CN114349877A - Sulfated arrowhead polysaccharide and preparation method and application thereof - Google Patents
Sulfated arrowhead polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN114349877A CN114349877A CN202210031854.0A CN202210031854A CN114349877A CN 114349877 A CN114349877 A CN 114349877A CN 202210031854 A CN202210031854 A CN 202210031854A CN 114349877 A CN114349877 A CN 114349877A
- Authority
- CN
- China
- Prior art keywords
- arrowhead
- polysaccharide
- sulfated
- spst
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- 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
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Diabetes (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Polymers & Plastics (AREA)
- Materials Engineering (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a sulfated arrowhead polysaccharide component, which contains 46.71 +/-2.31% of total sugar, 1.77 +/-0.11% of uronic acid, 12.22 +/-0.5% of sulfate and 1.01% of sulfate substitution degree; the sulfated arrowhead polysaccharide component consists of galacturonic acid, glucose, galactose and arabinose in a molar ratio of 1.00:19.54:3.51: 1.25; the sulfated arrowhead polysaccharide component has a molecular weight of 4.165X 105kDa; the surface of the sulfated arrowhead polysaccharide component presents hollow honeycomb-shaped aggregation spheres with different sizes, and the average height is 141.4 nm. The sulfated arrowhead polysaccharide component provided by the invention has a remarkable anticoagulation effect, and can be used for preparing functional products such as oral liquid, tablets, capsules and the like.
Description
Technical Field
The invention belongs to the field of preparation and application of natural products, and particularly relates to sulfated arrowhead polysaccharide and a preparation method and application thereof.
Background
Arrowheads (Sagittaria trifolia L.) are tubers of arrowheads of alisma, have a long medicinal history in China, and are recorded as bitter, sweet, slightly cold and nontoxic in the record of the famous medical records of the black taro book in Weijin early period; it can be used for treating diabetes, arthralgia, pyrexia, and qi invigorating. As one of special aquatic vegetables of the eight immortals in south China, the arrowhead is widely planted in Jiang Zhe areas of China, and the planting area is up to 20000 hectares. The arrowhead is rich in various nutrient components and bioactive substances such as starch, polysaccharide, terpenoids, vitamins, trace elements and the like. Among them, the arrowhead polysaccharide has a wide range of pharmacological actions such as anti-tumor, enhancing immunity, protecting liver, anti-oxidation, etc., and is the main functional component of arrowhead (Journal of the Science of Food and Agriculture,2021,101(8): 3085-.
Cardiovascular diseases (CVD) are the most fatal diseases worldwide. In China, about 2.9 hundred million CVD patients have higher death rate than malignant tumors, and account for more than 40 percent of death components of diseases. Thrombus is the "genuine" caused by CVD, and the imbalance between blood coagulation and anticoagulation results in hypercoagulability of blood to induce thrombus, which accelerates the CVD process, and anticoagulation is the basis and key for CVD prevention and treatment (Nature Reviews science 2011,8(9): 502-. Heparin and derivatives thereof are the most widely used anticoagulant drugs in clinical applications, and although they play an important role in CVD control, bleeding complications caused by excessive anticoagulation cannot be overcome (American Journal of clinical, 2018,35(9): 898-903). Therefore, the development of new anticoagulant drugs has been one of the most concerned problems in the biomedical field.
In recent years, the anticoagulant effect of plant polysaccharide has attracted attention, and polysaccharides such as medlar (Chinese food science report, 2015,15(10): 157-.
At present, the Research on the arrowhead polysaccharide focuses on the activity Research of extraction, separation and enhancement of the immunity of the organism, antioxidation, liver protection and the like (CN 108421279A; CN 108324729A; Carbohydrate Polymers,2020,229: 115355; Carbohydrate Polymers,2020,246: 116595; Food Research International,2020,136: 109345; Journal of Ethnopharmacology,2018,227: 237-. The invention provides a preparation method, structural characteristics and anticoagulant application of sulfated arrowhead polysaccharide, and provides a lead compound for development of novel anticoagulant drugs.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a sulfated arrowhead polysaccharide which has excellent anticoagulant activity, can remarkably prolong (P <0.01) TT, PT and APTT and reduce FIB concentration; the ability of prolonging TT is obviously higher than that of heparin with the same concentration, and the ability of prolonging PT and APTT is equivalent to that of heparin with the same concentration.
According to a first aspect of the present invention, there is provided a sulfated arrowhead polysaccharide obtained by sulfating an arrowhead polysaccharide with sulfamic acid; the sulfated arrowhead polysaccharide has a sulfate group substitution degree of 0.33-1.48.
According to a second aspect of the present invention, there is provided a process for the preparation of a sulfated sagittaria sagittifolia polysaccharide comprising the steps of: placing arrowhead polysaccharide and sulfamic acid in an organic solvent, and heating for reaction for a certain time; cooling to room temperature, quenching reaction with alkaline solution, adjusting pH of the reaction solution to neutrality, precipitating with ethanol to obtain precipitate, redissolving the precipitate with distilled water, dialyzing with running water, and freeze-drying to obtain PST with different degrees of substitution; for example, the specific steps may be: accurately weighing 100mg of arrowhead polysaccharide component PST, placing the weighed components in a round-bottom flask, respectively adding a certain amount of sulfamic acid according to different mass ratios, then adding 10mL of N, N-Dimethylformamide (DMF), turning on a magnetic stirrer, fully and uniformly mixing the components, and reacting for 3h at 80 ℃; cooling to room temperature, adding 1mol/L NaOH solution to quench the reaction, and adjusting the pH value to be neutral; adding 3 times volume of absolute ethyl alcohol, and precipitating at 4 ℃ for 12 h; redissolving the precipitate with distilled water, dialyzing with running water for 48h, and freeze-drying to obtain PST with different degrees of substitution;
preferably, the mass ratio of the arrowhead polysaccharide to the sulfamic acid is 1: 1-1: 8, and further preferably 1: 4;
preferably, the sulfated sagittaria sagittifolia polysaccharide has a degree of substitution of sulfate groups of 0.33 to 1.48.
Preferably, the mass ratio of the arrowhead polysaccharide component to the sulfamic acid is 1:4, and the obtained sulfated arrowhead polysaccharide has a sulfate group substitution degree of 1.01. The total sugar content of the prepared sulfated arrowhead polysaccharide is 46.71 +/-2.31 percent, the uronic acid content is 1.77 +/-0.11 percent, the sulfate group content is 12.22 +/-0.5 percent, and the substitution degree of the sulfate group is 1.01; the resulting sulfated sagittaria sagittifolia polysaccharide comprises monosaccharides, galacturonic acid in the following molar ratios: glucose: galactose: arabinose 1.00:19.54:3.51: 1.25; the sulfated arrowhead polysaccharide component has a molecular weight of 4.165X 105kDa; the surface of the sulfated arrowhead polysaccharide component presents hollow honeycomb-shaped aggregation spheres with different sizes, and the average height is 141.4 nm.
Preferably, the raw material arrowhead polysaccharide in the preparation process of the sulfated arrowhead polysaccharide is an isolated and purified arrowhead polysaccharide, and the preparation process comprises the following steps: :
1) extraction of arrowhead crude polysaccharide
Cleaning fresh arrowhead, slicing, drying, crushing and sieving to prepare arrowhead powder for later use; defatting to obtain rhizoma Sagittariae Sagittifoliae defatted powder; extracting the rhizoma Sagittariae Sagittifoliae defatted powder with hot water, precipitating with ethanol, and freeze drying the precipitate to obtain rhizoma Sagittariae Sagittifoliae crude polysaccharide; the method specifically comprises the following steps: cleaning fresh arrowhead, slicing, drying at 60 ℃ to constant weight, crushing, and sieving with a 60-mesh sieve to prepare arrowhead powder for later use; adding petroleum ether, degreasing in dark for 12h, and vacuum filtering to obtain rhizoma Sagittariae Sagittifoliae defatted powder; soaking rhizoma Sagittariae Sagittifoliae defatted powder in water at a liquid-to-material ratio of 40:1 for 4h, extracting with hot water at 85 deg.C for 1h, filtering, repeating for three times, mixing filtrates, and concentrating under reduced pressure to 1/5; adding 3 times volume of anhydrous ethanol, precipitating at 4 deg.C for 12 hr, and freeze drying the precipitate to obtain crude polysaccharide of rhizoma Sagittariae Sagittifoliae; the extraction rate is 9.93 +/-0.74%;
2) separation and purification of arrowhead crude polysaccharide
Deproteinizing the crude arrowhead polysaccharide by adopting a Sevag reagent, removing the residual Sevag reagent, dialyzing, and freeze-drying dialysate to obtain purified arrowhead polysaccharide; loading purified arrowhead polysaccharide to a Diethylaminoethyl (DEAE) cellulose-52 chromatographic column, eluting with deionized water (100 mg of arrowhead polysaccharide sample can be eluted with 100-150 mL of deionized water), collecting polysaccharide components eluted by the deionized water, concentrating, dialyzing for 3 times, and freeze-drying to obtain a arrowhead polysaccharide component PST;
the method comprises the following specific steps: deproteinizing 3 times with Sevag reagent (n-butanol: chloroform: 4: 1); dialyzing for 3 times (i.e. dialyzing with distilled water for 24h by using a dialysis bag with the molecular weight cutoff of 7000Da, and repeating for three times) after removing residual Sevag reagent; freeze-drying the dialyzate to obtain purified arrowhead polysaccharide; weighing 100mg of purified polysaccharide, dissolving in 20mL of deionized water, and loading onto a Diethylaminoethyl (DEAE) cellulose-52 chromatographic column (2.6 cm. times.60 cm); eluting with deionized water, concentrating, dialyzing with distilled water for 3 times, and freeze drying to obtain Sagittaria sagittifolia polysaccharide component PST; the PST contains 81.4 + -3.2% of total sugar and 2.97 + -0.3% of sulfate radical;
according to another aspect of the invention, the invention provides the use of the sulfated arrowhead polysaccharide in the preparation of an anticoagulant function product; can be used for preparing oral liquid, tablet, capsule, etc.
Preferably, the oral liquid contains sulfated arrowhead polysaccharide, erythritol, potassium sorbate and water.
Preferably, the tablet contains sulfated arrowhead polysaccharide, polyvinylpyrrolidone, calcium hydrogen phosphate, povidone K30, magnesium stearate and aerosil. Preferably, the capsule contains sulfated sagittaria sagittifolia polysaccharide and starch.
Compared with the prior art, the invention has the beneficial effects that:
the anticoagulant activity of the arrowhead polysaccharide component is obviously enhanced through sulfation modification, the sulfate group substitution degree of the sulfated arrowhead polysaccharide is 0.33-1.48, and especially the anticoagulant activity is the best when the sulfate group substitution degree is 1.01.
Secondly, when the sulfated arrowhead polysaccharide component provided by the invention is 5.00mg/mL, the thrombin time, the prothrombin time and the activated partial thromboplastin time can be obviously prolonged; reducing fibrinogen content; the effect of prolonging thrombin time is significantly higher than that of the positive control heparin at the same concentration.
Drawings
FIG. 1 ion exchange elution profile of sagittaria sagittifolia polysaccharide;
FIG. 2 is a high performance liquid chromatogram of SPST-3 monosaccharide composition (GalA, galacturonic acid; Glu, glucose; Gal, galactose; Arab, arabinose);
FIG. 3 shows a molecular weight distribution of SPST-3;
FIG. 4 is an infrared spectrum of SPST-3;
FIG. 5 is a scanning electron micrograph of SPST-3;
FIG. 6 atomic force microscopy images of SPST-3.
Detailed Description
The arrowhead is supplied by a vegetable base in rural areas of Jiangsu salt city, and the production place is east Taiwan city of salt city in Jiangsu province; monosaccharide standards, T-series glucan standards and the like are purchased from national drug group chemical reagents, Inc.; the Prothrombin Time (PT), the Thrombin Time (TT), the Activated Partial Thromboplastin Time (APTT) and the Fibrinogen (FIB) measuring kit required by the anticoagulant activity test are provided by Nanjing institute of bioengineering; the human H plasma is provided by Elissa biotechnology, Inc. of Jiangsu; the infrared spectrum is measured by a Fourier infrared spectrometer of Hongkong science and technology, and the model is FTIR-650; scanning Electron Microscope (SEM) was measured by HITACHI ultra high resolution field emission scanning electron microscope and was model number Regulus 8100; atomic Force Microscopy (AFM) was performed by Park atomic force microscopy, model No. NX 10; the plasma coagulation time was measured by a semi-automatic coagulation analyzer of the Poron medical treatment, model number PUN-2048. All experiments were performed in triplicate, data were expressed as mean ± SD, statistical analysis of the data was performed using t-test or ANOVA, P <0.05 considered statistically different.
Example 1 extraction of crude Sagittaria sagittifolia polysaccharide
Cleaning fresh arrowhead, slicing, drying at 60 ℃ to constant weight, crushing, and sieving with a 60-mesh sieve to prepare arrowhead powder for later use; adding petroleum ether, degreasing in dark for 12h, and vacuum filtering to obtain rhizoma Sagittariae Sagittifoliae defatted powder. Weighing 10g of arrowhead degreasing powder, soaking the arrowhead degreasing powder in water according to the liquid-material ratio of 40:1 for 4 hours, extracting the arrowhead degreasing powder with hot water at 85 ℃ for 1 hour, filtering the solution, repeating the extraction for three times, combining the filtrates, and concentrating the combined filtrates under reduced pressure to 1/5 of the original volume; adding 3 times volume of anhydrous ethanol, precipitating at 4 deg.C for 12 hr, and freeze drying to obtain 0.99 + -0.07 g of crude polysaccharide of rhizoma Sagittariae Sagittifoliae. The extraction rate was 9.93. + -. 0.74%. The extraction rate (%) of the crude polysaccharide is M (g)/M (g) x 100, M is the mass of the crude polysaccharide from arrowhead, and M is the mass of the defatted powder from arrowhead.
Example 2 purification and isolation of crude Sagittaria sagittifolia polysaccharide
Deproteinizing for 3 times by using Sevag reagent (calculated by volume ratio, n-butanol: chloroform: 4: 1); dialyzing for 3 times (with a dialysis bag with a molecular weight cutoff of 7000Da, distilled water for 24h, and repeating for three times) after removing residual Sevag reagent; freeze-drying the dialyzate to obtain purified arrowhead polysaccharide; weighing 100mg of purified polysaccharide, dissolving in 20mL of deionized water, and loading onto a Diethylaminoethyl (DEAE) cellulose-52 chromatographic column (2.6 cm. times.60 cm); sequentially carrying out gradient elution by using deionized water and 0.1-0.5 mol/L NaCl solution at the flow rate of 1mL/min and 5mL per tube, drawing an elution curve (figure 1), wherein experimental results show that the arrowhead polysaccharide mainly comprises neutral sugar (namely a main peak eluted by the deionized water); collecting polysaccharide component eluted with deionized water, concentrating, dialyzing for 3 times, and freeze drying to obtain Sagittaria sagittifolia polysaccharide component PST (PST); the total sugar content of PST measured by a phenol-sulfuric acid method is 81.4 +/-3.2 percent; the content of sulfate radical measured by barium chloride gelatin turbidimetry is 2.97 +/-0.3%.
Example 3 preparation of sulfated arrowhead polysaccharide component
Accurately weighing 100mg of PST, placing the PST in a round-bottom flask, respectively adding 100mg, 200mg, 400mg and 800mg of Sulfamic Acid (SA), then adding 10mL of N, N-Dimethylformamide (DMF), turning on a magnetic stirrer, fully and uniformly mixing the PST and the DMF, and reacting for 3 hours at 80 ℃; cooling to room temperature, adding 1mol/L NaOH solution to quench the reaction, and adjusting the pH value to be neutral; adding 3 times volume of absolute ethyl alcohol, and precipitating at 4 ℃ for 12 h; the precipitate was reconstituted with distilled water, dialyzed 3 times against distilled water, and freeze-dried to give sulfated PSTs, which were designated as SPST-1(PST: SA ═ 1:1), SPST-2(PST: SA ═ 1:2), SPST-3(PST: SA ═ 1:4), and SPST-4(PST: SA ═ 1:8), in that order.
Example 4 compositional analysis of sulfated PST
The total sugar content is measured by a phenol-sulfuric acid method; the content of uronic acid is measured by m-hydroxyl biphenyl method; measuring the content of sulfate groups by adopting a barium chloride gelatin turbidimetry; the substitution Degree (DS) of the sulfate group is 1.62 multiplied by S/(32-1.02 multiplied by S), and S is the percentage content of the sulfate group.
As shown in table 1, as the amount of sulfamic acid was increased, the total sugar and uronic acid content of sulfated PST was sequentially decreased; the content and the substitution degree of the sulfate group increase firstly and then decrease, and when the PST is 1:4, the content and the substitution degree of the sulfate group are maximum.
TABLE 1 composition analysis results of PST and its sulfated products
Example 5 anticoagulant Activity assay of sulfated Sagittaria sagittifolia polysaccharide component
Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Fibrinogen (FIB) are used as anticoagulant activity evaluation indexes; according to the result of preliminary experiment, the concentration range of the tested sample is designed to be 0.31mg-1～5.00mg.mL-1(ii) a Physiological water as blank control, 5.00mg.mL-1Heparin is a positive control; the experimental methods and procedures were referred to kit instructions.
As shown in Table 2, SPST-3 of 2.50mg/mL to 5.00mg/mL and SPST-4 of 5.00mg/mL can significantly prolong TT values (P <0.05, P <0.01) compared with physiological saline; when the concentration of SPST-3 is 5.00mg/mL, the TT value reaches 36.20 +/-1.24 s and is obviously higher (P <0.05) than that of heparin (30.8 +/-2.52 s) at the same concentration.
Compared with physiological saline, 5.00mg/mL of SPST-1, SPST-2 and SPST-4 can obviously prolong PT values (P is less than 0.05, and P is less than 0.01); 2.50 mg/mL-5.00 mg/mL of SPST-3 can prolong PT remarkably, and when the concentration of SPST-3 is 5.00mg/mL, the PT value reaches 38.0 +/-1.21 s, which is higher than that of heparin (32.6 +/-5.23 s) with the same concentration, but the difference is not significant (P > 0.05).
Compared with physiological saline, SPST-1 of 5.00mg/mL, SPST-2 of 1.25 mg/mL-5.00 mg/mL, SPST-3 of 0.63 mg/mL-5.00 mg/mL and SPST-4 of 0.63 mg/mL-5.00 mg/mL can obviously prolong the APTT value (P <0.05, P < 0.01); when the concentration of SPST-3 is 5.00mg/mL, the APTT value reaches 187 +/-9.60 s, which is higher than that of heparin with the same concentration (182 +/-2.77 s), but the difference is not significant (P > 0.05).
Compared with physiological saline, SPST-1, SPST-2 and SPST-4 with the concentration of 5.00mg/mL and SPST-3 with the concentration of 2.50 mg/mL-5.00 mg/mL can obviously reduce FIB content (P is less than 0.05, and P is less than 0.01); when the concentration of SPST-3 was 5.00mg/mL, the FIB content dropped to 156.3 + -13.4 mg/dL, but was still higher than that of the same concentration heparin (76.63 + -3.26 mg/dL).
The results in the table 1 show that the introduction of the sulfate group is beneficial to improving the anticoagulation activity of the PST, and the higher the substitution degree of the sulfate group is, the stronger the anticoagulation activity is within the substitution degree range of the sulfate group of 0.22-1.01; however, when the degree of substitution with sulfate groups increases to 1.48, the anticoagulant activity shows a downward trend. This shows that the degree of substitution of sulfate is the key to the regulation of PST anticoagulant activity, and the activity is highest at 1.01 (the corresponding sulfate is SPST-3), compared with normal saline, SPST-3 of 5.00mg/mL can significantly (P <0.01) prolong TT, PT and APTT and reduce FIB concentration; the ability of prolonging TT is obviously higher than that of heparin with the same concentration, the ability of prolonging PT and APTT is equivalent to that of heparin with the same concentration, but the ability of reducing FIB is not as good as that of heparin; the anticoagulant mechanism of SPST-3 involves activation of intrinsic, extrinsic, and common coagulation pathways and reduction of FIB content.
TABLE 2 influence of PST and its sulfated species on TT, PT, APTT, FIB
*P<0.05 and**P<0.01 represents comparison to saline group;#P<0.05 represents comparison to heparin group.
Example 6 further characterization of SPST-3
SPST-3 is considered to have higher anticoagulant activity, and the structure of the SPST-3 is further characterized.
And (3) monosaccharide composition determination: accurately weighing 2mg of SPST-3, putting the SPST-3 into a hydrothermal reactor, adding 2mL of 2mol/L trifluoroacetic acid (TFA), sealing, and hydrolyzing at 110-120 ℃ for 6 h; removing residual TFA, adding 200 mu L of 0.5mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) and 200 mu L of 0.3mol/L NaOH solution in sequence, mixing, and reacting at 70 ℃ for 1 h; after cooling, adding 0.3mol/L HCl solution to stop the reaction; extracting with dichloromethane for three times, and collecting a water layer to obtain PMP-derivatized monosaccharide; before sample injection, the sample is filtered by a 0.45 mu m microporous filter membrane, and the volume of the sample injection is 20 mu L. Instrument and chromatographic conditions: a Thermo Ultimate3000 high performance liquid chromatograph equipped with a Thermo U3000 diode array detector; the chromatographic column is a Supersil ODS2 column (5 μm, 4.6 mm. times.250 mm); mobile phase: PBS (pH 6.8): acetonitrile 74:26 (v/v); a flow rate; 1.0 mL/min; column temperature: 30 ℃; detector wavelength 245 nm; operating time: and 80 min.
And (3) measuring the molecular weight: preparing SPST-3 into a solution of 2 mg/mL; filtering the sample with 0.45 μm microporous membrane before sample injection, wherein the volume of the sample injection is 20 μ L; the apparatus is an Elite P230II high performance liquid chromatograph equipped with a refractive index detector, and the chromatographic column is Shodex SUGAR KS-804(8.0mm × 300mm) saccharide liquid chromatographic column; preparing a series of standard sugar solutions with different molecular weights by taking dextrans with different molecular weights as standard sugar, and establishing a three-order correction curve for polysaccharide molecular weight determination according to retention time and molecular weight values; chromatographic conditions were mobile phase: ultrapure water; flow rate: 1.0 mL/min; column temperature: 50 ℃; detector temperature: 35 ℃; operating time: and (3) 30 min.
Infrared spectrum determination: mixing 2mg of dried SPST-3 and 100mg of dried potassium bromide, tabletting, and measuring in FTIR-650 infrared spectrometer with scanning range of 4000cm-1～400cm-1。
Scanning electron microscope analysis: analyzing the morphology of SPST-3 by using a HITACHI ultrahigh resolution field emission scanning electron microscope, which comprises the following steps: taking SPST-3, pasting the SPST-3 on a carrying table of double-sided conductive adhesive by using a cotton swab, and blowing off non-sticky polysaccharide powder by using an ear washing ball. The sample is placed in an ion sputtering instrument for foil spraying treatment, the treated samples are sequentially placed under a scanning electron microscope, and the appearance of the sample is observed after the instrument is stably arranged.
Atomic force microscopy analysis: analyzing the appearance of SPST-3 by adopting a Park atomic force microscope, specifically comprising the following steps: uniformly coating the 0.5mg/L SPST-3 solution on the surface of a cover glass, airing, and placing on an atomic force microscope to scan and observe the molecular form of the cover glass; the image is obtained in a Contact mode, and the probe is Si3N4(cantilever length 200 μm, elastic modulus 0.12N/m), the morphological characteristics of the image were analyzed by NanoScope Analysis 1.9 software.
As shown in fig. 2, SPST-3 is composed of galacturonic acid, glucose, galactose, and arabinose in a molar ratio of 1.00:19.54:3.51: 1.25.
As shown in FIG. 3, SPST-3 belongs to the homogeneous polysaccharide, and has a molecular weight of 4.165X 105kDa。
As shown in FIG. 4, 3436cm was observed from the infrared spectrum of SPST-3-1A strong absorption peak exists nearby, which is caused by the stretching vibration of-OH; 2931cm-1The absorption peak is caused by C-H stretching or bending vibration; 2976cm-1Asymmetric stretching vibration of methyl CH; 2926cm-1Asymmetric stretching vibration of methylene CH; 1641cm-1Stretching vibration of carbonyl CO; 1397cm-1Stretching and vibrating CO; 1258cm-1、811cm-1Respectively asymmetric SO stretching vibration and C-O-S stretching vibration; 1152cm-1、1053cm-1Between 1000cm-1～1200cm-1The vibration is C-O-C glycosidic bond skeleton vibration or C-O-H bending vibration; 581cm-1Is the peak of absorption of pyranose ring. Infrared spectrum analysis shows that SPST-3 conforms to the structural characteristics of sulfur-containing polysaccharide.
As shown in FIG. 5, at the magnification of × 1k, the surface topography of SPST-3 shows criss-cross honeycomb grooves, the surface level is uneven and rough, and the formation of large-aperture wrinkle structure may be caused by the repulsive force of intermolecular sulfate groups.
As shown in FIG. 6, the SPST-3 molecular surface exhibits hollow honeycomb-shaped aggregated spheres with different sizes within a scanning range of 10X 10 μm, and the average height is 141.4nm, which is consistent with the observation result of a scanning electron microscope.
EXAMPLE 5 preparation of SPST-3 oral liquid
Adding 1.0g of SPST-3 into 100mL of purified water, stirring at room temperature until the SPST-3 is dissolved, respectively adding 0.1g of erythritol and 0.1g of potassium sorbate, stirring uniformly, canning, instantly sterilizing, bottling (10 mL/bottle), and sealing to obtain the SPST-3 oral liquid.
EXAMPLE 6 preparation of SPST-3 tablets
Mixing 1.0g of SPST-3, 150mg of polyvinylpyrrolidone and 1.0mg of calcium hydrogen phosphate, grinding, and sieving with a 100-mesh sieve; adding 5% polyvidone K30 in 95% ethanol solution while stirring to obtain soft material, and wet granulating; drying at 60 deg.C, sieving, grading, adding 1% magnesium stearate and 2% silica gel micropowder, mixing, and tabletting to obtain SPST-3 tablet.
EXAMPLE 7 preparation of SPST-3 capsules
Taking 1.0g of SPST-3 which is sieved by a 40-mesh sieve, spraying 95% ethanol according to the proportion of 1:1, mixing uniformly, adding 10% starch to prepare a soft material, sieving by a 20-mesh sieve for granulation, drying in an oven at 60 ℃, sieving by a 20-mesh sieve for granulation, filling in a No. 3 empty capsule under the environment that the relative humidity is lower than 65%, and obtaining the SPST-3 capsule.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A sulfated arrowhead polysaccharide, which is obtained by sulfating arrowhead polysaccharide with sulfamic acid; the sulfated arrowhead polysaccharide has a sulfate group substitution degree of 0.33-1.48.
2. The sulfated arrowhead polysaccharide of claim 1, wherein: the sulfated sagittaria sagittifolia polysaccharide has a degree of substitution of sulfate groups of 1.01.
3. A preparation method of sulfated arrowhead polysaccharide comprises the following steps: placing arrowhead polysaccharide and sulfamic acid in an organic solvent, and heating for reaction for a certain time; cooling to room temperature, quenching reaction with alkaline solution, adjusting pH of the reaction solution to neutral, precipitating with ethanol to obtain precipitate, re-dissolving the precipitate with distilled water, dialyzing with distilled water, and freeze-drying to obtain sulfated rhizoma Sagittariae Sagittifoliae polysaccharides with different degrees of substitution.
4. The method of claim 3, wherein: the weight ratio of the arrowhead polysaccharide component to the sulfamic acid is 1: 1-1: 8.
5. The method of claim 4, wherein: the sulfated arrowhead polysaccharide has a sulfate group substitution degree of 0.33-1.48.
6. The method of claim 5, wherein: the mass ratio of the arrowhead polysaccharide component to the sulfamic acid is 1:4, and the sulfate group substitution degree of the obtained sulfated arrowhead polysaccharide is 1.01.
7. The method of claim 5, wherein: the resulting sulfated sagittaria sagittifolia polysaccharide comprises monosaccharides, galacturonic acid in the following molar ratios: glucose: galactose: arabinose 1.00:19.54:3.51: 1.25; the sulfated arrowhead polysaccharide has a total sugar content of 46.71 + -2.31%, an uronic acid content of 1.77 + -0.11%, and a sulfate group content of 12.22 + -0.5%; the sulfated arrowhead polysaccharide has a molecular weight of 4.165 × 105kDa; the surface of the sulfated arrowhead polysaccharide presents hollow honeycomb-shaped aggregation spheres with different sizes, and the average height is 141.4 nm.
8. Use of a sulfated arrowhead polysaccharide prepared by any of the preparation methods of claims 3-7 for the preparation of an anticoagulant.
9. The use according to claim 8, wherein the anticoagulant drug is in the form of an oral liquid, a tablet or a capsule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210031854.0A CN114349877B (en) | 2022-01-12 | 2022-01-12 | Sulfated arrowhead polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210031854.0A CN114349877B (en) | 2022-01-12 | 2022-01-12 | Sulfated arrowhead polysaccharide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114349877A true CN114349877A (en) | 2022-04-15 |
| CN114349877B CN114349877B (en) | 2023-03-28 |
Family
ID=81109327
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210031854.0A Active CN114349877B (en) | 2022-01-12 | 2022-01-12 | Sulfated arrowhead polysaccharide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114349877B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543979A (en) * | 2003-11-18 | 2004-11-10 | 武汉理工大学 | Preparing process of anticoagulant konjak glucomanan sulfate |
| CN1651466A (en) * | 2004-12-31 | 2005-08-10 | 中国药科大学 | Sulphatization derivative of polysaccharide, its preparation method and use |
| CN103554297A (en) * | 2013-10-21 | 2014-02-05 | 兰州大学 | Preparation method and application of high-sulfation-degree radix hedysari polysaccharide sulfate |
| CN105995747A (en) * | 2016-06-20 | 2016-10-12 | 王佳宁 | Instant brewing arrowhead nutrition powder and preparation method thereof |
| CN113621085A (en) * | 2021-07-08 | 2021-11-09 | 新疆农业科学院农业机械化研究所 | Preparation method of dandelion root polysaccharide sulfation modification product and application of dandelion root polysaccharide sulfation modification product |
-
2022
- 2022-01-12 CN CN202210031854.0A patent/CN114349877B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543979A (en) * | 2003-11-18 | 2004-11-10 | 武汉理工大学 | Preparing process of anticoagulant konjak glucomanan sulfate |
| CN1651466A (en) * | 2004-12-31 | 2005-08-10 | 中国药科大学 | Sulphatization derivative of polysaccharide, its preparation method and use |
| CN103554297A (en) * | 2013-10-21 | 2014-02-05 | 兰州大学 | Preparation method and application of high-sulfation-degree radix hedysari polysaccharide sulfate |
| CN105995747A (en) * | 2016-06-20 | 2016-10-12 | 王佳宁 | Instant brewing arrowhead nutrition powder and preparation method thereof |
| CN113621085A (en) * | 2021-07-08 | 2021-11-09 | 新疆农业科学院农业机械化研究所 | Preparation method of dandelion root polysaccharide sulfation modification product and application of dandelion root polysaccharide sulfation modification product |
Non-Patent Citations (5)
| Title |
|---|
| 孔倩等: "南瓜水溶性多糖的制备及硫酸酯化初步研究", 《食品科学》 * |
| 尹馨梓等: "多糖硫酸酯化修饰及其抗凝血作用研究进展", 《天然产物研究与开发》 * |
| 朱焱等: "魔芋葡甘聚糖硫酸酯的制备及其体外抗凝血活性研究", 《武汉理工大学学报(信息与管理工程版)》 * |
| 赵龙等: "蒽酮-硫酸法测定慈姑中多糖的含量", 《食品研究与开发》 * |
| 陈小红: "响应面法优化茯苓多糖硫酸酯化合成工艺及其抗凝血活性分析", 《湖北农业科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114349877B (en) | 2023-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10835552B2 (en) | Method for preparing linseed polysaccharide having antiviral activity and immunological activity, and use of the linseed polysaccharide | |
| CN112851829B (en) | A fructus Lycii polysaccharide with blood lipid reducing effect | |
| CN111285939A (en) | Artemisia annua polysaccharide with effects of resisting oxidation and regulating intestinal flora and preparation method and application thereof | |
| CN115746156B (en) | Lycium barbarum polysaccharide with immunoregulatory function and preparation method thereof | |
| CN114163545B (en) | Lycium barbarum polysaccharide and application thereof in reducing blood sugar | |
| CN114591448B (en) | Mulberry and phellinus linteus fruiting body manna galactosan and preparation and application thereof | |
| CN114751997B (en) | Yellow large tea polysaccharide with anti-inflammatory activity, preparation method and application thereof, and anti-inflammatory pharmaceutical composition | |
| CN112457422A (en) | Preparation method of phlebopus portentosus polysaccharide | |
| CN114349877B (en) | Sulfated arrowhead polysaccharide and preparation method and application thereof | |
| CN107698695A (en) | Homogeneous polysaccharide with immunomodulatory action and preparation method thereof | |
| CN116570552A (en) | Carrier hydrogel for targeted slow release of probiotics and preparation method and application thereof | |
| CN113214412B (en) | Extraction method and application of acanthopanax fruit polysaccharide and polysaccharide | |
| Guo et al. | Comparative Structural Characterization of Jujube Polysaccharides by Different Levels of Purification | |
| CN113388046B (en) | Citron fruit polysaccharide and preparation method and application thereof | |
| CN108948223B (en) | Myrtle polysaccharide P1, its separation method and application in preparing hypolipidemic medicine | |
| CN112107590A (en) | Application of swim bladder-derived heparin mucopolysaccharide in preparation of angiogenesis inhibitor | |
| CN113801248B (en) | Sagittaria sagittifolia non-starch polysaccharide component and preparation method and application thereof | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof | |
| yan et al. | Hypolipidemic Effects of Mesona chinensis Benth Polysaccharides with Different Structures and Molecular Weights | |
| CN109400731A (en) | A kind of cold-water-soluble astragalus polyose and preparation method thereof and its extracorporeal anti-tumor application | |
| CN113527532B (en) | Long-chain algal polysaccharide and separation and purification method thereof | |
| CN115232225B (en) | Prepared rehmannia root homogeneous polysaccharide and preparation method and application thereof | |
| CN109206531B (en) | Polysaccharide P2, separation and purification method thereof and application thereof in preparation of hypolipidemic drugs | |
| CN111548429B (en) | Rana japonica oil polysaccharide component and application thereof | |
| Shukla et al. | Downstream process intensification for biotechnologically generated hyaluronic acid: Purification and characterization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |