CN116675782A - Method for extracting polygonatum odoratum polysaccharide and determination method thereof - Google Patents
Method for extracting polygonatum odoratum polysaccharide and determination method thereof Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 108
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 107
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 107
- 241001633680 Polygonatum odoratum Species 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 48
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 241000756042 Polygonatum Species 0.000 claims abstract description 22
- 238000001556 precipitation Methods 0.000 claims abstract description 9
- 238000003809 water extraction Methods 0.000 claims abstract description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 17
- 239000002244 precipitate Substances 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 13
- 238000007664 blowing Methods 0.000 claims description 11
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 11
- 238000005303 weighing Methods 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- 239000012192 staining solution Substances 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 5
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 4
- 229940088598 enzyme Drugs 0.000 claims description 4
- 239000008187 granular material Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 108090000526 Papain Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000011436 enzymatic extraction method Methods 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011344 liquid material Substances 0.000 claims description 3
- 238000000643 oven drying Methods 0.000 claims description 3
- 229940055729 papain Drugs 0.000 claims description 3
- 235000019834 papain Nutrition 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 3
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims description 2
- 239000001913 cellulose Substances 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 11
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 235000013305 food Nutrition 0.000 abstract description 5
- 239000013589 supplement Substances 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 12
- 229960001031 glucose Drugs 0.000 description 12
- 230000003544 deproteinization Effects 0.000 description 9
- 230000014759 maintenance of location Effects 0.000 description 6
- 210000002421 cell wall Anatomy 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000031971 Yin Deficiency Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
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- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 239000012086 standard solution Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for extracting polygonatum odoratum polysaccharide by a water extraction and alcohol precipitation method, a method for extracting polygonatum odoratum polysaccharide by an ultrasonic-assisted enzyme method, a method for measuring the sugar content of polygonatum odoratum polysaccharide and a method for measuring the protein content of polygonatum odoratum polysaccharide. The invention supplements the current proposal of industrialized production of the extracted and purified polygonatum polysaccharide, can perfect the application of the polygonatum polysaccharide in food by measuring the polygonatum polysaccharide, and can evaluate the advantages and disadvantages of the polygonatum polysaccharide extraction method more accurately by measuring the sugar and protein of the polygonatum polysaccharide.
Description
Technical Field
The invention relates to a method for extracting polygonatum odoratum polysaccharide and a determination method thereof.
Background
Rhizoma Polygonati Odorati is also called herba Hedyotidis Diffusae, and root of herba Hedyotidis Diffusae, which is the dry rhizome of rhizoma Polygonati Odorati of Liliaceae, and has the disadvantages of excessive heat, inability to shake, and bone and muscle formation. The black face is removed after long-term administration, the color is good, the body is moist, the weight is light, and the people are not old. The polygonatum polysaccharide is one of main active ingredients of polygonatum, and the research on the polygonatum polysaccharide is mainly focused on the aspects of extraction, purification, separation and the like at present, and has less structure-activity relationship and application research on the polygonatum polysaccharide. The polygonatum polysaccharide is natural and harmless, can be used for treating diabetes and hyperthyroidism yin deficiency, improving obesity and nonalcoholic fatty liver, resisting fatigue, aging, swelling and immunity adjustment, improving cognitive dysfunction and the like, and has great application potential in food health care. The polygonatum odoratum is used as a medicine and food dual-purpose substance, and the extracted polygonatum odoratum polysaccharide is required to be ensured not to cause any harm to human health when being directly applied to foods.
Disclosure of Invention
The invention aims to solve the technical problems and provide a method for extracting polygonatum odoratum polysaccharide and a determination method thereof, which supplement the current scheme for industrially producing the extracted and purified polygonatum odoratum polysaccharide, and can perfect the application of the polygonatum odoratum polysaccharide in food through the determination of the polygonatum odoratum polysaccharide.
The technical scheme adopted for solving the technical problems is as follows: the method for extracting the polygonatum polysaccharide by the water extraction and alcohol precipitation method comprises the following steps:
step one: cutting the cleaned fresh rhizoma polygonati into small blocks, putting the small blocks into a crucible, and then putting the crucible into an electrothermal blowing drying oven for drying; grinding dried rhizoma Polygonati Odorati into powder or granule, transferring rhizoma Polygonati Odorati into beaker, and adding 95% absolute ethanol to obtain yellow solution;
step two: then placing the beaker containing the yellow solution into an ultrasonic cleaner for extraction to obtain turbid yellow solution;
step three: filtering the turbid yellow solution with a semipermeable membrane under reduced pressure to obtain pale yellow solution and pale yellow powder, transferring the powder and the semipermeable membrane into a beaker together, adding water for dissolution, and condensing and refluxing the solution obtained by suction filtration;
step four: evaporating the condensed and reflowed solution by using a rotary evaporator to obtain a solution after rotary evaporation, transferring the solution into a beaker, adding 95% absolute ethyl alcohol, and standing to generate a precipitate;
step five: and (3) placing the beaker which is placed still to generate the sediment into a centrifuge, carrying out solid-liquid separation, taking out the sediment, and washing the sediment with 95% absolute ethyl alcohol to obtain the polygonatum polysaccharide.
Preferably, in the first step, 95% absolute ethanol is added into a beaker, and the liquid-to-material ratio of the absolute ethanol to the powdery or small granular polygonatum odoratum is 1:2.
preferably, the drying temperature of the electrothermal blowing drying oven in the first step is less than or equal to 55 ℃.
Preferably, the drying temperature of the electrothermal blowing drying oven in the first step is constant temperature 55 ℃ and the drying time is 9.5 hours.
Preferably, the extraction temperature of the ultrasonic cleaner in the second step is 55-60 ℃.
Preferably, the extraction temperature of the ultrasonic cleaner in the second step is 55 ℃, the extraction time is 15min, and a turbid yellow solution is obtained after repeating the steps twice.
Preferably, in the fourth step, the solution condensed and refluxed is evaporated by a rotary evaporator, the initial temperature is 40 ℃, the initial rotating speed is 50RPM, the temperature is gradually increased to 60RPM, and the temperature is gradually increased to 60 ℃ for 80 minutes.
Preferably, in the fifth step, the solution is placed in a centrifuge, and the rotational speed of the centrifuge is 2000rmp, and the time is 15min.
The ultrasonic-assisted enzymatic extraction method of the polygonatum odoratum polysaccharide comprises the following steps of:
step 1: cutting fresh rhizoma Polygonati Odorati into small pieces, and oven drying in electrothermal air drying oven;
step 2: crushing the dried polygonatum odoratum in the step 1 in a crusher, and filtering the crushed polygonatum odoratum in a beaker by using a 24-mesh sieve;
step 3: adding a Hac-NaAc buffer solution and cellulose decomposing enzyme into a beaker, placing the beaker with the polygonatum odoratum solution into an ultrasonic cleaner for extraction, and standing the extracted polygonatum odoratum polysaccharide solution;
step 4: pouring the rhizoma polygonati odorati polysaccharide solution extracted after standing into a centrifuge tube, and putting the centrifuge tube into the centrifuge for centrifugation;
step 5: extracting the centrifuged precipitate into a beaker, and placing the beaker into an electrothermal blowing drying box for drying to obtain the polygonatum polysaccharide.
Preferably, the drying temperature of the electrothermal blowing drying oven is 55 ℃.
Preferably, the temperature of extraction of the beaker with the rhizoma Polygonati Odorati solution in step 3 in the ultrasonic cleaner is 35 ℃.
The method for measuring the sugar content of the polygonatum odoratum polysaccharide comprises the following steps of:
step A1: dissolving rhizoma Polygonati Odorati polysaccharide obtained by the method for extracting rhizoma Polygonati Odorati polysaccharide in beaker, and adding into 100ml volumetric flask to fix volume to obtain rhizoma Polygonati Odorati polysaccharide solution;
step A2: preparing six volumetric flasks, respectively weighing 1ml, 2ml, 3ml, 4ml, 5m and 6ml of rhizoma Polygonati Odorati polysaccharide solution, adding into the volumetric flask, and adding distilled water to constant volume;
step A3: weighing 0.0700g of anthrone, preparing 85% concentrated sulfuric acid, and putting the anthrone into the prepared concentrated sulfuric acid;
step A4: respectively adding 1ml of the prepared polygonatum odoratum polysaccharide solution in the step A2 into six test tubes, respectively adding 5ml of the anthrone-concentrated sulfuric acid solution prepared in the step A2 into the six test tubes, heating in a water bath kettle at 100 ℃, and finally cooling in a refrigerator;
step A5: preparing a glucose solution, measuring absorbance of the glucose solution at 626nm wavelength, drawing a relation curve of absorbance and glucose, obtaining a relation equation of absorbance and glucose according to the relation curve of absorbance and glucose, measuring absorbance of the cooled polygonatum odoratum polysaccharide solution in the six test tubes in the step A4 at 626nm wavelength, and obtaining the concentration of the cooled polygonatum odoratum polysaccharide solution in the six test tubes according to the relation equation of absorbance and glucose.
The method for measuring the protein content of the polygonatum odoratum polysaccharide comprises the following steps of:
step B1: preparing a polygonatum odoratum polysaccharide solution with the concentration of 0.1% by using the polygonatum odoratum polysaccharide obtained by the method for extracting the polygonatum odoratum polysaccharide, adding 0.2% papain, and adding sodium acetate as an acetic acid buffer solution to adjust the PH of the polygonatum odoratum polysaccharide solution to be 7; heating and sterilizing at high temperature after constant-temperature water bath to generate white flocculent precipitate, and filtering the white flocculent precipitate to remove the precipitate;
step B2: pouring the filtrate into a 75ml crucible, placing the crucible in a magnetic stirrer, placing a magnet until the filtrate is evaporated to dryness, collecting the crystals obtained after the evaporation to dryness, grinding the crystals into powder, and sealing and preserving the powder;
step B3: 0.0500G of Coomassie brilliant blue G250 is weighed, 25mL of 95% absolute ethyl alcohol is used for dissolution, then 50mL of 85% (W/V) phosphoric acid is added, distilled water is used for constant volume to 500mL, and the concentration is 0.01%, so that a Marc brilliant blue staining solution is obtained;
step B4: weighing 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of deproteinized polygonatum polysaccharide solution, respectively placing the above materials in colorimetric tubes 1-6, and adding distilled water to 1.0mL; respectively adding 5mL of coomassie brilliant blue staining solution, uniformly mixing, standing, and taking distilled water as a blank control after the same treatment;
step B5: preparing a bovine serum albumin solution, measuring absorbance of the bovine serum albumin solution at 595nm wavelength by an ultraviolet-visible spectrophotometer, obtaining an absorbance-protein relation curve, obtaining an absorbance-protein relation equation according to the absorbance-protein relation curve, measuring absorbance of the rhizoma polygonati officinalis polysaccharide protein content in the colorimetric tubes 1-6 at 595nm wavelength, and obtaining the rhizoma polygonati officinalis polysaccharide protein content in the colorimetric tubes 1-6 according to the absorbance-protein relation equation.
The invention has the beneficial effects that: the invention optimizes the extraction process of the polygonatum odoratum polysaccharide, explores the optimal condition for extracting the polygonatum odoratum polysaccharide by an ultrasonic-enzyme auxiliary method, improves the current method for extracting the polygonatum odoratum polysaccharide, greatly shortens the extraction time and greatly improves the extraction rate; by measuring the sugar content and the protein removal rate of the polygonatum polysaccharide, the advantages and disadvantages of the polygonatum polysaccharide extraction method can be more accurately evaluated.
Drawings
FIG. 1 is a graph of anhydrous glucose standard in an embodiment of the present invention.
FIG. 2 is a graph of protein standards in an embodiment of the invention.
FIG. 3 is a graph showing the relationship between protein removal rate and polysaccharide retention rate before deproteinization in the examples of the present invention.
FIG. 4 is a graph showing the relationship between protein removal rate and polysaccharide retention rate after deproteinization in the examples of the present invention.
Detailed Description
The invention is further described below with reference to the drawings and embodiments.
Example 1
The method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation comprises the following steps: weighing 5.0g of clean fresh rhizoma polygonati officinalis, cutting into small pieces, placing into a crucible, and placing the crucible into an electrothermal blowing drying oven for drying, wherein the constant temperature is 55 ℃ and the drying time is 9.5 hours. Grinding dried rhizoma Polygonati Odorati in grinding body for 10min, grinding into powder or granule, transferring the powder or granule into beaker, adding 95% absolute ethanol into the beaker, and mixing the materials at a ratio of 1:2 to obtain yellow solution. Extracting the beaker in an ultrasonic cleaner at 55deg.C for 15min, and repeating for two times to obtain turbid yellow solution; the mixture was filtered under reduced pressure through a semipermeable membrane to obtain a pale yellow solution and pale yellow powder, the powder and the semipermeable membrane were transferred together into a 500ml beaker, 500ml of water was added for dissolution, and the solution obtained by suction filtration was subjected to condensation reflux. The distilled solution was evaporated with a rotary evaporator at an initial temperature of 40℃and an initial rotation speed of 50RPM, gradually increased to 60RPM, and gradually increased to 60℃for 80 minutes to obtain 100ml of the solution. Transferring the solution into a 500ml beaker, adding 100ml of 95% absolute ethanol, and standing to generate a precipitate; the solution was placed in a centrifuge at 2000rmp for 15min, solid-liquid separation, and the precipitate was removed and washed with 95% absolute ethanol.
The results were: the liquid-material ratio is 1:2, ultrasonic treatment is carried out for 30min, and the ultrasonic temperature is 55 ℃, thus obtaining 1.0424g of polygonatum odoratum crude polysaccharide, and the extraction rate is 20.84%.
The principle of example 1 is as follows: since plant polysaccharides are part of the cell wall structure, the extraction method depends on the cell wall structure. The basic extraction rule is to break the cell wall from the outer layer to the inner layer using mild extraction conditions such as pH or temperature without changing the polysaccharide structure.
The equipment required for example 1, which is known in the art, is available directly from commercial sources.
Example 2
The ultrasonic-assisted enzymatic extraction method of polygonatum odoratum polysaccharide comprises the following steps: cutting fresh rhizoma Polygonati Odorati into small pieces, and oven drying in electrothermal air drying oven at 55deg.C for 5 hr. Pulverizing dried rhizoma Polygonati Odorati in pulverizer, and filtering with 24 mesh sieve to 100ml small beaker. Weighing 5.0g of crushed polygonatum powder, adding the powder into a 250ml small beaker, adding a Hac-NaAc buffer solution into the solution at a liquid-material ratio of 51.75ml/g, adding 0.1g of weighed cellulolytic enzyme into the solution, placing the small beaker with the polygonatum solution into an ultrasonic cleaner for extraction, and setting the temperature to be 35 ℃ for 30min; standing the extracted rhizoma Polygonati Odorati polysaccharide solution for one day. And pouring the rest rhizoma polygonati odorati polysaccharide solution into a centrifuge tube at about 1/2 of the position, and putting the centrifuge tube into the centrifuge for centrifugation at 4000 speed for 10min. Extracting the centrifuged precipitate into a small 250ml beaker, putting the small beaker into an electrothermal blowing drying oven for drying, and weighing the dried polygonatum odoratum polysaccharide 3.2652g when the temperature is constant at 50 ℃ and the time is 11.
The results were: when the ultrasonic temperature is 35 ℃ and the ultrasonic time is 30min, 3.2652g of the polygonatum odoratum crude polysaccharide is obtained, and the extraction rate is 65.3%.
The principle of example 2 is as follows: the ultrasonic assisted extraction method utilizes the vibration breaking action of ultrasonic waves on cell walls, and simultaneously utilizes cavitation to strengthen the release and diffusion of polysaccharide in cells.
The equipment required for example 2, which is known in the art, is available directly from commercial sources.
Example 3
The method for measuring the sugar content of the polygonatum odoratum polysaccharide comprises the following steps: dissolving rhizoma Polygonati Odorati polysaccharide 0.10g in beaker, and adding into 100ml volumetric flask to fix volume to obtain rhizoma Polygonati Odorati polysaccharide solution. Six volumetric flasks were prepared, and 1ml, 2ml, 3ml, 4ml, 5m, 6ml of Polygonatum odoratum polysaccharide solution was measured separately, added to the volumetric flask and distilled water was added to the volumetric flask. 0.0700g of anthrone is weighed and 85% concentrated sulfuric acid is prepared; placing anthrone into prepared concentrated sulfuric acid; and respectively taking 1ml of prepared polygonatum odoratum polysaccharide solution, and adding into a test tube. Then, 5ml of an anthrone-concentrated sulfuric acid solution was added to each of the six test tubes. Heating in a water bath at 100deg.C for ten minutes, and cooling in a refrigerator for 10min.
In this example, the method for preparing 85% concentrated sulfuric acid comprises: 30ml of 18mol/L concentrated sulfuric acid was diluted with distilled water to 35ml.
As shown in FIG. 1, a glucose solution was prepared, and absorbance was measured at 626 nm. Drawing a relation curve of absorbance and glucose to obtain an absorbance and glucose relation equation y=0.7958x+0.0624, R 2 = 0.9851. Wherein y represents absorbance, x represents solution concentration, R 2 Representing the degree of fitting of the linear regression equation.
And measuring absorbance of the cooled polygonatum odoratum polysaccharide solution at 626nm wavelength according to the relation curve and equation of absorbance and glucose, and calculating the concentration of the cooled polygonatum odoratum polysaccharide solution in six test tubes according to the relation equation of absorbance and glucose.
Example 4
The method for measuring the protein content of the polygonatum odoratum polysaccharide comprises the following steps of:
step B1: deproteinization: preparing a polygonatum odoratum polysaccharide solution with the concentration of 0.1%, adding 0.2% papain, adding sodium acetate as an acetic acid buffer solution to adjust the PH of the polygonatum odoratum polysaccharide solution to be 7, heating in a constant-temperature water bath at 60 ℃ for 120min, heating in the water bath at 100 ℃ for 5min, generating white flocculent precipitate, filtering, and filtering the precipitate;
step B2: and (3) evaporating and crystallizing: pouring the filtrate into a 75ml crucible, placing the crucible in a magnetic stirrer, placing a magnet, setting the parameter temperature to 300 ℃ until the filtrate is evaporated to dryness, collecting the crystals obtained after the evaporation to dryness, grinding the crystals into powder, and hermetically preserving the powder;
step B3: preparation of coomassie brilliant blue staining solution: precisely weighing 0.0500G of Coomassie brilliant blue G250, dissolving with 25mL of 95% absolute ethanol, adding 50mL of 85% (W/V) phosphoric acid, and fixing the volume to 500mL with distilled water with the concentration of 0.01%;
step B4: drawing a protein standard curve: precisely weighing 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of deproteinized rhizoma polygonati officinalis polysaccharide standard solution, respectively placing the solution into colorimetric tubes 1-6, adding distilled water to 1.0mL, respectively adding 5mL of coomassie brilliant blue staining solution, and uniformly mixing. Standing for 3min, and taking distilled water as blank control after the same treatment. Preparing bovine serum albumin solution, measuring at 595nm wavelength with ultraviolet-visible spectrophotometer, measuring with radix Polygonati Odorati polysaccharide solution concentration as abscissa and absorbance value as ordinate, and drawing absorbance and bovine serum albumin standard curve as shown in figure 2 to obtain equation y=0.0796x+0.0624, R 2 ＝0.9851。
As shown in fig. 3 and 4, the protein content of the polygonatum odoratum polysaccharide in the colorimetric tubes 1-6 is measured at 595nm wavelength, the protein content of the polygonatum odoratum polysaccharide in the colorimetric tubes 1-6 is obtained according to an equation of the relation between the absorbance and the protein, the protein content in a sample is calculated by comparing the absorbance with a bovine serum albumin standard curve, and the protein removal rate and the polysaccharide retention rate are calculated. The calculation of the results can be obtained, and the calculation formulas of the protein removal rate and the polysaccharide retention rate before deproteinization are as follows: y=0.7427x+1.2413, r 2 ＝0.9606，R 2 Represents the fitting degree of a linear regression equation, y represents absorbance, and x represents solutionConcentration.
After deproteinization, the calculation formula of protein removal rate and polysaccharide retention rate is as follows: y=0.5507 x+1.3495, r 2 = 0.9902. Wherein R is 2 Representing the fitting degree of a linear regression equation, wherein y represents absorbance, and x represents solution concentration; protein removal rate (%) = (content before deproteinization-content after deproteinization)/content before deproteinization×100%; retention of polysaccharide (%) = content of polysaccharide after deproteinization/content of polysaccharide before deproteinization x 100%.
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the description, each embodiment is described in a progressive manner, and each embodiment is mainly described by the differences from other embodiments, so that the same similar parts among the embodiments are mutually referred.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The method for extracting polygonatum polysaccharide by water extraction and alcohol precipitation is characterized by comprising the following steps of: the method comprises the following steps:
step one: cutting the cleaned fresh rhizoma polygonati into small blocks, putting the small blocks into a crucible, and then putting the crucible into an electrothermal blowing drying oven for drying; grinding dried rhizoma Polygonati Odorati into powder or granule, transferring rhizoma Polygonati Odorati into beaker, and adding 95% absolute ethanol to obtain yellow solution;
step two: then placing the beaker containing the yellow solution into an ultrasonic cleaner for extraction to obtain turbid yellow solution;
step three: filtering the turbid yellow solution with a semipermeable membrane under reduced pressure to obtain pale yellow solution and pale yellow powder, transferring the powder and the semipermeable membrane into a beaker together, adding water for dissolution, and condensing and refluxing the solution obtained by suction filtration;
step four: evaporating the condensed and reflowed solution by using a rotary evaporator to obtain a solution after rotary evaporation, transferring the solution into a beaker, adding 95% absolute ethyl alcohol, and standing to generate a precipitate;
step five: and (3) placing the beaker which is placed still to generate the sediment into a centrifuge, carrying out solid-liquid separation, taking out the sediment, and washing the sediment with 95% absolute ethyl alcohol to obtain the polygonatum polysaccharide.
2. The method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation according to claim 1, wherein the method is characterized in that: in the first step, 95% absolute ethyl alcohol and powdery or granular polygonatum odoratum are added into a beaker, wherein the liquid-material ratio is 1:2.
3. the method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation according to claim 1, wherein the method is characterized in that: and in the first step, the drying temperature of the electrothermal blowing drying oven is less than or equal to 55 ℃.
4. The method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation according to claim 3, wherein the method is characterized in that: the drying temperature of the electrothermal blowing drying oven in the step one is constant temperature 55 ℃ and the drying time is 9.5 hours.
5. The method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation according to claim 1, wherein the method is characterized in that: in the second step, the extraction temperature of the ultrasonic cleaner is 55-60 ℃.
6. The method for extracting polygonatum odoratum polysaccharide by water extraction and alcohol precipitation according to claim 5, wherein the method is characterized in that: in the second step, the extraction temperature of the ultrasonic cleaner is 55 ℃, the extraction time is 15min, and a turbid yellow solution is obtained after repeating the steps twice.
7. The ultrasonic assisted enzymatic extraction method of polygonatum odoratum polysaccharide is characterized by comprising the following steps of: the method comprises the following steps:
step 1: cutting fresh rhizoma Polygonati Odorati into small pieces, and oven drying in electrothermal air drying oven;
step 2: crushing the dried polygonatum odoratum in the step 1 in a crusher, and filtering the crushed polygonatum odoratum in a beaker by using a 24-mesh sieve;
step 3: adding a Hac-NaAc buffer solution and cellulose decomposing enzyme into a beaker, placing the beaker with the polygonatum odoratum solution into an ultrasonic cleaner for extraction, and standing the extracted polygonatum odoratum polysaccharide solution;
step 4: pouring the rhizoma polygonati odorati polysaccharide solution extracted after standing into a centrifuge tube, and putting the centrifuge tube into the centrifuge for centrifugation;
step 5: extracting the centrifuged precipitate into a beaker, and placing the beaker into an electrothermal blowing drying box for drying to obtain the polygonatum polysaccharide.
8. The method for extracting polygonatum odoratum polysaccharide by using an ultrasonic-assisted enzymatic method according to claim 7, wherein the method is characterized in that: the temperature of the beaker with the polygonatum odoratum solution extracted in the step 3 in an ultrasonic cleaner is 35 ℃.
9. The method for measuring the sugar content of the polygonatum odoratum polysaccharide is characterized by comprising the following steps of: the method comprises the following steps:
step A1: dissolving rhizoma Polygonati Odorati polysaccharide obtained by the method for extracting rhizoma Polygonati Odorati polysaccharide according to any one of claims 1-8 in beaker, and adding into 100ml volumetric flask to fix volume to obtain rhizoma Polygonati Odorati polysaccharide solution;
step A2: preparing six volumetric flasks, respectively weighing 1ml, 2ml, 3ml, 4ml, 5m and 6ml of rhizoma Polygonati Odorati polysaccharide solution, adding into the volumetric flask, and adding distilled water to constant volume;
step A3: weighing 0.0700g of anthrone, preparing 85% concentrated sulfuric acid, and putting the anthrone into the prepared concentrated sulfuric acid;
step A4: respectively adding 1ml of the prepared polygonatum odoratum polysaccharide solution in the step A2 into six test tubes, respectively adding 5ml of the anthrone-concentrated sulfuric acid solution prepared in the step A2 into the six test tubes, heating in a water bath kettle at 100 ℃, and finally cooling in a refrigerator;
step A5: preparing a glucose solution, measuring absorbance of the glucose solution at 626nm wavelength, drawing a relation curve of absorbance and glucose, obtaining a relation equation of absorbance and glucose according to the relation curve of absorbance and glucose, measuring absorbance of the cooled polygonatum odoratum polysaccharide solution in the six test tubes in the step A4 at 626nm wavelength, and obtaining the concentration of the cooled polygonatum odoratum polysaccharide solution in the six test tubes according to the relation equation of absorbance and glucose.
10. The method for measuring the protein content of the polygonatum odoratum polysaccharide is characterized by comprising the following steps of: the method comprises the following steps:
step B1: preparing a polygonatum odoratum polysaccharide solution with a concentration of 0.1% by using the polygonatum odoratum polysaccharide obtained by the method for extracting polygonatum odoratum polysaccharide according to any one of claims 1-8, adding 0.2% papain, and adding sodium acetate as an acetic acid buffer solution to adjust the PH of the polygonatum odoratum polysaccharide solution to be 7; heating and sterilizing at high temperature after constant-temperature water bath to generate white flocculent precipitate, and filtering the white flocculent precipitate to remove the precipitate;
step B2: pouring the filtrate into a 75ml crucible, placing the crucible in a magnetic stirrer, placing a magnet until the filtrate is evaporated to dryness, collecting the crystals obtained after the evaporation to dryness, grinding the crystals into powder, and sealing and preserving the powder;
step B3: 0.0500G of coomassie brilliant blue G250 is weighed, 25mL of 95% absolute ethyl alcohol is used for dissolution, 50mL of 85% phosphoric acid is added, distilled water is used for constant volume to 500mL, and the concentration is 0.01%, so that a masian brilliant blue staining solution is obtained;
step B4: weighing 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of deproteinized polygonatum polysaccharide solution, respectively placing the above materials in colorimetric tubes 1-6, and adding distilled water to 1.0mL; respectively adding 5mL of coomassie brilliant blue staining solution, uniformly mixing, standing, and taking distilled water as a blank control after the same treatment;
step B5: preparing a bovine serum albumin solution, measuring absorbance of the bovine serum albumin solution at 595nm wavelength by an ultraviolet-visible spectrophotometer, obtaining an absorbance-protein relation curve, obtaining an absorbance-protein relation equation according to the absorbance-protein relation curve, measuring absorbance of the rhizoma polygonati officinalis polysaccharide protein content in the colorimetric tubes 1-6 at 595nm wavelength, and obtaining the rhizoma polygonati officinalis polysaccharide protein content in the colorimetric tubes 1-6 according to the absorbance-protein relation equation.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557842A (en) * | 2004-02-02 | 2004-12-29 | 中南大学湘雅二医院 | Process for extracting polygonatum polysaccharides, preparing process for medical preparation and use thereof |
| CN101580551A (en) * | 2009-06-23 | 2009-11-18 | 宽甸玉竹农副产品有限公司 | Method for extracting polygonatum odoratum polysaccharide |
| CN101585885A (en) * | 2009-06-16 | 2009-11-25 | 西北农林科技大学 | Method for preparing polygonatum odoratum polysaccharide |
| CN108359023A (en) * | 2018-03-02 | 2018-08-03 | 北京国康本草物种生物科学技术研究院有限公司 | A kind of extracting method and fragrant solomonseal rhizome polyoses extract of fragrant solomonseal rhizome polyoses |
| CN112125983A (en) * | 2020-09-30 | 2020-12-25 | 华南理工大学 | Water-soluble polygonatum odoratum polysaccharide, sulfated polygonatum odoratum polysaccharide thereof, and preparation method and application of water-soluble polygonatum odoratum polysaccharide |
| CN114560957A (en) * | 2022-04-12 | 2022-05-31 | 湖南明舜制药有限公司 | Method for extracting polygonatum odoratum polysaccharide |
-
2023
- 2023-05-17 CN CN202310556655.6A patent/CN116675782A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557842A (en) * | 2004-02-02 | 2004-12-29 | 中南大学湘雅二医院 | Process for extracting polygonatum polysaccharides, preparing process for medical preparation and use thereof |
| CN101585885A (en) * | 2009-06-16 | 2009-11-25 | 西北农林科技大学 | Method for preparing polygonatum odoratum polysaccharide |
| CN101580551A (en) * | 2009-06-23 | 2009-11-18 | 宽甸玉竹农副产品有限公司 | Method for extracting polygonatum odoratum polysaccharide |
| CN108359023A (en) * | 2018-03-02 | 2018-08-03 | 北京国康本草物种生物科学技术研究院有限公司 | A kind of extracting method and fragrant solomonseal rhizome polyoses extract of fragrant solomonseal rhizome polyoses |
| CN112125983A (en) * | 2020-09-30 | 2020-12-25 | 华南理工大学 | Water-soluble polygonatum odoratum polysaccharide, sulfated polygonatum odoratum polysaccharide thereof, and preparation method and application of water-soluble polygonatum odoratum polysaccharide |
| CN114560957A (en) * | 2022-04-12 | 2022-05-31 | 湖南明舜制药有限公司 | Method for extracting polygonatum odoratum polysaccharide |
Non-Patent Citations (4)
| Title |
|---|
| 仇宏伟: "超声波辅助酶法提取玉竹多糖工艺研究", 青岛农业大学学报(自然科学版), vol. 37, no. 01, 13 January 2020 (2020-01-13), pages 38 - 42 * |
| 熊庆娥: "植物生理学实验教程", vol. 01, 31 August 2003, 四川科学技术出版社, pages: 87 * |
| 田启建 等: "湘西主要特色药用植物栽培与利用", vol. 01, 31 May 2015, 西南交通大学出版社, pages: 447 * |
| 边宝林 等: "山药专论", vol. 01, 31 January 2013, 中医古籍出版社, pages: 127 * |
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