CN115124629B - Preparation and application of seaweed polysaccharide calcium - Google Patents
Preparation and application of seaweed polysaccharide calcium Download PDFInfo
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- CN115124629B CN115124629B CN202210007874.4A CN202210007874A CN115124629B CN 115124629 B CN115124629 B CN 115124629B CN 202210007874 A CN202210007874 A CN 202210007874A CN 115124629 B CN115124629 B CN 115124629B
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- polysaccharide
- undaria pinnatifida
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 78
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 78
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 241001474374 Blennius Species 0.000 title description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title description 2
- 239000011575 calcium Substances 0.000 title description 2
- 229910052791 calcium Inorganic materials 0.000 title description 2
- 241001261506 Undaria pinnatifida Species 0.000 claims abstract description 80
- 241000220289 Pedunculata Species 0.000 claims abstract description 20
- 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 abstract description 16
- 239000008103 glucose Substances 0.000 claims abstract description 16
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- 230000000694 effects Effects 0.000 claims abstract description 11
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- 229910021641 deionized water Inorganic materials 0.000 claims description 6
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- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 5
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 5
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 5
- 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 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
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- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 claims description 4
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- JCZPMGDSEAFWDY-SQOUGZDYSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide Chemical compound NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO JCZPMGDSEAFWDY-SQOUGZDYSA-N 0.000 claims description 3
- 150000001450 anions Chemical class 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
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- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
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- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 claims 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract description 6
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- 235000013376 functional food Nutrition 0.000 abstract 1
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- 241000199919 Phaeophyceae Species 0.000 description 4
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 235000011148 calcium chloride Nutrition 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
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- 150000002772 monosaccharides Chemical class 0.000 description 3
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- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
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- 235000013305 food Nutrition 0.000 description 2
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- 239000002808 molecular sieve Substances 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
- 239000003888 alpha glucosidase inhibitor Substances 0.000 description 1
- AEMOLEFTQBMNLQ-BKBMJHBISA-N alpha-D-galacturonic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-BKBMJHBISA-N 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
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- 230000002155 anti-virotic effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
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Images
Classifications
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the research field of algal polysaccharides, and discloses undaria pinnatifida pedunculata polysaccharide UPPS-1 with alpha-glucosidase activity inhibition function, and a preparation method and application thereof. UPPS-1 is uniform polysaccharide obtained by extracting Undaria pinnatifida stalk with calcium chloride solution, separating and purifying with ion exchange column and gel column, and has weight average molecular weight of 174.88kDa. Through the research of alpha-glucosidase activity, UPPS-1 inhibits alpha-glucosidase on intestinal mucosa, so that the speed of decomposing starch into glucose is reduced, the absorption of glucose by small intestine is reduced and delayed, and the blood glucose abnormality of diabetics is improved. The polysaccharide of the invention can be used as a potential diabetes inhibitor and used for developing functional foods or medicines.
Description
Technical Field
The invention relates to a preparation method and application of seaweed extract.
Background
Brown algae contains multiple nutritional components such as polysaccharide, protein, vitamins, minerals, etc. The polysaccharide is a high molecular compound with various biological activities such as antioxidation, anti-tumor, immunoregulation, antivirus and the like, so the polysaccharide has application value in drug development.
The undaria pinnatifida stem is a stem segment part of undaria pinnatifida, is a large-scale economic brown algae, and is mainly distributed in Liaoning, shandong, jiangsu, zhejiang and other places in China, and belongs to Phaeophyta, phaeophyta. The water content is large, the tissue is hard, the color is strong brown, and the taste is good, so that the food can be widely used for food cooking. The undaria pinnatifida has good effects on resisting virus, resisting tumor, reducing blood pressure, regulating immunity, treating cardiovascular and cerebrovascular diseases, and the like. At present, the extraction and separation and the biological activity of undaria pinnatifida pedunculata polysaccharide are not studied intensively.
Disclosure of Invention
The primary aim of the invention is to provide undaria pinnatifida pedunculata polysaccharide with diabetes regulating function.
Another object of the present invention is to provide a method for preparing the above polysaccharide with diabetes-modulating undaria pinnatifida stems; the invention takes the undaria pinnatifida peduncle entity as a research object, separates and purifies the undaria pinnatifida peduncle entity through calcium chloride leaching and alcohol precipitation by an ion exchange column and a molecular sieve, researches the biological activity of the undaria pinnatifida peduncle entity, and analyzes the molecular weight and monosaccharide composition of undaria pinnatifida peduncle polysaccharide.
It is still another object of the present invention to provide the use of the above-mentioned undaria pinnatifida stalk polysaccharide for regulating diabetes.
The aim of the invention is achieved by the following technical scheme:
a Undaria pinnatifida pedunculata polysaccharide UPPS-1 with weight average molecular weight of 174.880kDa for regulating diabetes
The sugar content of the undaria pinnatifida pedunculata polysaccharide is 37.95%.
The Updown stem polysaccharide UPPS-1 mainly comprises mannuronate, aminoglucose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and fucose with a molar ratio of 4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01.
The preparation method of the undaria pinnatifida peduncle polysaccharide comprises the following steps:
(1) Desalting the undaria pinnatifida, sun-drying and pulverizing into powder.
(2) Adding CaCl2 solution for extraction, collecting supernatant, and concentrating under reduced pressure.
(3) Precipitating the concentrated solution with ethanol, decolorizing, deproteinizing, dialyzing, and lyophilizing to obtain crude polysaccharide.
(4) Preparing crude polysaccharide into solution, eluting with ion exchange column, tracking and monitoring the collected target peak product by phenol sulfuric acid method, concentrating, dialyzing, and freeze drying to obtain crude component.
(5) Further separating the crude component by a gel column, tracking and monitoring a target peak product by a phenol sulfuric acid method, concentrating, dialyzing, and freeze-drying to obtain a finely divided component.
The step (1) is specifically carried out according to the following steps: washing salt on the surface of the undaria pinnatifida stem with tap water, replacing water every 12 hours, soaking for three days, draining, naturally drying in the sun, and crushing to obtain the undaria pinnatifida stem powder.
The step (2) is specifically carried out according to the following steps: mixing the undaria pinnatifida stalk powder with the feed liquid ratio of 1 according to the mol/LCaCl2 of 0.15: 15 Extracting at 70deg.C for 3 hr. Collecting filtrate, and concentrating under reduced pressure to obtain concentrated solution of Undaria pinnatifida stalk.
The step (3) is specifically carried out according to the following steps: precipitating the concentrated solution of Undaria pinnatifida stem with 80% ethanol overnight, centrifuging, collecting precipitate, re-dissolving with a small amount of deionized water, decolorizing with 717 anion resin at 55deg.C for 12 hr, removing protein by Sevage method (chloroform: n-butanol=4:1) for more than three times, and removing white floccules at boundary of two phases. And (3) performing dialysis at 3500Da for 48 hours, and performing freeze drying to obtain crude polysaccharide.
The step (4) is specifically carried out according to the following steps: dissolving the undaria pinnatifida stem crude polysaccharide with deionized water, preparing a polysaccharide solution with the concentration of 30mg/mL, separating the polysaccharide solution by a DEAE-52 ion exchange column, eluting by using 0, 0.5, 1 and 2mol/LNacl solutions in sequence, controlling the flow rate at 1mL/min, eluting for 10 min/tube, tracking and monitoring by adopting a phenol sulfuric acid method, and collecting a main peak part according to absorbance. Collecting the eluted component of 0.5mol/LNacl solution, concentrating, dialyzing with 3500Da dialysis bag for 48 hr, and freeze drying to obtain crude Undaria pinnatifida stalk polysaccharide UPPS-1.
The step (5) is specifically carried out according to the following steps: dissolving the crude polysaccharide of the undaria pinnatifida peduncles with pure water, preparing polysaccharide solution with the concentration of 30mg/mL, separating by a Sephadex-G100 gel column, eluting by using pure water, controlling the flow rate to be 0.5mL/min, eluting for 10 min/tube, tracking and monitoring by a phenol sulfuric acid method, and collecting a main peak part according to absorbance. Collecting pure water eluting component, concentrating under reduced pressure, dialyzing with 3500Da dialysis bag for 48 hr, and lyophilizing to obtain refined Undaria pinnatifida stalk polysaccharide UPPS-1.
The application of the undaria pinnatifida pedunculata polysaccharide UPPS-1 in preparing and regulating diabetes mellitus.
The alpha-glucosidase inhibitor can reduce the speed of decomposing starch into glucose by inhibiting alpha-glucosidase on intestinal mucosa, thereby reducing and delaying the absorption of glucose by small intestine and improving the abnormal blood sugar of diabetes patients.
In the method, compared with the traditional water solvent extraction method, the undaria pinnatifida pedunculata polysaccharide reduces the generation of algin through calcium chloride extraction, and improves the solubility.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention takes the undaria pinnatifida peduncles as a research object, prepares the undaria pinnatifida peduncles polysaccharide UPPS-1 through extracting by a calcium chloride solution, separating and purifying by a DEAE-52 ion exchange column and a molecular sieve, and determines the specific activity application of the undaria pinnatifida peduncles polysaccharide UPPS-1.
2. Compared with the traditional water solvent extraction method, the extraction method of the undaria pinnatifida pedunculata peduncles polysaccharide has the advantages that partial algin is removed by using a calcium chloride solution, and the solubility of the polysaccharide is improved.
3. The weight average molecular weight of the Undaria pinnatifida pedunculata polysaccharide UPPS-1 is 174.880kDa, and the Undaria pinnatifida pedunculata polysaccharide UPPS-1 mainly comprises mannuronate, glucosamine, rhamnose, glucuronic acid, galacturonate, glucose, galactose and fucose.
4. The Undaria pinnatifida stem polysaccharide UPPS and UPPS-1 can inhibit alpha-glucosidase within the concentration range of 100-1000 mug/mL, and slow down the decomposition speed of starch into glucose, thereby inhibiting diabetes.
Drawings
FIG. 1 is a chromatography elution diagram of undaria pinnatifida pedunculata polysaccharide through a DEAE-52 ion exchange column;
FIG. 2 is a chromatographic elution diagram of undaria pinnatifida pedunculata polysaccharide through a Sephadex-G100 gel column;
FIG. 3 is an ultraviolet spectrogram of Undaria pinnatifida stalk polysaccharide UPPS-1;
FIG. 4 is an infrared spectrum of Undaria pinnatifida pedunculata polysaccharide UPPS-1;
FIG. 5 is a standard sugar peak time table;
FIG. 6 is a standard sugar HPLC plot;
FIG. 7 is a diagram showing the monosaccharide composition of Undaria pinnatifida stalk polysaccharide UPPS-1;
FIG. 8 is a graph showing the effect of Undaria pinnatifida stem polysaccharide UPPS, UPPS-1 on inhibition of α -glucosidase;
Detailed Description
The invention is described in further detail below with reference to the drawings and the specific examples, but the embodiments of the invention are not limited thereto. Unless otherwise specified, the reagents, apparatus and methods employed in the present invention are those conventionally commercially available in the art and conventional methods of use.
Example 1
Washing salt on the surface of the undaria pinnatifida stem with tap water, replacing water every 12 hours, soaking for three days, draining, naturally drying in the sun, and crushing to obtain undaria pinnatifida stem powder;
mixing the undaria pinnatifida stalk powder with the feed liquid ratio of 1 according to the mol/LCaCl2 of 0.15: 15 Extracting at 70deg.C for 3 hr. Collecting filtrate, concentrating under reduced pressure to obtain concentrated solution of Undaria pinnatifida stalk;
precipitating the concentrated solution of Undaria pinnatifida stem with 80% ethanol overnight, centrifuging, collecting precipitate, re-dissolving with a small amount of deionized water, decolorizing with 717 anion resin at 55deg.C for 12 hr, removing protein by Sevage method (chloroform: n-butanol=4:1) for more than three times, and removing white floccules at boundary of two phases. Dialyzing for 48h at 3500Da, and lyophilizing to obtain crude polysaccharide;
dissolving the undaria pinnatifida stem crude polysaccharide with deionized water, preparing a polysaccharide solution with the concentration of 30mg/mL, separating the polysaccharide solution by a DEAE-52 ion exchange column, eluting by using 0, 0.5, 1 and 2mol/LNacl solutions in sequence, controlling the flow rate at 1mL/min, eluting for 10 min/tube, tracking and monitoring by adopting a phenol sulfuric acid method, and collecting a main peak part according to absorbance. Collecting the eluting component of 0.5mol/LNacl solution, concentrating, dialyzing with 3500Da dialysis bag for 48 hr, and lyophilizing to obtain crude Undaria pinnatifida stalk polysaccharide UPPS-1;
dissolving the crude polysaccharide of the undaria pinnatifida peduncles with pure water, preparing polysaccharide solution with the concentration of 30mg/mL, separating by a Sephadex-G100 gel column, eluting by using pure water, controlling the flow rate to be 0.5mL/min, eluting for 10 min/tube, tracking and monitoring by a phenol sulfuric acid method, and collecting a main peak part according to absorbance. Collecting pure water eluting component, concentrating under reduced pressure, dialyzing with 3500Da dialysis bag for 48 hr, and lyophilizing to obtain refined Undaria pinnatifida stalk polysaccharide UPPS-1;
determination of Undaria pinnatifida pedunculata polysaccharide UPPS-1 sugar content
10mg of glucose is weighed, the volume is fixed to 100mL, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mL of glucose solution are removed, and distilled water is added to the volume to 1mL. Phenol is dissolved in a water bath kettle at 60 ℃, and 5mL of phenol is taken to be constant volume to 100mL. 1mL of 5% phenol solution was added and the mixture was shaken well. 5mL of concentrated sulfuric acid is added, the mixture is shaken well, reacted at room temperature and kept stand for 30min. Absorbance was measured at 490 nm. And drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance value as an ordinate. The undaria pinnatifida pedunculata polysaccharide UPPS-1 is prepared into a solution with the concentration of 1mg/mL, and 1mL is taken and detected according to a phenol sulfuric acid method. The UPPS-1 sugar content was calculated to be 37.95% according to the standard curve y= 6.6798x-0.034 (r2=0.9997).
Ultraviolet spectrum analysis of Undaria pinnatifida stalk UPPS-1
A certain amount of the obtained undaria pinnatifida pedunculata polysaccharide is dissolved in distilled water, the concentration is 1mg/mL, the undaria pinnatifida pedunculata polysaccharide is scanned on a spectrophotometer, the wavelength range is 200-800nm, the result is shown in figure 3, and no characteristic absorption peaks exist at 260nm and 280nm, so that the undaria pinnatifida pedunculata polysaccharide does not contain nucleic acid and protein.
Infrared spectrum analysis of Undaria pinnatifida stalk UPPS-1
Mixing 2mg of the obtained undaria pinnatifida peduncle polysaccharide with 50mg of dry potassium bromide powder, fully grinding, tabletting, and carrying out infrared spectrum scanning within 4000-400cm < -1 >, wherein the result is shown in figure 4, and the characteristic absorption peak of saccharides appears at 3444.20cm < -1 >, which is generated by O-H stretching vibration. An absorption peak is formed at 2930.90cm < -1 >, which is generated by C-H stretching vibration. The absorption peak at 1420.07cm-1 is the C-O stretching vibration. The absorption peak of the asymmetric stretching vibration of c=o at 1643.99cm-1 was found to be a carboxyl group in UPPS and to contain uronic acid. An asymmetric stretching vibration of S=O is arranged at 1243.76cm < -1 >, an asymmetric stretching vibration of C-O-S is arranged at 821.28cm < -1 >, and UPPS possibly contains a sulfuric acid group. The expansion and contraction vibration of C-C is 1055.07cm < -1 >.
Monosaccharide composition analysis of Undaria pinnatifida peduncles UPPS-1
10mg of the Undaria pinnatifida stem UPPS-1 obtained by the above method is hydrolyzed with 1mol/L trifluoroacetic acid at 110 ℃ for 6 hours, and then is derivatized with 0.5mol/LPMP methanol solution, and the derivative is analyzed on an Agilent 1260 high performance liquid chromatograph.
Chromatographic conditions: BDS HYPERSIL C chromatographic column (5 μm. Times.4.6 mm. Times.250 mm), detection wavelength 250nm, column temperature 30 ℃, flow rate 0.8mL/min, UV detector, mobile phase phosphate buffer: acetonitrile (87:17).
Wherein the standard substance is: glucose, galactose, fucose, glucuronic acid, glucosamine, galactosamine, mannose, rhamnose, xylose, galacturonic acid, arabinose. The results are shown in FIGS. 5 and 6.
As shown by the chromatographic detection result, the undaria pinnatifida pedunculata polysaccharide mainly comprises mannuronic acid, aminoglucose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and fucose in a molar ratio of 4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01.
Determination of alpha-glucosidase Activity of Undaria Pinnatifida Stem UPPS-1
0.2U/mL of alpha-glucosidase, 0.3mL of polysaccharide samples with different concentrations and 0.4mL of polysaccharide samples with different concentrations are incubated in a constant-temperature water bath at 37 ℃ for 10min, 5mmol/LPNPG solution and 0.3mL of the polysaccharide samples are added, the constant-temperature water bath at 37 ℃ is continued for 20min, at the wavelength of 405nm, the absorbance value is determined to be Ai,0.2mol/L (ph=6.8) of phosphate buffer is used for replacing the samples to determine that the absorbance is A0, and the phosphate buffer is used for replacing the enzyme to be Aj. The formula of the inhibition rate of alpha-glucosidase is:
t= [1- (Ai-Aj)/A0 ]. Times.100%, and the results are shown in FIG. 6, the undaria pinnatifida stem polysaccharide UPPS and UPPS-1 show good inhibition rate at 100-1000 ug/mL.
Claims (6)
1. A undaria pinnatifida pedunculata polysaccharide UPPS-1 with alpha-glucosidase activity, which is characterized in that: the weight average molecular weight is 174.88kDa; the undaria pinnatifida stem polysaccharide UPPS-1 consists of mannuronate, aminoglucose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and fucose with the molar ratio of 4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01.
2. The undaria pinnatifida stem polysaccharide UPPS-1 with alpha-glucosidase activity according to claim 1, wherein: the sugar content of the undaria pinnatifida pedunculate polysaccharide UPPS-1 is 37.95%.
3. The method for preparing undaria pinnatifida stem polysaccharide UPPS-1 according to any one of claims 1-2, comprising the following steps:
(1) Desalting the undaria pinnatifida stems, sun-drying and pulverizing into powder;
(2) Adding CaCl 2 Extracting the solution, collecting supernatant, concentrating under reduced pressureShrinking;
(3) Precipitating the concentrated solution with ethanol, decolorizing, deproteinizing, dialyzing, and lyophilizing to obtain crude polysaccharide;
(4) Preparing crude polysaccharide into solution, eluting with ion exchange column, tracking and monitoring the collected target peak product by phenol sulfuric acid method, concentrating, dialyzing, and freeze drying to obtain crude component;
(5) Further separating the crude component by a gel column, tracking and monitoring a target peak product by a phenol sulfuric acid method, concentrating, dialyzing, and freeze-drying to obtain a finely divided component;
the step (4) is specifically carried out according to the following steps: dissolving the undaria pinnatifida stem crude polysaccharide with deionized water, preparing a polysaccharide solution with the concentration of 30mg/mL, separating by a DEAE-52 ion exchange column, eluting by using 0, 0.5, 1 and 2mol/LNacl solutions in sequence, controlling the flow rate at 1mL/min, controlling the eluting time at 10 min/pipe, adopting a phenol sulfuric acid method for tracking and monitoring, collecting a main peak part according to absorbance, collecting an eluting component of the 0.5mol/LNacl solution, concentrating, dialyzing for 48h by using 3500Da dialysis bags, and freeze-drying to obtain the undaria pinnatifida stem polysaccharide UPPS-1;
the step (5) is specifically carried out according to the following steps: dissolving the crude undaria pinnatifida stem polysaccharide with pure water, preparing polysaccharide solution with concentration of 30mg/mL, separating by a Sephadex-G100 gel column, eluting by pure water, controlling the flow rate at 0.5mL/min, eluting for 10 min/tube, tracking and monitoring by a phenol sulfuric acid method, collecting main peak parts according to absorbance, respectively collecting pure water eluting components, concentrating under reduced pressure, dialyzing for 48h by using 3500Da dialysis bags, and freeze-drying to obtain the subdivided undaria pinnatifida stem polysaccharide UPPS-1.
4. The method for preparing undaria pinnatifida stem polysaccharide UPPS-1 according to claim 3, wherein the method comprises the following steps:
the step (1) is specifically carried out according to the following steps: washing salt on the surface of the undaria pinnatifida stem with tap water, replacing water every 12 hours, soaking for three days, draining, naturally drying in the sun, and crushing to obtain undaria pinnatifida stem powder;
the step (2) is specifically carried out according to the following steps: mixing the powder of Undaria pinnatifida stalk with 0.15mol/LCaCl 2 Extracting at 70deg.C for 3 hr at feed-liquid ratio of 1:15, collecting filtrate, and concentrating under reduced pressureShrinking to obtain concentrated solution of undaria pinnatifida stems;
the step (3) is specifically carried out according to the following steps: precipitating the concentrated solution of the undaria pinnatifida stem with 80% ethanol overnight, centrifuging, collecting precipitate, re-dissolving with a small amount of deionized water, decolorizing with 717 anion resin at 55deg.C for 12h, removing proteins by Sevage method for more than three times until no white floccules exist at the boundary of two phases, dialyzing for 48h at 3500Da, and lyophilizing to obtain crude polysaccharide, wherein chloroform/n-butanol=4:1.
5. Use of undaria pinnatifida stem polysaccharide UPPS-1 with alpha-glucosidase activity according to any one of claims 1-2 for preparing diabetes inhibitor drugs.
6. The use according to claim 5, characterized in that: the diabetes inhibitor medicine has the effects of inhibiting alpha-glucosidase on intestinal mucosa, slowing down the speed of decomposing starch into glucose, reducing and delaying the absorption of glucose by small intestine, and improving the activity of abnormal blood sugar of diabetes patients.
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