respectively collecting a precipitate and the supernatant II, and operating the precipitate according to (1) to obtain a 50% ethanol precipitation component PSPW-2. (3) And in the same way, continuously and slowly adding ethanol into the supernatant II until the mass fraction is 70%, so as to obtain supernatant III and 70% of an alcohol precipitation component PSPW-3. (4) Continuously and slowly adding ethanol into the supernatant III until the mass fraction is 80%, and obtaining 80% of an ethanol precipitation component PSPW-4 by the same method. The yields (%) of the respective components were calculated separately.
1.3.2 physical and chemical Properties study of the alcohol precipitation Components
1.3.2.1 sugar content determination
The phenol-concentrated sulfuric acid method is adopted to analyze the sugar content of each alcohol precipitation component, and the specific operation method is as follows: (1) Precisely weighing a proper amount of glucose reference substance, placing in a volumetric flask, and adding distilled water to prepare glucose reference substance solution with mass concentration of 0.1 mg/mL. Accurately sucking 0-1.2 mL of reference solution into a graduated test tube, adding distilled water to 2mL, sequentially adding 50 mu L of 80% phenol solution and 2.5mL of concentrated sulfuric acid, standing at 60 ℃ for 20min, mixing uniformly, taking a 0-tube as a blank tube, measuring an ultraviolet absorbance at 490nm, taking sugar content (mg) as an abscissa X, taking absorbance (A) as an ordinate Y, drawing a corresponding standard curve, and establishing a regression equation. (2) And respectively precisely weighing a proper amount of each polysaccharide sample in a volumetric flask, and adding distilled water to prepare a polysaccharide sample solution with the mass concentration of 1 mg/mL. Accurately sucking 0.2mL of each group of polysaccharide water solution into a graduated test tube, adding distilled water to 2mL, and performing the rest operation in the same way as 1.3.2.1 (1), measuring the light absorption value, and calculating the sugar content in each alcohol precipitation component sample according to the light absorption value.
1.3.2.2 protein content determination
The method adopts a Coomassie brilliant blue method to measure and determine the protein content in each alcohol precipitation component sample of PSPW, and comprises the following specific operation methods: (1) 50mg of Coomassie brilliant blue G-250 is precisely weighed and dissolved in 25.0mL of 90% ethanol and 50.0mL of phosphoric acid solution, and distilled water is added to fix the volume to 500mL for later use. (2) Accurately weighing a proper amount of chicken albumin reference substance, placing the chicken albumin reference substance into a volumetric flask, and adding distilled water to prepare a chicken albumin reference substance solution with the mass concentration of 1.0 mg/mL. Respectively precisely absorbing 0-1.2 mL of reference substance solution into a graduated test tube, adding distilled water to 1mL, sequentially adding 5mL of coomassie brilliant blue solution, uniformly mixing, standing for 5min, taking 0 tube as a blank tube, measuring ultraviolet absorbance at 595nm, taking standard protein content (mug) as an abscissa X, taking a corresponding absorbance value (A) as an ordinate Y, drawing a standard curve, and establishing a regression equation. (3) Accurately sucking 0.3mL of each polysaccharide sample solution, adding distilled water to 1mL, sequentially adding 5mL from 1.3.2.2 (2), performing the same operation, measuring absorbance, and calculating protein content of each alcohol precipitation component sample according to absorbance value.
1.3.2.3 molecular weight determination
The molecular weight of each component polysaccharide was determined by High Performance Gel Permeation Chromatography (HPGPC) using the following procedure: (1) Dextran references (5, 12, 25, 50, 80, 150 ku) with different molecular weights are respectively precisely weighed, a reference solution with the mass concentration of 2.0mg/mL is prepared by using a 0.02M phosphate buffer (pH 6.0), and the reference solution is filtered by using a 0.45 mu M microporous filter membrane. The chromatographic column TSK-GEL G4000 PWXL (7.5 mm. Times.30.0 cm), mobile phase 0.02M phosphate buffer (pH 6.0), sample volume 20. Mu.L and flow rate 0.3mL/min were measured using a high performance liquid chromatograph, differential refractive index detector. And drawing a standard curve by taking chromatographic column retention time RT as an abscissa X and taking the molecular weight log Lg (Mw) of the glucan reference substance as an ordinate Y, and establishing a regression equation. (2) The polysaccharide samples of each component are weighed, phosphate buffer solution is used for preparing a solution with the concentration of 2mg/mL, a microporous filter membrane with the thickness of 0.45 mu m is used for filtering, the measurement is carried out according to the condition of 1.3.2.3 (1), and the molecular weight of the polysaccharide samples of each component is calculated according to the retention time of the samples.
1.3.2.4 monosaccharide composition determination
The monosaccharide composition in each sample was determined by the method established by Jacob Lehrfeld et al, and the specific procedure was as follows: (1) Respectively precisely weighing polysaccharide samples 2mg in a reaction bottle, adding 2mol/L trifluoroacetic acid 2mL, sealing, hydrolyzing at 121deg.C for 2 hr, vacuum pumping to neutrality, and treating with NaCO 3 Acetylation and NaBH 4 Neutral sugar and acid sugar were detected simultaneously by gas chromatograph after reduction. Chromatographic conditions: rtx-50 capillary column (30.0mX0.25mm X0.25 μm) with a temperature program of: 180 ℃ (2 min) to 6 ℃/min,210 ℃ to 0.3 ℃/min,215 ℃ to 6 ℃/min, and 240 ℃ (30 min). Air and nitrogen are respectively used as carrier gases, the flow rate of the carrier gases is 0.88mL/min, and the split ratio is 19:1, the pressure is 110kPa, and the detector is a hydrogen flame ionization detector. (2) Accurately weighing 10mg of fucose, rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid standard monosaccharides, preparing a nine-sugar standard sample according to 1.3.2.4 (1), and detecting by a gas chromatograph.
1.3.2.5 Infrared Spectrometry
Weighing 2mg of polysaccharide sample of each component, adding 100mg of KBr dry powder, uniformly mixing, fully grinding in an agate mortar, tabletting, and adopting a Fourier transform infrared spectrometer to carry out 4000-400 cm -1 Is measured for each component sample.
1.3.2.6 appearance and morphology observations
And (3) taking a proper amount of polysaccharide samples of all the alcohol precipitation components, placing the polysaccharide samples on a sample stage, placing the sample stage in an ion sputtering instrument, plating a layer of conductive gold film, and observing the sample by adopting a scanning electron microscope.
1.3.3 study of the immunocompetence of the alcohol precipitation fractions
1.3.3.1 effects on RAW264.7 cell proliferation
(1) RAW264.7 cell culture: RAW264.7 cell culture method in reference (research on physicochemical properties and biological Activity of polysaccharide and its components purified by fractional precipitation Guiping, university of northwest, 2018), taking frozen RAW264.7 cells, placing into a water bath of 37deg.C for 1-2min, carefully blowing the frozen solution with a pipette, transferring into a sterile centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1mL of complete culture medium of DMEM, gently blowing uniformly, transferring into a culture dish, and placing into 5% CO 2 Culturing in a cell culture box at 37 ℃. When the cell density reaches 80-90%, discarding the upper culture solution, washing the cells with 1-2mL of PBS buffer solution, discarding PBS, digesting the cells with 1mL of trypsin solution, adding 1mL of complete culture medium to stop digestion, lightly blowing the cells by a liquid-transfering device until the cells are completely detached from a culture dish, transferring the cell suspension into a sterile centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1mL of DMEM complete culture medium, lightly blowing the cells uniformly, and passaging at a ratio of 1:2-1:3 for later use.
(2) Taking RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to 1×10 5 Inoculating 100 μl of each well into 96-well plate, placing at 37deg.C, 5% CO 2 Culturing for 12h in an incubator, discarding the supernatant, respectively adding polysaccharide solutions of various components with different mass concentrations (50-800 mug/mL), taking a blank culture medium as a blank control, adding 100 mugMTT (0.5 mg/mL) solution into each hole after continuous culturing for 24h, continuously culturing for 4h, adding MTT solution into each hole, washing cells with 100 mugLPBS, discarding PBS, adding 100 mug of DMSO solution into each hole, oscillating for 5min, detecting the absorbance at 570nm by an enzyme-labeled instrument, calculating the cell viability, setting 5 duplicate holes for each group of concentration, and controlling the cell viability (%) = Abs sample/Abs to be multiplied by 100%.
1.3.3.2RAW264.7 cell phagocytic capacity assay
The cell treatment method comprises the following steps of 1.3.3.1, adding 5-100 μg/mL of polysaccharide solution, taking blank medium as blank control, LPS (10 μg/mL) as positive control, and 5% CO at 37deg.C 2 Culturing in incubator for 24 hr, adding 0.1% culture solution into each wellCulturing the neutral red solution for 4 hours in a culture box at 37 ℃, discarding the neutral red solution, washing cells 3-4 times by using 100 mu L of PBS (phosphate buffer solution) for each hole until the washing solution is colorless, discarding the PBS, adding 100 mu L of cell lysate (acetic acid: ethanol=1:1) into each hole, continuously culturing for 1 hour in the culture box, shaking and mixing uniformly, detecting the light absorption value at 540nm by an enzyme-labeling instrument, calculating the phagocytic capacity of the cells on the neutral red, setting 5 compound holes for each group, and obtaining a phagocytic index=abs sample/Abs blank multiplied by 100%.
1.3.3.3 Effect on RAW264.7 cell acid phosphatase Activity
According to the cell treatment method, 1.3.3.1, 5-100 mug/mL of polysaccharide solutions of all components are respectively added, 5 compound holes are formed in each group, a serum-free culture medium is used as a blank control, LPS (10 mug/mL) is used as a positive control, the culture is continued for 24 hours, the culture solution is discarded after 24 hours, 25 mug of Triton X-100 (1%) and 150 mug of p-nitrophenylphosphoric acid solution (1 mg/mL) are added, the culture is continued for 1 hour, 50 mug L3.0M NaOH solution is added to each hole, the mixture is uniformly mixed by shaking at a constant temperature of 37 ℃, an enzyme label instrument is used for measuring the absorbance at 405nm, and the acid phosphatase activity=abs sample/Abs blank multiplied by 100%.
1.3.3.4 Effect on NO secretion by RAW264.7 cells
Cell treatment method is as 1.3.3.1, taking logarithmic phase cell, regulating cell concentration, inoculating into 96-well plate, each well having 100 μl, 37deg.C, 5% CO 2 Culturing in an incubator for 12h, discarding the supernatant, washing cells for 1-2 times by using PBS, respectively adding polysaccharide solutions (25-200 mug/mL) of each component with different mass concentrations, taking a serum-free culture medium as a blank control, taking LPS as a positive control, setting 5 compound wells for each group of concentration, culturing in the incubator for 24h, sucking 50 mu L of cell supernatant of each well into a new 96-well plate, respectively adding 50 mu L of Griess reagent A under the dark condition, vibrating and incubating for 10min at the constant temperature of 37 ℃, adding 50 mu L of Griess reagent B into each well, vibrating for 10min at the constant temperature of 37 ℃, and measuring the absorbance value at 540nm by using an enzyme-labeled incubator. Simultaneously adding NaNO with different concentrations on a 96-well plate 2 Adding 50 mu L of Griess reagent A and Griess reagent B respectively as standard solutions for reacting for 10min, measuring absorbance at 540nm by using an enzyme-labeled instrument, taking NO secretion (mu mol/L) as abscissa X, taking absorbance (A) as ordinate Y, and drawing standard curve of NO contentLine, NO secretion was calculated from cell supernatant uptake.
1.3.4 data analysis
The experiment adopts average value standard deviationThe experimental data are shown for statistical analysis using SPSS 19.0 and plotted using graphpad prism 7.0 software. Significance level at P<0.05 is a significant difference.
2 results and analysis
2.1 physicochemical Properties of the various alcohol precipitation Components of PSPW
2.1.1 measurement of the yields, sugar content and protein content of the respective alcohol precipitated components, as shown in Table 1, the yields of the respective components were as follows: PSPW-2 (1.48%) > PSPW-3 (1.03%) > PSPW-4 (0.71%) > PSPW-1 (0.49%). From the sugar content and the protein content results, the rhizoma polygonati water-soluble polysaccharide content PSPW-3 (57.82 +/-0.16%) > PSPW-4 (57.44 +/-1.23%) > PSPW-2 (45.89 +/-0.97%) > PSPW-1 (43.21 +/-0.38%); rhizoma Polygonati protein content PSPW-1 (34.33+ -1.72%) > PSPW-4 (33.82 + -0.54%) > PSPW-2 (24.71+ -0.26%) > PSPW-3 (20.08+ -0.72%). From this, it was found that each of the above components was glycoprotein complex.
TABLE 1 physicochemical Properties of the various alcohol precipitation Components of PSPW
2.1.2 monosaccharide composition determination
The GC gas phase chromatogram of the monosaccharide composition of each component is shown in figure 1. From the results, the monosaccharide composition and the ratio of the components are obviously different, and each component contains galactose with higher ratio. As can be seen from Table 2, PSPW-1 monosaccharide composition mainly comprises galactose (52.75%) and galacturonic acid (30.15%), the other 3 components contain trace galacturonic acid, and the acidic sugar is concentrated and distributed in 30% ethanol precipitation component; the monosaccharide composition in PSPW-3 was predominantly galactose (79.3%) and mannose (15.33%). PSPW-4 monosaccharide composition was based on galactose (56.43%), mannose (36.28%) and glucose (7.29%).
TABLE 2PSPW fractional alcohol precipitation of the monosaccharide composition of the fractions
Note that: "-", is not detected.
2.1.3 molecular Mass determination
The molecular mass regression equation was lg (Mw) = -0.1742RT+9.7715 (R) 2 = 0.9921). As shown in fig. 2, the PSPW alcohol precipitation components have a uniform and symmetrical peak on the HPLC spectrum, and the molecular mass of each alcohol precipitation component gradually decreases as the volume fraction of ethanol gradually increases. Substituting retention time of each component sample into regression equation to calculate and obtain molecular masses of PSPW-1, PSPW-2, PSPW-3 and PSPW-4 of 2.26X10 respectively 4 、2.24×10 4 、1.15×10 4 And 6.58×10 3 u。
2.1.4 IR Spectrometry of samples
As shown in figure 3 of the specification, the related literature combined with the infrared characteristics of polysaccharide can be known from 4000-400 cm -1 The general absorption characteristic peaks of polysaccharide exist in each component in the range. At 3400cm -1 The vicinity of the material has wide strong absorption peak, and the 3447.01cm < -1 > is a hydroxyl O-H telescopic vibration absorption peak; 2931.04cm -1 C-H stretching vibration absorption peak of methyl, methylene or methine; at 1750.66cm -1 A telescopic vibration absorption peak of carbonyl c=o; 1649.36cm -1 A polysaccharide hydroxyl group O-H flexural vibration absorption peak; at 1438.69cm -1 An absorption peak appears, which is presumed to be an N-H bond bending stretching vibration absorption peak, indicating that each component is glycoprotein complex; at 1163.7cm -1 The carbon-oxygen C-O absorption peak on the sugar ring; 1072.85cm -1 The position is an alcohol hydroxyl O-H variable angle vibration absorption peak; at 890.76cm -1 The characteristic absorption peak of the beta-glycosidic bond shows that each component is beta-polysaccharide.
2.1.5 appearance and morphology observations of the Components
Scanning electron microscopy has been widely used in recent years for observing the appearance of polysaccharides as an effective tool for observing the appearance of samples. As shown in FIG. 4, the microscopic morphology of each alcohol precipitation component is mainly in the form of regular flakes, wherein the surfaces of PSPW-2 and PSPW-3 are smoother; PSPW-1 and PSPW-4 have a rough surface, possibly associated with a high proportion of proteins contained therein.
2.2 immunocompetence of the alcohol precipitation fractions
2.2.1 Effect of alcohol precipitation Components on RAW264.7 cell viability
As can be seen from the description of the figure 5 (a), each alcohol precipitation component of PSPW (rhizoma polygonati water-soluble polysaccharide) has a promoting effect on the proliferation ability of RAW264.7 cells and a certain dose dependency in the mass concentration range of 50-800 mug/mL, the PSPW can obviously promote the cell viability (P < 0.05) in the range of 400-800 mug/mL, and each alcohol precipitation component can obviously promote the proliferation ability (P < 0.05) of RAW264.7 cells in the range of 200-800 mug/mL. At high doses (800. Mu.g/mL), PSPW and PSPW-1-4 cell viability were 115.09.+ -. 2.90%, 113.16.+ -. 1.90%, 120.39.+ -. 2.37%, 137.62.+ -. 2.12% and 132.58.+ -. 1.67%, respectively. The cell viability of each component was found to be: PSPW-3> PSPW-4> PSPW-2> PSPW-1. When the mass concentration of each group of samples is lower than 100 mug/mL, the RAW264.7 cell viability is not affected (P is more than 0.05), and the subsequent experiment selects samples with the mass concentration of 12.5-100 mug/mL to carry out each index measurement.
2.2.2 Effect of alcohol precipitation Components on phagocytic Capacity of RAW264.7 cells
Referring to the description, FIG. 5 (b), there was no significant effect on RAW264.7 cell phagocytic activity (P > 0.05) when PSPW mass concentration was less than 10. Mu.g/mL, compared to the blank group. Within the range of 10-100 mug/mL, PSPW-1, PSPW-2 and PSPW-4 can obviously promote phagocytic capacity of RAW264.7 cells and have certain dose dependency (P < 0.05); in the range of 5-100 mug/mL, compared with a blank group, each dose group of PSPW-3 can obviously promote the phagocytic capacity (P < 0.05) of RAW264.7 cells. PSPW-3 was found to be the strongest in promoting phagocytosis of neutral red by macrophages when compared to the remaining groups.
2.2.3 Effect of alcohol precipitation Components on RAW264.7 cell acid phosphatase Activity
As shown in the figure 5 (c) of the specification, compared with a blank group, in the mass concentration range of 5-100 mug/mL, each alcohol precipitation component of the rhizoma polygonati water-soluble polysaccharide can improve the activity of RAW264.7 cell acid phosphatase and has certain dose dependency; within the range of 10-100 mug/mL, PSPW and PSPW-1, PSPW-2 and PSPW-4 can promote the activity of cell phosphatase and have concentration dependence. PSPW-3 can obviously promote the activity (< 0.05) of RAW264.7 cell acid phosphatase at the mass concentration of 5 mug/mL, and the phosphatase activity is 1.38 times of that of a blank group when the mass concentration reaches 100 mug/mL. The comparison among the alcohol precipitation components shows that PSPW-3 has the strongest regulation effect on RAW264.7 cell acid phosphatase.
2.2.4 Effect of alcohol precipitation Components on NO secretion by RAW264.7 cells
As shown in the attached figure 5 (d) of the specification, compared with a blank group, in the mass concentration range of 5-100 mug/mL, each component of the rhizoma polygonati water-soluble polysaccharide can obviously increase the NO secretion of RAW264.7 cells and has certain dose dependency. Compared with the blank group, the amounts of NO secretion of cells in the PSPW, PSPW-1, PSPW-2, PSPW-3 and PSPW-4 groups are increased by 36.70%, 35.36%, 41.90%, 59.11% and 44.89%, respectively, wherein the promotion effect by PSPW-3 is most remarkable. From the above results, it was found that PSPW-3 has the strongest immunoregulatory activity.
Example 2
Extracting polysaccharide from rhizoma polygonati: extracting water-soluble polysaccharide in rhizoma polygonati prepared by wine according to a polysaccharide extraction method established in the earlier stage of the subject group to obtain water-soluble polysaccharide in rhizoma polygonati prepared by wine;
separation and purification of polygonatum sibiricum polysaccharide: weighing the water-soluble polysaccharide of rhizoma polygonati prepared by using the wine, adding double distilled water for dissolving to enable the mass concentration to be 0.5%, slowly adding a certain volume of ethanol solution to enable the mass fraction of ethanol in the solution to reach 30%, standing for 10 hours at 4 ℃, centrifuging, respectively collecting sediment and supernatant I, adding double distilled water for dissolving the sediment, discarding the sediment, and freeze-drying the supernatant to obtain a 30% water-soluble polysaccharide alcohol precipitation component PSPW-1;
taking supernatant I in the step (II), continuously adding ethanol to a mass fraction of 50%, standing at 4 ℃ for 8-16 h, centrifuging, respectively collecting precipitate and supernatant II, performing related operations on the precipitate as in the step (II), and freeze-drying to obtain 50% of rhizoma polygonati water-soluble polysaccharide alcohol precipitation component PSPW-2;
taking the supernatant II in the step, adding ethanol to the supernatant II until the mass fraction is 70%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain a supernatant III and a 70% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-3;
and fifthly, taking the supernatant III in the step, adding ethanol to the mass fraction of 80%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain an 80% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-4.
Example 3
Extracting polysaccharide from rhizoma polygonati: extracting water-soluble polysaccharide in rhizoma polygonati prepared by wine according to a polysaccharide extraction method established in the earlier stage of the subject group to obtain water-soluble polysaccharide in rhizoma polygonati prepared by wine;
separation and purification of polygonatum sibiricum polysaccharide: weighing the water-soluble polysaccharide of rhizoma polygonati prepared by using the wine, adding double distilled water for dissolving to enable the mass concentration to be 2%, slowly adding a certain volume of ethanol solution to enable the mass fraction of ethanol in the solution to be 30%, standing for 10 hours at 5 ℃, centrifuging, respectively collecting sediment and supernatant I, adding double distilled water for dissolving the sediment, discarding the sediment, and freeze-drying the supernatant to obtain a 30% water-soluble polysaccharide alcohol precipitation component PSPW-1 of the rhizoma polygonati;
taking supernatant I in the step (II), continuously adding ethanol to a mass fraction of 50%, standing at 2 ℃ for 8-16 h, centrifuging, respectively collecting precipitate and supernatant II, performing related operations on the precipitate as in the step (II), and freeze-drying to obtain 50% of rhizoma polygonati water-soluble polysaccharide alcohol precipitation component PSPW-2;
taking the supernatant II in the step, adding ethanol to the supernatant II until the mass fraction is 70%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain a supernatant III and a 70% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-3;
and fifthly, taking the supernatant III in the step, adding ethanol to the mass fraction of 80%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain an 80% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-4.
Example 4
Extracting polysaccharide from rhizoma polygonati: extracting water-soluble polysaccharide in rhizoma polygonati prepared by wine according to a polysaccharide extraction method established in the earlier stage of the subject group to obtain water-soluble polysaccharide in rhizoma polygonati prepared by wine;
separation and purification of polygonatum sibiricum polysaccharide: weighing the water-soluble polysaccharide of rhizoma polygonati prepared by using the wine, adding double distilled water for dissolving to enable the mass concentration to be 1.5%, slowly adding a certain volume of ethanol solution to enable the mass fraction of ethanol in the solution to reach 30%, standing at 6 ℃ for 16 hours, centrifuging, respectively collecting sediment and supernatant I, adding double distilled water for dissolving the sediment, discarding the sediment, and freeze-drying the supernatant to obtain a 30% water-soluble polysaccharide alcohol precipitation component PSPW-1;
taking the supernatant I in the step II, continuously adding ethanol until the mass fraction is 50%, standing for 10 hours at 6 ℃, centrifuging, respectively collecting a precipitate and a supernatant II, performing related operations on the precipitate according to the same steps, and performing freeze drying to obtain a 50% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-2;
taking the supernatant II in the step, adding ethanol to the supernatant II until the mass fraction is 70%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain a supernatant III and a 70% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-3;
and fifthly, taking the supernatant III in the step, adding ethanol to the mass fraction of 80%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain an 80% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-4.
The rhizoma polygonati water-soluble polysaccharide extract prepared by the preparation method of the embodiment 1-4 is detected by adopting methods such as Gas Chromatography (GC), high Performance Gel Permeation Chromatography (HPGPC), fourier transform infrared spectroscopy (FT-IR) and the like:
the rhizoma polygonati polysaccharide extract comprises the following raw material medicines in percentage by mole: 50-85% of galactose, 0-40% of galacturonic acid, 2-40% of mannose, 2-15% of rhamnose, 1-8% of glucose and 0-5% of arabinose.
The rhizoma polygonati extract also comprises trace amounts of the following components: 0 to 0.1 percent of fucose, 0 to 0.1 percent of xylose, 0 to 0.1 percent of glucuronic acid.
Finally, it should be noted that: the invention is not limited to the specific embodiments described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art should, in light of the present disclosure, make any changes, equivalents, and modifications that are within the spirit and scope of the present invention.
Claims (9)
1. A preparation method of a rhizoma polygonati polysaccharide extract, which is characterized by comprising the following steps:
preparing water-soluble polysaccharide of rhizoma polygonati prepared by wine:
adding yellow wine into rhizoma polygonati crude products according to a volume ratio of 5:1, stirring uniformly, moistening for 10-12 hours, steaming for 4h at 120 ℃ in an autoclave, steaming to the inside and outside for moistening black, taking out, slicing, drying in a baking oven at 60 ℃ to obtain rhizoma polygonati wine prepared decoction pieces, crushing, sieving with a 100-mesh sieve to obtain rhizoma polygonati wine prepared decoction piece powder, adding 10 times of distilled water, extracting at 80 ℃ for 2 times, filtering the extracting solution each time for 2h, merging 2 times of extracting solutions, recovering filtrate, adding 4 times of absolute ethyl alcohol, precipitating with alcohol for 12 hours at 4 ℃ for filtering, recovering alcohol, adding distilled water for redissolution, deproteinizing, centrifuging, discarding precipitate, placing supernatant in a 3000Da dialysis bag for dialysis, concentrating the dialyzate under reduced pressure, and freeze-drying to obtain rhizoma polygonati water-soluble polysaccharide;
separation and purification of polygonatum sibiricum polysaccharide: weighing the water-soluble polysaccharide of rhizoma polygonati prepared by using the wine, adding double distilled water for dissolving to enable the mass concentration to be 0.5-2%, slowly adding a certain volume of ethanol solution to enable the mass fraction of the ethanol in the solution to be 30%, standing for 8-16 h at the temperature of 2-6 ℃, centrifuging, respectively collecting sediment and supernatant I, adding double distilled water for dissolving the sediment, discarding the sediment, and freeze-drying the supernatant to obtain a 30% water-soluble polysaccharide alcohol sediment component PSPW-1;
taking supernatant I in the step II, continuously adding ethanol until the mass fraction is 50%, standing at 2-6 ℃ for 8-16 h, centrifuging, respectively collecting precipitate and supernatant II, performing related operation on the precipitate as in the step II, and freeze-drying to obtain 50% of rhizoma polygonati water-soluble polysaccharide alcohol precipitation component PSPW-2;
taking the supernatant II in the step, adding ethanol to the supernatant II until the mass fraction is 70%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain a supernatant III and a 70% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-3;
and fifthly, taking the supernatant III in the step, adding ethanol to the mass fraction of 80%, performing related operations on precipitation according to the same steps, and freeze-drying to obtain an 80% polygonatum water-soluble polysaccharide alcohol precipitation component PSPW-4.
2. The method of claim 1, wherein the centrifugal speed is 8000-12000 r/min and the centrifugal time is 8-12 min.
3. The method for preparing the rhizoma polygonati polysaccharide extract according to claim 1, wherein the freeze-drying conditions in the step (a) are as follows: the sample solution is placed at the temperature of minus 75 ℃ to minus 85 ℃ for 2 to 4 hours, the freezing temperature of a freeze dryer is minus 50 ℃ to minus 60 ℃, the temperature of a frozen shelf in a cold trap chamber is minus 20 ℃ to minus 30 ℃, the sublimation drying temperature is 45 ℃ to 55 ℃, the analysis drying temperature is 30 ℃ to 40 ℃, and the analysis time is 7 to 9 hours.
4. A rhizoma polygonati polysaccharide extract prepared by the preparation method of the rhizoma polygonati polysaccharide extract of claim 1, which is characterized by comprising the following raw materials in percentage by mole: galactose 52.75%, galacturonic acid 30.15%, mannose 4.48%, rhamnose 7.37%, glucose 2.33%, arabinose 2.92%.
5. A rhizoma polygonati polysaccharide extract prepared by the preparation method of the rhizoma polygonati polysaccharide extract of claim 1, which is characterized by comprising the following raw materials in percentage by mole: 82.25% of galactose, 0.02% of galacturonic acid, 2.22% of mannose, 11.01% of rhamnose, 1.57% of glucose and 2.95% of arabinose.
6. A rhizoma polygonati polysaccharide extract prepared by the preparation method of the rhizoma polygonati polysaccharide extract of claim 1, which is characterized by comprising the following raw materials in percentage by mole: 79.3% of galactose, 0.05% of galacturonic acid, 15.33% of mannose, 2.92% of rhamnose, 2.45% of glucose and 0.01% of arabinose.
7. A rhizoma polygonati polysaccharide extract prepared by the preparation method of the rhizoma polygonati polysaccharide extract of claim 1, which is characterized by comprising the following raw materials in percentage by mole: 56.43% galactose, 0.03% galacturonic acid, 36.28% mannose, 0.01% rhamnose, 7.29% glucose and 0.02% arabinose.
8. The use of the extract of Polygonatum sibiricum polysaccharide as claimed in any one of claims 4 to 7, wherein the extract of Polygonatum sibiricum polysaccharide is used for preparing functional food additives.
9. The use of the extract of Polygonatum sibiricum polysaccharide as claimed in any one of claims 4 to 7, wherein the extract of Polygonatum sibiricum polysaccharide is used for preparing a food additive exhibiting an immunomodulating effect.
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