CN111138556A - Antiallergic sulfated polysaccharide and preparation method and application thereof - Google Patents
Antiallergic sulfated polysaccharide and preparation method and application thereof Download PDFInfo
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- CN111138556A CN111138556A CN202010024128.7A CN202010024128A CN111138556A CN 111138556 A CN111138556 A CN 111138556A CN 202010024128 A CN202010024128 A CN 202010024128A CN 111138556 A CN111138556 A CN 111138556A
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- sulfated polysaccharide
- antiallergic
- polysaccharide
- sulfated
- exchange resin
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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
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
-
- 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
- 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
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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
Abstract
The invention relates to an antiallergic sulfated polysaccharide, a preparation method and application thereof, wherein the antiallergic sulfated polysaccharide is prepared by taking sulfated polysaccharide as a raw material, removing divalent metal ions, and has the characteristics of low molecular weight and high content of sulfate groups, the molecular weight of the antiallergic sulfated polysaccharide is 600Da-1000Da, and the retention degree of the sulfate groups relative to the raw material sulfated polysaccharide is more than or equal to 95 mass%. The anti-allergic sulfated polysaccharide prepared by the invention has simple production process and high production efficiency, and the obtained sulfated polysaccharide has strong anti-allergic activity and is very suitable for developing anti-allergic medicaments or functional foods.
Description
Technical Field
The invention relates to the field of biological agents, in particular to an antiallergic sulfated polysaccharide, and a preparation method and application thereof.
Background
With the increasing number of food allergic people, food allergic diseases have become an important public health problem. Studies have shown that approximately 8% of children in the united states are allergic to food (Gupta et al, 2011); the proportion of food allergies in european populations is up to 6% (nwau et al, 2014); the incidence of food allergies in australian children 1 year old reaches 11% (Peters et al, 2017). In our country, the prevalence of food allergy has increased from 3.5% in 1999 to 7.7% in 2009 (Hu et al, 2010). However, the drugs for food allergy in the market are mainly antihistamines, such as chlorpheniramine, loratadine, promethazine and the like, which are usually used for treating symptoms and root causes and have strong drug resistance. Therefore, the development of safe and effective anti-food allergy drugs or functional foods with natural active ingredients has become an urgent need.
In recent years, the antiallergic activity of sulfated polysaccharides from seaweed has been attracting attention. For example, Gracilaria verrucosa sulfated polysaccharides are effective in inhibiting scylla paramamosain (TM) -induced food allergy symptoms (Liu et al, 2016); intraperitoneal injection of fucosan in mice can reduce the production of serum IgE of mice allergic to lactalbumin (OVA) (Yanase et al, 2009); eucheuma sulphated oligosaccharides can promote Treg polarization and inhibit the transcription and expression of TH2 cytokines, thereby effectively inhibiting TM-induced food allergy in mice (Yoshida et al, 2005). However, the structural characteristics of high sulfate groups endow the polyanionic system characteristics of the algal sulfated polysaccharides, so that the sulfated polysaccharides become a foundation bed for adsorbing metal ions in the ocean. Because of the enrichment of metal ions, the activity of sulfated polysaccharide can not be fully exerted, namely lower anti-allergic activity.
Disclosure of Invention
The invention aims to overcome the problem of low antiallergic activity of the existing sulfated polysaccharides, and provides an antiallergic sulfated polysaccharide which is modified and processed by adopting common sulfated polysaccharide as a raw material, wherein the common sulfated polysaccharide is derived from marine brown algae (such as kelp, sargassum fusiforme, gulfweed, undaria pinnatifida, acanga, gloiopeltis, carrageen, and the like), marine red algae (such as red wool algae, eucheuma, gracilaria, laver, gelidium amansii, porphyra, echinococcus, seaweed, partridge, and the like) and microsphericium algae, and has higher antiallergic activity after divalent metal ions (activated by sulfate groups) are removed, the molecular weight is reduced, and the retention degree of the sulfate groups is improved.
The invention also provides a preparation method of the antiallergic sulfated polysaccharide, which has low cost and high efficiency and can obtain the sulfated polysaccharide with high antiallergic activity.
The invention also protects the application of the antiallergic sulfated polysaccharide in preparing antiallergic drugs or antiallergic functional foods. The lowest effective dose of the antiallergic medicine which is orally taken and effectively exerts the antiallergic effect is 10mg/kg according to the mass of the sulfated polysaccharide.
Earlier researches of the invention find that the ash content of the seaweed sulfated polysaccharide can reach more than 20 percent, wherein the contents of calcium, manganese and magnesium elements respectively reach 8155, 6633 and 6215 mg/kg. After the metal ions are combined with sulfate groups of the polysaccharide, the bioactivity of the polysaccharide is seriously inactivated. Therefore, the metal ions in the algal sulfated polysaccharides must be effectively removed, and the bioactivity of the polysaccharides must be fully activated, so that the product quality is effectively improved.
In addition, studies have shown that immune dysfunction in food allergy patients is closely related to the intestinal flora. Most antigens in the gastrointestinal tract are derived from the metabolites of food and its intestinal microorganisms, which can reduce the immune tolerance of the body to allergens by promoting the production of antigen-specific Treg cells. In addition, some gut microbes stimulate gut DCs to activate B cells, thereby producing specific IgA antibodies. The applicant found that the low-molecular sulfated polysaccharides with high sulfated groups have selective inhibition effect on microorganisms compared with the low-molecular sulfated polysaccharides with low sulfated groups (the content of sulfated groups is less than or equal to 15%) and the high-polymeric sulfated polysaccharides with high sulfated groups: can effectively inhibit Escherichia coli for promoting food allergy, and has effect in promoting intestinal probiotic (such as Bacillus bifidus). Therefore, the implementation of the effective degradation of the sulfated polysaccharides of the seaweed and the improvement of the retention degree of sulfate groups can effectively improve the improvement effect of the sulfated polysaccharides on intestinal flora, thereby further improving the antiallergic activity of the sulfated polysaccharides.
Therefore, the key to develop the high antiallergic activity sulfated polysaccharide is to remove divalent metal ions (sulfate group activation), reduce molecular weight and improve the retention degree of the sulfate group. However, the preparation process having the above three elements (low divalent metal ion, low molecular weight and high retention of sulfuric acid group) at the same time is difficult to obtain because the three elements are related to each other, and under the condition that one element is achieved, another element tends to be shifted in the opposite direction to the target. For example, the removal of metal ions brings about the breaking of chemical bonds, which also results in the loss of sulfate groups, i.e. a reduction in the content of sulfate groups.
The inventors found that the metal ion bonded to the sulfuric acid group was well dissociated only under the condition of pH 2 to 3, but a large amount of the sulfuric acid group was dropped under the acidic condition. Further research shows that the sulfate group combined on the polysaccharide has good heat resistance and can effectively realize the molecular weight fracture of the polysaccharide under high-temperature conditions. The method utilizes the metal chelation and pH regulation of weak acid to control the pH of a reaction system to be 4-5, so as to fully avoid the acidic range causing the shedding of sulfate groups, and simultaneously utilizes the hydrolysis of high temperature on polysaccharide and the effect of promoting the dissociation of metal ions to realize the synchronous activation of the hydrolysis of sulfated polysaccharide and sulfate groups.
The specific scheme is as follows:
an antiallergic sulfated polysaccharide is prepared by taking sulfated polysaccharide as a raw material, removing divalent metal ions, and has the characteristics of low molecular weight and high sulfate group content, wherein the molecular weight of the antiallergic sulfated polysaccharide is 600Da-1000Da, and the retention degree of sulfate groups relative to the raw material sulfated polysaccharide is more than or equal to 95 mass%.
Further, the removal rate of at least one ion of calcium ions, magnesium ions or manganese ions is more than or equal to 99 mass percent relative to the raw material sulfated polysaccharide.
Further, the content of sulfate groups of the antiallergic sulfated polysaccharide is more than or equal to 10 mass%, and the sulfate groups are bonded to L-fucose or D-galactose.
Further, the content of glucose in the monosaccharide composition of the antiallergic sulfated polysaccharide is less than or equal to 5 mass percent;
optionally, the content of sulfate groups in the antiallergic sulfated polysaccharide is more than or equal to 10 mass%.
The invention also provides a preparation method of the antiallergic sulfated polysaccharide, which comprises the following steps:
s1: carrying out solid-state infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder to obtain sulfated polysaccharide wet powder, and placing the sulfated polysaccharide wet powder in an infrared drying oven for heating to obtain pre-degraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, and then a composite chelating agent prepared by S2 is added to adjust the pH value; then heating to obtain low molecular weight sulfated polysaccharide solution;
s4: performing static adsorption and purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained in the step S3 by using the anion exchange resin, filtering, further washing the anion exchange resin by using deionized water, and fully removing metal ions and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, and directly spray-drying the filtrate after filtering to obtain the antiallergic sulfated polysaccharide.
Further, the sulfated polysaccharide dry powder in step S1 is a dry powder obtained by extracting sulfated polysaccharide from at least one of marine brown algae, marine red algae or microspherococcus, and drying;
optionally, the sulfated polysaccharide content in the sulfated polysaccharide wet powder in the step S1 is 10 to 30 mass%;
optionally, in the step S1, the wet sulfated polysaccharide powder is made into powder dough, the powder dough is flattened into a cake skin with the thickness of 0.05-0.15cm, the cake skin is flatly laid on a clean stainless steel tray, and then the tray is placed in an infrared drying oven, so that the distance between the cake skin and an infrared lamp is kept at 8-12 cm; 2500-.
Further, in the step S2, citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate are mixed according to the mass ratio of 2-4:2-4:1-2:1: 1;
optionally, in step S3, dissolving the pre-degraded sulfated polysaccharide dry powder in deionized water, wherein the mass ratio of the pre-degraded sulfated polysaccharide to the deionized water is 10-20%, and then adding a compound chelating agent, wherein the mass ratio of the compound chelating agent to the polysaccharide solution is 0.1-0.3%;
optionally, adjusting the pH to 4-5 in step S3;
optionally, in step S3, steam heating is used to hydrolyze at 100 ℃ and 130 ℃ for 5-15 minutes, preferably atmospheric steam heating at 100 ℃ for 10 minutes.
Further, in step S4, the low molecular weight sulfated polysaccharide solution obtained in step S3 is statically adsorbed for 0.5 to 1.5 hours by using DEAE-52 cellulose anion exchange resin, filtered, and the anion exchange resin is further washed by deionized water to sufficiently remove metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin, soaking the anion exchange resin in 0.5-1.5mol/L KCl solution for 0.5-1.5h to desorb the low molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly drying the filtrate by spray to obtain the antiallergic sulfated polysaccharide.
The invention also provides a preparation method of the antiallergic sulfated polysaccharide, and the prepared antiallergic sulfated polysaccharide has the characteristics of molecular weight of 600Da-1000Da, removal of divalent metal ions, low molecular weight and high content of sulfate groups; the preferred molecular weight is 600Da to 800 Da.
The invention also protects the application of the antiallergic sulfated polysaccharide in preparing antiallergic drugs or antiallergic functional foods.
Has the advantages that:
(1) the solid infrared pre-degradation technology of sulfated polysaccharide is adopted, and the infrared degradation efficiency is further improved by properly adding water; by pre-degradation, the viscosity of the sulfated polysaccharide is greatly reduced, and the concentration of the sulfated polysaccharide solution is increased by 15% (w/v), so that the production efficiency is effectively improved, and the production cost is reduced; through pre-degradation, the hydrolysis time in a weak acid environment is also greatly reduced, so that the retention degree of polysaccharide sulfate groups is improved.
(2) The steam hydrolysis-ion chelation synchronous technology is adopted, the pH value of the system is kept at 4-5, preferably 4.5, the sugar chain breakage can be rapidly realized, the divalent metal ions bonded on the sulfate group can be dissociated, and the dissociated metal ions are adsorbed under the action of the chelating agent, so that the effects of sulfate polysaccharide molecule degradation and divalent metal ion dissociation are synchronously realized, and the antiallergic activity of the low-molecular sulfate polysaccharide is fully activated; from the production angle, the infrared pre-degradation-weak acid assisted high-temperature secondary degradation coupling technology is adopted, the obtained low-molecular sulfated polysaccharide product has lower molecular weight (600Da-1000Da), replaces a high-pressure high-temperature degradation technology, and has the advantages of high safety and low energy consumption.
(3) According to the static adsorption and purification technology of the anion exchange resin, the DEAE-52 cellulose is directly placed in a container, an aqueous solution rich in sulfated polysaccharide is poured for static adsorption, and after filtration and cleaning, the KCl with the concentration of 1mol/L is preferably adopted for soaking and desorption to release the sulfated polysaccharide adsorbed on the resin, so that the effects of separating the chelating agent combined with metal ions, neutral sugar generated by hydrolysis of the sulfated polysaccharide and other impurities (such as protein) are achieved. The technology replaces the traditional column chromatography technology, greatly improves the purification efficiency, reduces the production cost, and is suitable for industrial production.
In a word, the solid infrared pre-degradation and high-temperature degradation-ion chelation synchronization technology adopted by the invention has the advantages of low cost and high efficiency, and is suitable for industrial production. In addition, natural and efficient antiallergic active substances are products urgently needed in the current market, and the natural and efficient antiallergic active substances have very important social and economic benefits for realizing high-value utilization of algal polysaccharides.
Drawings
In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.
FIG. 1 is a flow chart of a process for preparing antiallergic sulfated polysaccharides according to an embodiment of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.
Example 1
Preparation of antiallergic fucoidan, referring to fig. 1, the procedure was as follows:
s1, solid infrared pre-degradation of fucosan: adding deionized water into fucosan (sulfuric acid group is 35.4 mass%; contents of calcium, magnesium and manganese elements are 12000mg/kg, 6540mg/kg and 10500mg/kg respectively) dry powder from herba Zosterae Marinae, wherein the water content is 20 mass%, preparing fucosan wet powder dough into cake skin with thickness of 0.1cm, spreading on clean stainless steel tray, and placing the tray in infrared drying oven to keep distance between the cake skin and infrared lamp at 10 cm; heating at 3000w for 1h to obtain the pre-degraded fucosan dry powder.
S2, preparation of a composite chelating agent: mixing citric acid, polyaspartic acid, tartaric acid, sodium Ethylene Diamine Tetracetate (EDTA) and sodium pyrophosphate according to a mass ratio of 3:3:2:1:1 to prepare the composite chelating agent.
S3, polysaccharide hydrolysis and sulfate group synchronous activation: dissolving the pre-degraded fucosan dry powder prepared by S1 in deionized water, wherein the polysaccharide concentration is 15% (g/mL); continuously adding 0.2 percent (g/mL) of composite chelating agent into the polysaccharide solution to ensure that the pH value of the polysaccharide solution is 4.5; then heating and hydrolyzing for 10 minutes by adopting normal pressure steam (100 ℃) to obtain the low molecular weight fucosan solution.
S4, static adsorption and purification by anion exchange resin: statically adsorbing the low molecular weight fucosan solution obtained from S3 for 1h by using DEAE-52 cellulose anion exchange resin, filtering, further washing the resin with deionized water, and fully removing metal ions and other impurities adsorbed by a chelating agent; and then soaking the resin in 1mol/L KCl solution for 1h to desorb the low molecular fucosan adsorbed on the resin, filtering, and directly spray-drying the filtrate to obtain the low molecular fucosan dry powder.
Through test analysis, the content of sulfate groups in the obtained low-molecular fucosan is 34.7 mass percent, and the retention rate of the sulfate groups is 98 percent; the sulfate group is bonded to the L-fucose; the glucose content in the monosaccharide composition was 1.5 mass%; the average molecular weight is 602 Da; the contents of calcium, magnesium and manganese elements in the low molecular weight fucosan are respectively 48mg/kg, 39.24mg/kg and 84mg/kg, and the removal rates are respectively 99.6%, 99.4% and 99.2%.
Example 2
The preparation method of the antiallergic sulfated galactan comprises the following steps:
s1, solid infrared pre-degradation of sulfuric acid galactan: adding deionized water into dried powder of galactan sulfate (sulfate group is 11.2 mass percent, and the contents of calcium, magnesium and manganese elements are 9870mg/kg, 4820mg/kg and 8960mg/kg respectively) from Eucheuma, wherein the water addition amount is 20 mass percent, preparing the galactan sulfate wet powder into cake skin with the thickness of 0.1cm, flatly laying the cake skin on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the polysaccharide cake skin and an infrared lamp at 10 cm; 3000w is selected and heated for 1h to obtain the pre-degraded sulfated galactan dry powder.
S2, preparation of a composite chelating agent: mixing citric acid, polyaspartic acid, tartaric acid, sodium Ethylene Diamine Tetracetate (EDTA) and sodium pyrophosphate according to a mass ratio of 3:3:2:1:1 to prepare the composite chelating agent.
S3, polysaccharide hydrolysis and sulfate group synchronous activation: dissolving the pre-degraded sulfated galactan dry powder prepared by S1 in deionized water, wherein the polysaccharide concentration is 15% (g/mL); continuously adding 0.2 percent (g/mL) of composite chelating agent into the polysaccharide solution to ensure that the pH value of the polysaccharide solution is 4.5; then heating and hydrolyzing for 10 minutes by using normal pressure steam (100 ℃) to obtain the low molecular weight sulfuric acid galactan solution.
S4, static adsorption and purification by anion exchange resin: statically adsorbing the low-molecular-weight sulfuric acid galactan solution obtained from the step S3 for 1 hour by using DEAE-52 cellulose anion exchange resin, filtering, further washing the resin with deionized water, and fully removing metal ions and other impurities adsorbed by a chelating agent; and then soaking the resin in 1mol/L KCl solution for 1h to desorb the low molecular sulfuric galactan adsorbed on the resin, filtering, and directly spray-drying the filtrate to obtain the low molecular sulfuric galactan dry powder.
Through test analysis, the content of sulfate groups in the obtained low-molecular sulfated galactan is 10.86 mass percent, and the retention rate of the sulfate groups is 97 percent; the sulfate group is bonded to D-galactose; the glucose content in the monosaccharide composition was 4.3 mass%; the average molecular weight is 940 Da; the contents of calcium, magnesium and manganese elements in the low molecular weight sulfated galactan are respectively 29.61mg/kg, 33.74mg/kg and 53.76mg/kg, and the removal rates are respectively 99.7%, 99.3% and 99.4%.
Example 3
The preparation method of the antiallergic sulfated polysaccharide comprises the following steps:
s1: performing solid infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder (the sulfated polysaccharide dry powder is dry powder obtained by extracting sulfated polysaccharide from microspherococcus algae and drying) to obtain sulfated polysaccharide wet powder, wherein the sulfated polysaccharide content in the sulfated polysaccharide wet powder is 25% by mass, preparing the wet powder into powder balls, flattening the powder balls into cake skins with the thickness of 0.12cm, flatly paving the cake skins on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the cake skins and an infrared lamp at 10 cm; 2800w is selected for the infrared drying oven, and the infrared drying oven is heated for 1h to obtain pre-degraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate according to a mass ratio of 3:3:2:1:1 to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, the mass ratio of the pre-degraded sulfuric acid polysaccharide to the deionized water is 16% (g/mL), then the compound chelating agent prepared by S2 is added, the mass ratio of the compound chelating agent to the polysaccharide solution is 0.1% (g/mL), and the pH value is adjusted to be 4.5; then heating by adopting steam, and heating and hydrolyzing for 10 minutes at 125 ℃ to obtain a low-molecular sulfated polysaccharide solution;
s4: performing static adsorption purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained from S3 for 1h by using DEAE-52 cellulose anion exchange resin, filtering, further washing the anion exchange resin with deionized water, and fully removing metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin, soaking the anion exchange resin in 1.2mol/L KCl solution for 0.8h to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly spray-drying the filtrate to obtain the antiallergic sulfated polysaccharide.
Example 4
The preparation method of the antiallergic sulfated polysaccharide comprises the following steps:
s1: performing solid infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder (the sulfated polysaccharide dry powder is dry powder obtained by extracting sulfated polysaccharide from marine brown algae and drying) to obtain sulfated polysaccharide wet powder, wherein the sulfated polysaccharide content in the sulfated polysaccharide wet powder is 15% by mass, preparing the wet powder into powder balls, flattening the powder balls into cake skins with the thickness of 0.10cm, flatly laying the cake skins on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the cake skins and an infrared lamp at 10 cm; heating the infrared drying oven at 3000w for 1h to obtain pre-degraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate according to a mass ratio of 4:4:2:1:1 to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, the mass ratio of the pre-degraded sulfuric acid polysaccharide to the deionized water is 12% (g/mL), then the compound chelating agent prepared by S2 is added, the mass ratio of the compound chelating agent to the polysaccharide solution is 0.2% (g/mL), and the pH value is adjusted to be 4.5; then heating by adopting steam, and heating and hydrolyzing for 8 minutes at 120 ℃ to obtain a low-molecular sulfated polysaccharide solution;
s4: performing static adsorption purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained from S3 for 1h by using DEAE-52 cellulose anion exchange resin, filtering, further washing the anion exchange resin with deionized water, and fully removing metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin, soaking the anion exchange resin in 1mol/L KCl solution for 1h to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly spray-drying the filtrate to obtain the antiallergic sulfated polysaccharide.
Example 5
The preparation method of the antiallergic sulfated polysaccharide comprises the following steps:
s1: performing solid infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder (the sulfated polysaccharide dry powder is dry powder obtained by extracting sulfated polysaccharide from marine red algae and drying) to obtain sulfated polysaccharide wet powder, wherein the sulfated polysaccharide content in the sulfated polysaccharide wet powder is 30% by mass, preparing the wet powder into powder balls, flattening the powder balls into cake skins with the thickness of 0.05cm, flatly laying the cake skins on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the cake skins and an infrared lamp at 12 cm; 3500w of the infrared drying oven is selected and heated for 0.5h to obtain predegraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate according to a mass ratio of 2:2:2:1:1 to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, the mass ratio of the pre-degraded sulfuric acid polysaccharide to the deionized water is 10% (g/mL), then the compound chelating agent prepared by S2 is added, the mass ratio of the compound chelating agent to the polysaccharide solution is 0.1% (g/mL), and the pH value is adjusted to be 4; then heating and hydrolyzing for 15 minutes at 110 ℃ by adopting steam to obtain a low-molecular sulfated polysaccharide solution;
s4: performing static adsorption purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained from S3 for 1.5h by using DEAE-52 cellulose anion exchange resin, filtering, further washing the anion exchange resin with deionized water, and sufficiently removing metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin, soaking the anion exchange resin in 1.5mol/L KCl solution for 0.5h to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly spray-drying the filtrate to obtain the antiallergic sulfated polysaccharide.
Example 6
The preparation method of the antiallergic sulfated polysaccharide comprises the following steps:
s1: performing solid infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder (the sulfated polysaccharide dry powder is dry powder obtained by extracting sulfated polysaccharide from marine brown algae and marine red algae and drying) to obtain sulfated polysaccharide wet powder, wherein the sulfated polysaccharide content in the sulfated polysaccharide wet powder is 10 mass percent, preparing the wet powder into powder balls, flattening the powder balls into cake skins with the thickness of 0.15cm, flatly paving the cake skins on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the cake skins and an infrared lamp at 8 cm; 2500w is selected for the infrared drying oven, and the infrared drying oven is heated for 1.5h to obtain pre-degraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate according to a mass ratio of 3:3:1:1:1 to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, the mass ratio of the pre-degraded sulfuric acid polysaccharide to the deionized water is 20% (g/mL), then the compound chelating agent prepared by S2 is added, the mass ratio of the compound chelating agent to the polysaccharide solution is 0.3% (g/mL), and the pH value is adjusted to be 5; then heating by adopting steam, and heating and hydrolyzing for 5 minutes at 130 ℃ to obtain a low-molecular sulfated polysaccharide solution;
s4: performing static adsorption purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained from S3 for 0.5h by using DEAE-52 cellulose anion exchange resin, filtering, further washing the anion exchange resin with deionized water, and sufficiently removing metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin, soaking the anion exchange resin in 0.5mol/L KCl solution for 1.5h to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly spray-drying the filtrate to obtain the antiallergic sulfated polysaccharide.
Comparative example 1
Fucosan (35.4 wt% sulfate group; and 12000mg/kg, 6540mg/kg, and 10500mg/kg of Ca, Mg, and Mn, respectively) derived from Laminaria japonica was directly used for antiallergic evaluation.
Comparative example 2
The sulfated galactan derived from Eucheuma (sulfate group 11.2 wt%; calcium, magnesium, and manganese contents of 9870mg/kg, 4820mg/kg, and 8960mg/kg, respectively) was used directly for antiallergic evaluation.
Comparative example 3
Antiallergic fucoidan was prepared according to the method of example 1, except that the metal ion removal was not performed, and the weak acid chelating agent was substituted with HCl solution, and the pH of the system was adjusted to 4.5, and the specific steps were as follows:
s1, solid infrared pre-degradation of fucosan: adding deionized water into fucosan (sulfuric acid group is 35.4 mass%; contents of calcium, magnesium and manganese elements are 12000mg/kg, 6540mg/kg and 10500mg/kg respectively) dry powder from herba Zosterae Marinae, wherein the water content is 20 mass%, preparing fucosan wet powder dough into cake skin with thickness of 0.1cm, spreading on clean stainless steel tray, and placing the tray in infrared drying oven to keep distance between the cake skin and infrared lamp at 10 cm; heating at 3000w for 1h to obtain the pre-degraded fucosan dry powder.
S2, polysaccharide hydrolysis: dissolving the pre-degraded fucosan dry powder prepared by S1 in deionized water, wherein the polysaccharide concentration is 15% (g/mL); continuously adding 0.4mol/L HCl solution into the polysaccharide solution to ensure that the pH value of the polysaccharide solution is 4.5; then heating and hydrolyzing for 10 minutes by adopting normal pressure steam (100 ℃) to obtain the low molecular weight fucosan solution.
Through test analysis, the content of the sulfate group of the obtained low-molecular fucosan is 33.9 mass percent, and the retention rate of the sulfate group is 95.76 percent; the sulfate group is bonded to the L-fucose; the glucose content in the monosaccharide composition was 1.5 mass%; average molecular weight of 643 Da; the contents of calcium, magnesium and manganese in the low molecular fucosan are 11980mg/kg, 6537mg/kg and 10495mg/kg respectively.
Comparative example 4
An anti-persulfate galactan was prepared as in example 1, except that no metal ion removal was performed, the weak acid chelating agent was replaced with HCl solution, and the pH of the system was adjusted to 4.5, as follows:
s1, solid infrared pre-degradation of sulfuric acid galactan: adding deionized water into dried powder of galactan sulfate (sulfate group is 11.2 mass percent, and the contents of calcium, magnesium and manganese elements are 9870mg/kg, 4820mg/kg and 8960mg/kg respectively) from Eucheuma, wherein the water addition amount is 20 mass percent, preparing the galactan sulfate wet powder into cake skin with the thickness of 0.1cm, flatly laying the cake skin on a clean stainless steel tray, and then putting the tray into an infrared drying box to keep the distance between the polysaccharide cake skin and an infrared lamp at 10 cm; 3000w is selected and heated for 1h to obtain the pre-degraded sulfated galactan dry powder.
S2, polysaccharide hydrolysis: dissolving the pre-degraded sulfated galactan dry powder prepared by S1 in deionized water, wherein the polysaccharide concentration is 15% (g/mL); continuously adding 0.4mol/L HCl solution into the polysaccharide solution to ensure that the pH value of the polysaccharide solution is 4.5; then heating and hydrolyzing for 10 minutes by using normal pressure steam (100 ℃) to obtain the low molecular weight sulfuric acid galactan solution.
Through test analysis, the content of sulfate groups in the obtained low-molecular sulfated galactan is 10.77 percent by mass, and the retention rate of the sulfate groups is 96.12 percent; the sulfate group is bonded to D-galactose; the glucose content in the monosaccharide composition was 4.3 mass%; average molecular weight is 929 Da; the contents of calcium, magnesium and manganese elements in the low molecular weight sulfated galactan are 9740mg/kg, 4808mg/kg and 8949mg/kg respectively.
Example 3: antiallergic activity of low-molecular sulfated polysaccharide
And (3) testing a sample: examples 1 to 2 low-molecular sulfated polysaccharides, comparative examples 1 to 4 sulfated polysaccharides, and low-molecular sulfated polysaccharides.
Experimental animals and breeding environment: SPF grade ICR male mice, weighing 20 liters and 0.3g, were purchased from Shanghai Spiker laboratory animals, Inc. The basic feed ingredients of the mice are as follows: 64% of carbohydrate, 21% of protein, 4% of fat, 5% of fiber and 6% of water. The feed is purchased from Shanghai Si Laike laboratory animals, Inc. The temperature of the breeding room is 23 +/-2 ℃, and the relative humidity is 55 +/-5%.
Grouping experiments: SPF grade ICR male mice were acclimatized for one week and fed freely. One week later, the animals were randomly divided into 20 groups, i.e., normal group, model group, sample group (ratio 1-4 and examples 1-2, and divided into three dose groups of low, medium and high), and 5 animals were tested per group. The low dose is 10mg/kg, the medium dose is 25mg/kg, and the high dose is 50 mg/kg.
Molding: except for the normal group, mice in the other groups were intraperitoneally injected with 200. mu.L of allergen solution (OVA: aluminum adjuvant: PBS: 1:1 mixed solution) at a dose of 100. mu.g/mouse on days 0 and 14. From day 28 onwards, model and sample group mice were gavaged with 50mg of OVA every 3 days for a total of 8 gavages; the groups of samples were gazed at different doses from day 41 to day 49. On day 50, mice were sacrificed by neck-breaking after blood was taken from the eyeballs, and serum was collected and used for allergy testing, with the results shown in table 1.
TABLE 1 evaluation table of antiallergic activity of sulfated polysaccharides
From the results in table 1, it is understood that the low-molecular sulfated polysaccharide which was hydrolyzed and subjected to metal ion removal had good antiallergic activity. Compared with a model group, the low-molecular fucan and the low-molecular polygalactosulfate obtained in the examples 1 and 2 show better antiallergic effect at a dose of 10mg/kg, and the OVA-specific IgE, IgG1 and IgG2a are remarkably reduced. Control 1 and control 2 are untreated high polymeric fucans and sulfated galactans, both of which show no significant anti-allergic effect at low doses, with a slight decrease in OVA-specific IgE, IgG1, IgG2a at high doses. The comparison groups 3 and 4 are low molecular weight fucosan and low molecular weight sulfated galactan which are hydrolyzed but not subjected to metal ion removal, and both low molecular weight sulfated polysaccharides have certain antiallergic activity under low dosage, but the antiallergic effect is obviously lower than that of the examples 1 and 2. Therefore, the preparation method disclosed by the invention is used for degrading the molecular weight of the sulfated polysaccharide and removing the divalent metal ions of the sulfated polysaccharide, so that the antiallergic activity of the sulfated polysaccharide is effectively improved. In view of the fact that the sulfated polysaccharide obtained by the preparation method has good antiallergic effect, the process technology is suitable for industrial production, and the obtained low molecular sulfated polysaccharide can be used for developing antiallergic drugs and functional foods.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. An antiallergic sulfated polysaccharide, which is characterized in that: the antiallergic sulfated polysaccharide is prepared by taking sulfated polysaccharide as a raw material, removing divalent metal ions, and has the characteristics of low molecular weight and high content of sulfate groups, wherein the molecular weight of the antiallergic sulfated polysaccharide is 600Da-1000Da, and the retention degree of the sulfate groups relative to the raw material sulfated polysaccharide is more than or equal to 95 mass%.
2. The antiallergic sulfated polysaccharide according to claim 1, wherein: compared with the raw material sulfated polysaccharide, the removal rate of at least one ion of calcium ions, magnesium ions or manganese ions is more than or equal to 99 mass percent.
3. The antiallergic sulfated polysaccharide according to claim 1 or 2, which is characterized in that: the content of sulfate groups in the antiallergic sulfated polysaccharide is more than or equal to 10 mass%, and the sulfate groups are combined on L-fucose or D-galactose.
4. The antiallergic sulfated polysaccharide according to claim 1 or 2, which is characterized in that: the content of glucose in monosaccharide composition of the antiallergic sulfated polysaccharide is less than or equal to 5 mass percent.
5. A method for preparing the antiallergic sulfated polysaccharide as claimed in any one of claims 1 to 4, wherein the method comprises the steps of: the method comprises the following steps:
s1: carrying out solid-state infrared pre-degradation on sulfated polysaccharide, adding deionized water into sulfated polysaccharide dry powder to obtain sulfated polysaccharide wet powder, and placing the sulfated polysaccharide wet powder in an infrared drying oven for heating to obtain pre-degraded sulfated polysaccharide dry powder;
s2: preparing a composite chelating agent, and mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate to prepare the composite chelating agent;
s3: polysaccharide hydrolysis and sulfuric acid group synchronous activation are carried out, pre-degraded sulfuric acid polysaccharide dry powder prepared by S1 is dissolved in deionized water, and then a composite chelating agent prepared by S2 is added to adjust the pH value; then heating to obtain low molecular weight sulfated polysaccharide solution;
s4: performing static adsorption and purification on anion exchange resin, performing static adsorption on the low-molecular-weight sulfated polysaccharide solution obtained in the step S3 by using the anion exchange resin, filtering, further washing the anion exchange resin by using deionized water, and fully removing metal ions, neutral sugars and impurities adsorbed by the anion exchange resin; and then desorbing the anion exchange resin to desorb the low-molecular sulfated polysaccharide adsorbed on the anion exchange resin, and directly spray-drying the filtrate after filtering to obtain the antiallergic sulfated polysaccharide.
6. The method for preparing antiallergic sulfated polysaccharide as claimed in claim 5, wherein: step S1, the sulfated polysaccharide dry powder is dry powder obtained by extracting sulfated polysaccharide from at least one of marine brown algae, marine red algae or microspherococcus, and drying;
optionally, the sulfated polysaccharide content in the sulfated polysaccharide wet powder in the step S1 is 10 to 30 mass%;
optionally, in the step S1, the wet sulfated polysaccharide powder is made into powder dough, the powder dough is flattened into a cake skin with the thickness of 0.05-0.15cm, the cake skin is flatly laid on a clean stainless steel tray, and then the tray is placed in an infrared drying oven, so that the distance between the cake skin and an infrared lamp is kept at 8-12 cm; 2500-.
7. The method for preparing antiallergic sulfated polysaccharide as claimed in claim 5, wherein: in the step S2, mixing citric acid, polyaspartic acid, tartaric acid, sodium ethylene diamine tetracetate and sodium pyrophosphate according to the mass ratio of 2-4:2-4:1-2:1: 1;
optionally, in step S3, dissolving the pre-degraded sulfated polysaccharide dry powder in deionized water, wherein the mass ratio of the pre-degraded sulfated polysaccharide to the deionized water is 10-20%, and then adding a compound chelating agent, wherein the mass ratio of the compound chelating agent to the polysaccharide solution is 0.1-0.3%;
optionally, adjusting the pH to 4-5 in step S3;
optionally, in step S3, steam heating is used to hydrolyze at 100 ℃ and 130 ℃ for 5-15 minutes, preferably atmospheric steam heating at 100 ℃ for 10 minutes.
8. The method for preparing antiallergic sulfated polysaccharide as claimed in claim 5, wherein: in the step S4, the low molecular weight sulfated polysaccharide solution obtained in the step S3 is statically adsorbed for 0.5 to 1.5 hours by adopting DEAE-52 cellulose anion exchange resin, and is filtered, the anion exchange resin is further washed by deionized water, and metal ions, neutral sugar and impurities adsorbed by the anion exchange resin are fully removed; and then desorbing the anion exchange resin, soaking the anion exchange resin in 0.5-1.5mol/L KCl solution for 0.5-1.5h to desorb the low molecular sulfated polysaccharide adsorbed on the anion exchange resin, filtering, and directly drying the filtrate by spray to obtain the antiallergic sulfated polysaccharide.
9. The antiallergic sulfated polysaccharide produced by the method for producing an antiallergic sulfated polysaccharide according to any one of claims 5 to 8, wherein the method comprises the steps of: the molecular weight of the antiallergic sulfated polysaccharide is 600Da-1000Da, divalent metal ions are removed, and the antiallergic sulfated polysaccharide has the characteristics of low molecular weight and high content of sulfate groups.
10. The use of the antiallergic sulfated polysaccharide of claim 9, which is characterized in that: can be used for preparing antiallergic drugs or antiallergic functional food.
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