Preparation method and application of Sargassum fusiformis polysaccharide-iron complex

Abstract

The present invention belongs to the technical field of polysaccharide complexes, and in particular to a method for preparing and applying a polysaccharide-iron complex of Sargassum fusiformis. The present invention adopts Sargassum fusiformis polysaccharide as a glycoside, and reacts it with a trivalent iron compound to prepare a polysaccharide-iron complex of Sargassum fusiformis, and through the optimization of process conditions, the iron content of the prepared polysaccharide-iron complex of Sargassum fusiformis is effectively improved, and the biological activity of the polysaccharide-iron complex of Sargassum fusiformis is further enhanced. The polysaccharide-iron complex of Sargassum fusiformis prepared by the present invention has good dissolution performance, small side effects, high stability, good solubility, and is easier to be absorbed by the body compared with traditional iron supplements. In addition, the polysaccharide-iron complex of Sargassum fusiformis has antioxidant activity in vitro, and has strong antioxidant activity against hydroxyl radicals. The preparation process of the present invention is simple, the source of raw materials is rich, and it can be used for preventing or treating iron deficiency anemia, and provides a new choice for a new iron supplement.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide complexes, and in particular relates to a preparation method and application of a Sargassum fusiformis polysaccharide-iron complex. Background Art

[0002] Sargassum fusiforme is a perennial warm-temperate algae that grows mainly in the southeastern coast of China and the Bohai Sea, as well as in Japan, North Korea and South Korea. It has been used as a traditional medicine and food in Asia for centuries. Sargassum fusiforme is rich in polysaccharides, proteins and trace elements, and plays an important role in anti-oxidation, anti-aging, anti-tumor, anti-virus, anti-fatigue, growth and development, and immunity. Sargassum fusiforme polysaccharides (SFPS) are the main active ingredients in Sargassum fusiforme, and have multiple physiological activities such as lowering blood sugar, lowering blood lipids, anti-cancer, antioxidant and anti-tumor. It has high health value and can be used as an additive in the special medical food industry.

[0003] Iron is an essential trace element in the human body and an important component of hemoglobin, myoglobin and various enzymes in human blood. When the body is iron deficient or iron utilization is impaired, hemoglobin synthesis will be reduced and the amount of hemoglobin in newly formed red blood cells will be insufficient. Severe iron deficiency will not only cause anemia, but also cause a lack of iron-containing enzymes in the body, leading to cell respiration disorders, affecting the function of tissues and organs, and clinically causing dysfunction of the gastrointestinal tract, circulatory, nervous and other systems. Iron deficiency and iron deficiency anemia are the most common malnutrition in the world today, and are also a micronutrient deficiency problem that needs to be solved in my country. The common first- and second-generation iron supplements on the market today, such as ferrous sulfate and ferrous fumarate, contain free iron ions. The valent iron complexes that enter the body will produce free radicals, thereby causing cell membrane damage. At the same time, the oral absorption and utilization rate of traditional iron supplements is low, which can easily lead to a series of side effects, such as gastrointestinal adverse reactions, such as nausea and vomiting, and the traditional iron supplements have a long course of treatment, making it difficult for patients to adhere to the full course of treatment, thus affecting the patient's compliance with medication. At present, the more ideal iron supplement is a composite blood-enriching preparation with trivalent iron as the core and polysaccharide as the complex. 3+ The polysaccharide-iron organic complex formed by the ligand not only has suitable complex stability as an iron supplement, and has no or little irritation to the gastrointestinal tract, but also has a variety of biological activities after releasing iron, and is a component beneficial to the body and can be absorbed and utilized. Therefore, it is a very promising oral iron supplement.

[0004] Polysaccharides can control the slow release of iron and prevent rapid hydrolysis and dissolution of iron. Studies have shown that polysaccharide iron complexes have high stability and high bioavailability. At the same time, polysaccharide iron complexes have higher safety than ferrous sulfate. In addition, polysaccharide iron complexes are complexes of polysaccharides and iron ions. There are no free iron ions. The toxic side effects on the human body are minimal or non-toxic, and they are effective iron supplements in the body. In view of these excellent properties, polysaccharide iron complexes have been recognized as potential drugs for the treatment of anemia. The development of a new, efficient and safe polysaccharide iron supplement has huge market prospects. However, no researchers have studied the synthesis of Sargassum fusiforme polysaccharide iron complex (SFIC) using Sargassum fusiforme polysaccharide. Therefore, the study of the preparation method of Sargassum fusiforme polysaccharide iron complex (SFIC) is of great significance. Summary of the invention

[0005] The main purpose of the present invention is to provide a preparation method and application of a Sargassum polysaccharide-iron complex, so as to solve the defects of existing iron supplement preparations such as low absorption and utilization rate, multiple side effects, poor stability and low iron content.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0007] A method for preparing a Sargassum polysaccharide-iron complex comprises the following steps:

[0008] (S.1) Extraction of Sargassum fusiformis polysaccharides by hot water extraction;

[0009] (S.2) Dissolve the Sargassum fusiformis polysaccharide and the chelating agent obtained in step (S.1) in water, stir and heat in a water bath, slowly add a sodium hydroxide solution dropwise and adjust the pH value of the solution, then add an aqueous solution of a trivalent iron compound dropwise and maintain the pH value of the solution constant, and stop adding the sample when reddish-brown insoluble matter appears in the solution, continue heating in a water bath, centrifuge while hot after the reaction is completed, filter and take the supernatant, obtain a mixed solution and add anhydrous ethanol for alcohol precipitation, centrifuge, filter, wash and dry to obtain the Sargassum fusiformis polysaccharide-iron complex.

[0010] Studies have shown that polysaccharides and Fe 3+ The polysaccharide iron complex is formed by oxygen bridge or hydroxyl bridge. 3+ Reduced to Fe by gastric acid 2+After being absorbed and utilized by the body, the complex formed by the complexation of high-valent iron and polysaccharide has the advantages of high utilization, fast absorption, good coordination stability, and no damage to the body. It is mild and non-irritating to physiological organs such as the esophagus, intestines and stomach, and has few side effects. After the polysaccharide releases iron as a ligand, it has multiple biological activities such as regulating the body's immunity, lowering blood pressure, and anti-oxidation, which can be absorbed and utilized by the body. The complex formed by the complexation of high-valent iron and polysaccharide has a dual effect as an iron supplement.

[0011] The present invention uses hot water extraction to extract Sargassum polysaccharide as the main raw material from Sargassum fusiformis, which is a medicinal and edible herb. The Sargassum material source is rich, which is helpful to promote and apply in industrial production. At the same time, the present invention uses Sargassum polysaccharide as a glycoside, and it is reacted with a trivalent iron compound to prepare Sargassum polysaccharide-iron complex, and the iron content of the prepared Sargassum polysaccharide-iron complex is effectively improved by the optimization of process conditions, and the biological activity of Sargassum polysaccharide-iron complex is further enhanced. The Sargassum polysaccharide-iron complex prepared by the present invention has good dissolution performance, small side effects, high stability, good solubility, and is more easily absorbed by the body compared with traditional iron supplements. In addition, Sargassum polysaccharide-iron complex has antioxidant activity in vitro, and has strong antioxidant activity to hydroxyl radicals. The preparation process of the present invention is simple, the raw material source is rich, and it can be used for preventing or treating iron deficiency anemia, and provides a new choice for novel iron supplements.

[0012] Preferably, the complexing agent is any one or more combinations of trisodium citrate, sodium alginate and sodium tartrate.

[0013] Preferably, the ferric iron compound is ferric chloride.

[0014] Preferably, the mass ratio of the Sargassum fusiformis polysaccharide to the complexing agent is 1 to 8:1.

[0015] When the mass ratio of Sargassum polysaccharide to the complexing agent is lower than 1:1, the content of Sargassum polysaccharide is too little, which may lead to insufficient combination of Sargassum polysaccharide and the complexing agent, resulting in low production of Sargassum polysaccharide-iron complex. At the same time, the content of Sargassum polysaccharide is too little, which may lead to limited reaction between the Sargassum polysaccharide and the complexing agent, making it difficult to form a stable complex structure, further affecting the antioxidant properties and other functions of the Sargassum polysaccharide-iron complex.

[0016] When the mass ratio of Sargassum polysaccharide to the complexing agent is higher than 8:1, the content of Sargassum polysaccharide is too much, and Sargassum polysaccharide cannot be completely converted into Sargassum polysaccharide-iron complex, which easily leads to a decrease in the reaction yield, further reducing the yield of Sargassum polysaccharide-iron complex. At the same time, too much Sargassum polysaccharide easily makes the subsequent purification and separation operation steps difficult, thereby reducing the purity of the Sargassum polysaccharide-iron complex.

[0017] Preferably, the molar concentration of the aqueous solution of the ferric iron compound is 0.1 to 10 mol / L.

[0018] When the molar concentration of the aqueous solution of the trivalent iron compound is lower than 0.1 mol / L, it cannot provide sufficient Fe 3+ The complex reaction is carried out with Sargassum polysaccharide, resulting in insufficient iron content in the generated Sargassum polysaccharide-iron complex. When the molar concentration of the aqueous solution of the ferric iron compound is higher than 10 mol / L, the content of the ferric iron compound is too high, which easily leads to a decrease in the solubility of the generated Sargassum polysaccharide-iron complex, further affecting its biological activity. At the same time, too much ferric iron compound reacts with the active functional groups in the Sargassum polysaccharide, resulting in a decrease in the stability of the generated Sargassum polysaccharide-iron complex.

[0019] Preferably, the reaction temperature of the water bath heating in step (S.2) is 50-90°C.

[0020] When the reaction temperature of the water bath heating in step (S.2) is lower than 50°C, the reaction rate slows down, which can easily lead to a decrease in reaction efficiency. At the same time, if the reaction temperature is too low, it is easy to cause incomplete reaction and even the formation of by-products, further affecting the purity of the Sargassum polysaccharide-iron complex. When the reaction temperature of the water bath heating in step (S.2) is higher than 90°C, it will cause the decomposition of Sargassum polysaccharide, causing its structure to be destroyed, thereby affecting its functionality. Excessively high reaction temperatures will also reduce the activity of the chelating agent or even inactivate it, thereby affecting the formation of the Sargassum polysaccharide-iron complex.

[0021] Preferably, the specific steps of adjusting the pH of the solution in step (S.2) are as follows:

[0022] Slowly drip a sodium hydroxide solution with a mass ratio of 10-30% and adjust the solution pH to 7-12.

[0023] Preferably, the specific steps of the alcohol precipitation treatment in step (S.2) are as follows:

[0024] Anhydrous ethanol is added until the anhydrous ethanol accounts for 50-90% of the total volume of the mixed solution, and then placed at 0-16°C for alcohol precipitation overnight.

[0025] Preferably, the specific steps of extracting Sargassum fusiformis polysaccharide by hot water extraction in step (S.1) are as follows:

[0026] The Sargassum fusiformis is homogenized by adding deionized water at a solid-liquid ratio of 1-2:1-20, the homogenate is heat-insulated and soaked, and then filtered. The filter residue is heat-insulated and soaked in hot water at a solid-liquid ratio of 0.5-1:1-5, and the operation is repeated twice. The filtrate is collected, concentrated, and dried to powder to obtain Sargassum fusiformis polysaccharide.

[0027] When extracting Sargassum polysaccharide from Sargassum fusiformis by hot water extraction, when the solid-liquid ratio of Sargassum fusiformis to deionized water is lower than 1:20, the polysaccharide components in Sargassum fusiformis may not be fully dissolved and released into the water, which may easily lead to reduced extraction effect and prolonged extraction time. When the solid-liquid ratio of Sargassum fusiformis to deionized water is higher than 2:1, it is easy to cause the concentration of Sargassum polysaccharide in the extract to decrease, reduce the activity and effect of the extract, thereby affecting the quality and yield of the final extraction of Sargassum polysaccharide. When the solid-liquid ratio of the filter residue to hot water is lower than 0.5:5, the amount of hot water is too much, which is easy to cause difficulty in solvent concentration, further leading to reduced extraction efficiency of Sargassum polysaccharide. When the solid-liquid ratio of the filter residue to hot water is higher than 1:1, the amount of hot water is too little, and it is difficult to fully extract the effective ingredients from the filter residue, resulting in a reduced extraction yield. At the same time, too high a solid-liquid ratio is easy to cause the filter residue to be more viscous, difficult to separate and purify, and increase the difficulty of subsequent process treatment.

[0028] The application of the preparation method of the Sargassum fusiformis polysaccharide-iron complex as described above in iron-supplementing health products for combating iron-deficiency anemia.

[0029] Therefore, the present invention has the following beneficial effects:

[0030] (1) The present invention uses hot water extraction to extract Sargassum fusiformis polysaccharide from Sargassum fusiformis, which is both medicinal and edible, as the main raw material. The Sargassum fusiformis material is abundant in source, which is helpful for popularization and application in industrial production;

[0031] (2) The present invention uses Sargassum fusiformis polysaccharide as a glycoside and reacts it with a trivalent iron compound to prepare a Sargassum fusiformis polysaccharide-iron complex. By optimizing the process conditions, the iron content of the prepared Sargassum fusiformis polysaccharide-iron complex is effectively increased, and the biological activity of the Sargassum fusiformis polysaccharide-iron complex is further enhanced;

[0032] (3) The Sargassum polysaccharide-iron complex prepared by the present invention has good dissolution performance, small side effects, high stability, good solubility, and is easier to be absorbed by the body compared with traditional iron supplements. In addition, the Sargassum polysaccharide-iron complex has antioxidant activity in vitro and has strong antioxidant activity against hydroxyl radicals;

[0033] (4) The preparation process of the present invention is simple and the raw material source is abundant, and it can be used to prevent or treat iron deficiency anemia, providing a new choice for new iron supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the in vitro simulated gastrointestinal iron release of Sargassum fusiformis polysaccharide-iron complex.

[0035] Figure 2 Schematic diagram comparing the turbidity levels of Sargassum fusiformis polysaccharide-iron complex and FeCl3.

[0036] Figure 3Schematic diagram of the sodium hydroxide titration curve of Sargassum fusiformis polysaccharide-iron complex and FeCl3.

[0037] Figure 4 Schematic diagram of the activity of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide-iron complex and ascorbic acid in scavenging DPPH free radicals.

[0038] Figure 5 Schematic diagram of the activity of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide-iron complex and ascorbic acid in scavenging ABTS free radicals.

[0039] Figure 6 Schematic diagram of the hydroxyl radical scavenging activities of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide-iron complex and ascorbic acid.

[0040] Figure 7 Schematic diagram of the superoxide anion radical scavenging activities of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide-iron complex and ascorbic acid. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for preparing a Sargassum polysaccharide-iron complex comprises the following steps:

[0044] (S.1) Extraction of Sargassum fusiformis polysaccharides by hot water extraction:

[0045] The Sargassum fusiformis was homogenized by adding deionized water at a solid-liquid ratio of 1:10, the homogenate was heat-insulated and soaked for 1 hour, and then filtered with a filter bag. The filter residue was added with 70°C water at a solid-liquid ratio of 1:1 and heat-insulated and soaked for 1 hour. The operation was repeated twice, and the filtrate was collected. The filtrate was concentrated by membrane and rotary evaporation to 1 / 20 of the original volume, and then dried in a 60°C oven for 4 hours until it was powdery, thereby obtaining Sargassum fusiformis polysaccharide;

[0046] (S.2) Synthesis of Sargassum polysaccharide-iron complex:

[0047] Weigh 2g of the Sargassum polysaccharide obtained in step (S.1) and 0.5g of trisodium citrate and dissolve them in 60mL of water, place them in a 70°C water bath and heat them in a water bath while stirring continuously. Slowly drip 20% NaOH solution and adjust the pH of the solution to 9, then add 2mol / LFeCl3 solution dropwise, and continuously adjust the pH of the solution to maintain the solution at pH = 9. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70°C for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution and add anhydrous ethanol until anhydrous ethanol accounts for 80% of the total volume of the mixed solution, and place the solution at 4°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a freeze-dried powder of Sargassum polysaccharide-iron complex.

[0048] Example 2

[0049] The difference between this embodiment and embodiment 1 is that:

[0050] This example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 0.5 g of the polysaccharide of Sargassum fusiformis obtained in step (S.1) and 0.5 g of trisodium citrate are weighed and dissolved in 60 mL of water. The rest is the same as in Example 1.

[0051] Example 3

[0052] The difference between this embodiment and embodiment 1 is that:

[0053] This example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 4 g of the polysaccharide of Sargassum fusiformis obtained in step (S.1) and 0.5 g of trisodium citrate are weighed and dissolved in 60 mL of water. The rest is the same as in Example 1.

[0054] Example 4

[0055] The difference between this embodiment and embodiment 1 is that:

[0056] This embodiment provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein a 0.1 mol / LFeCl3 solution is added dropwise in step (S.2). The rest is the same as in Example 1.

[0057] Example 5

[0058] The difference between this embodiment and embodiment 1 is that:

[0059] This embodiment provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein a 10 mol / LFeCl3 solution is added dropwise in step (S.2). The rest is the same as in Example 1.

[0060] Example 6

[0061] The difference between this embodiment and embodiment 1 is that:

[0062] This example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein the reaction temperature of water bath heating in step (S.2) is 50° C. The rest is the same as in Example 1.

[0063] Example 7

[0064] The difference between this embodiment and embodiment 1 is that:

[0065] This example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein the reaction temperature of water bath heating in step (S.2) is 90° C. The rest is the same as in Example 1.

[0066] Example 8

[0067] The difference between this embodiment and embodiment 1 is that:

[0068] The present embodiment provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 10% NaOH solution is slowly dripped and the pH of the solution is adjusted to 7, and then 2 mol / L FeCl3 solution is added dropwise, and the pH of the solution is continuously adjusted to maintain the solution at pH=7.

[0069] Example 9

[0070] The difference between this embodiment and embodiment 1 is that:

[0071] The present embodiment provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 30% NaOH solution is slowly dripped and the pH of the solution is adjusted to 12, and then 2 mol / L FeCl3 solution is added dropwise, and the pH of the solution is continuously adjusted to maintain the solution at pH = 12.

[0072] Example 10

[0073] A method for preparing a Sargassum polysaccharide-iron complex comprises the following steps:

[0074] (S.1) Extraction of Sargassum fusiformis polysaccharides by hot water extraction:

[0075] The Sargassum fusiformis was homogenized by adding deionized water at a solid-liquid ratio of 1:20, the homogenate was heat-insulated and soaked for 1 hour, and then filtered with a filter bag. The filter residue was added with 70°C water at a solid-liquid ratio of 0.5:5 and heat-insulated and soaked for 1 hour. The operation was repeated twice, and the filtrate was collected. The filtrate was concentrated by membrane and rotary evaporation to 1 / 20 of the original volume, and then dried in a 60°C oven for 4 hours until it was powdery, thereby obtaining Sargassum fusiformis polysaccharide;

[0076] (S.2) Synthesis of Sargassum polysaccharide-iron complex:

[0077] Weigh 2g of the Sargassum polysaccharide obtained in step (S.1) and 0.5g of trisodium citrate and dissolve them in 60mL of water, place them in a 60℃ water bath and heat them in a water bath while stirring continuously. Slowly drip 15% NaOH solution and adjust the pH of the solution to 8, then add 1mol / LFeCl3 solution dropwise, and continuously adjust the pH of the solution to maintain the solution at pH=8. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 60℃ for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution and add anhydrous ethanol until anhydrous ethanol accounts for 50% of the total volume of the mixed solution, and place the solution at 0℃ for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a freeze-dried powder of Sargassum polysaccharide-iron complex.

[0078] Embodiment 11

[0079] A method for preparing a Sargassum polysaccharide-iron complex comprises the following steps:

[0080] (S.1) Extraction of Sargassum fusiformis polysaccharides by hot water extraction:

[0081] The Sargassum fusiformis was homogenized by adding deionized water at a solid-liquid ratio of 2:1, the homogenate was heat-insulated and soaked for 1 hour, and then filtered with a filter bag. The filter residue was added with 70°C water at a solid-liquid ratio of 1:1 and heat-insulated and soaked for 1 hour. The operation was repeated twice, and the filtrate was collected. The filtrate was concentrated by membrane and rotary evaporation to 1 / 20 of the original volume, and then dried in a 60°C oven for 4 hours until it was powdery, thereby obtaining Sargassum fusiformis polysaccharide;

[0082] (S.2) Synthesis of Sargassum polysaccharide-iron complex:

[0083] Weigh 2g of the Sargassum polysaccharide obtained in step (S.1) and 0.5g of trisodium citrate and dissolve them in 60mL of water, place them in a water bath at 80°C and heat them with constant stirring. Slowly drip 25% NaOH solution and adjust the pH of the solution to 11, then add 3mol / LFeCl3 solution dropwise, and constantly adjust the pH of the solution to maintain the solution at pH = 11. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 80°C for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution and add anhydrous ethanol until anhydrous ethanol accounts for 90% of the total volume of the mixed solution, and place the solution at 16°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a freeze-dried powder of Sargassum polysaccharide-iron complex.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 1 is:

[0086] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 0.25 g of the polysaccharide of Sargassum fusiformis obtained in step (S.1) and 0.5 g of trisodium citrate are weighed and dissolved in 60 mL of water. The rest is the same as in Example 1.

[0087] Comparative Example 2

[0088] The difference between this comparative example and Example 1 is:

[0089] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 4.5 g of the polysaccharide of Sargassum fusiformis obtained in step (S.1) and 0.5 g of trisodium citrate are weighed and dissolved in 60 mL of water. The rest is the same as in Example 1.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 1 is:

[0092] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein a 0.05 mol / LFeCl3 solution is added dropwise in step (S.2). The rest is the same as in Example 1.

[0093] Comparative Example 4

[0094] The difference between this comparative example and Example 1 is:

[0095] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), a 11 mol / LFeCl3 solution is added dropwise. The rest is the same as in Example 1.

[0096] Comparative Example 5

[0097] The difference between this comparative example and Example 1 is:

[0098] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein the reaction temperature of water bath heating in step (S.2) is 45° C. The rest is the same as in Example 1.

[0099] Comparative Example 6

[0100] The difference between this comparative example and Example 1 is:

[0101] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein the reaction temperature of water bath heating in step (S.2) is 95° C. The rest is the same as in Example 1.

[0102] Comparative Example 7

[0103] The difference between this comparative example and Example 1 is:

[0104] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 8% NaOH solution is slowly dripped and the pH of the solution is adjusted to 6, and then 2 mol / L FeCl3 solution is added dropwise, and the pH of the solution is continuously adjusted to maintain the solution at pH=6.

[0105] Comparative Example 8

[0106] The difference between this comparative example and Example 1 is:

[0107] This comparative example provides a method for preparing a polysaccharide-iron complex of Sargassum fusiformis, wherein in step (S.2), 35% NaOH solution is slowly dripped and the pH of the solution is adjusted to 13, and then 2 mol / L FeCl3 solution is added dropwise, and the pH of the solution is continuously adjusted to maintain the solution at pH=13.

[0108] Comparative Example 9

[0109] A method for preparing Sargassum fusiformis polysaccharide comprises the following steps:

[0110] Extraction of Sargassum fusiformis polysaccharide by hot water extraction:

[0111] Deionized water was added to the Sargassum fusiformis at a solid-liquid ratio of 1:10 for homogenization, the homogenate was heat-insulated and soaked for 1 hour, and then filtered with a filter bag. The filter residue was added to 70°C water at a solid-liquid ratio of 1:1 and heat-insulated and soaked for 1 hour. The operation was repeated twice, and the filtrate was collected. The filtrate was concentrated by membrane and rotary evaporation to 1 / 20 of the original volume, and then dried in a 60°C oven for 4 hours until it was powdery to obtain Sargassum fusiformis polysaccharide.

[0112]

Performance test

[0113] The Sargassum fusiformis polysaccharide-iron complexes prepared according to the preparation methods of Examples 1 to 9 and Comparative Examples 1 to 8, respectively, and the Sargassum fusiformis polysaccharide prepared according to the preparation method of Comparative Example 9 were subjected to the following tests.

[0114] [Test on iron release of Sargassum fusiformis polysaccharide-iron complex in simulated gastrointestinal tract in vitro]

[0115] Artificial gastric juice and intestinal juice were prepared according to the Chinese Pharmacopoeia. Preparation of artificial gastric juice: Take 11.7 mL of concentrated hydrochloric acid, add deionized water to dilute to 50 mL to obtain a dilute hydrochloric acid solution. Take 16.4 mL of dilute hydrochloric acid solution, add 10 g of pepsin, dissolve in deionized water and make up to 100 mL. Preparation of artificial intestinal juice: Weigh 6.8 g of potassium dihydrogen phosphate, add 500 mL of deionized water to dissolve, and adjust the pH of the solution to 6.8 with 0.4% NaOH solution. Add 10 g of pancreatic enzyme, dilute and dissolve in deionized water and make up to 1000 mL. Prepare 0.5 mg / mL Sargassum fusiformis polysaccharide iron complex solution as the sample solution. Add 5.0 M HCl to adjust the pH of the sample solution to 2.0, and add 0.85 mL of artificial gastric juice to the sample solution. Stir the sample at 37 ° C for 2 h using a constant temperature magnetic stirrer. After the gastric juice reaction was completed, 1.0M NaHCO3 solution was added to adjust the pH of the sample solution to 6, and 4.25mL of artificial intestinal fluid was added. The pH of the sample solution was adjusted to 7 with 1.0M NaOH, and 120mM NaCl and 5mM KCl were added to 13.4mL. Then, the mixture was stirred at 37°C for 3h. During the entire reaction, the stirring speed was 150rpm, and 1.5mL of sample was taken out every 20min. The sample solution was filtered through a 0.22μm filter membrane and centrifuged at 4000r / min for 20min. The iron content in the supernatant of the sample solution was determined by o-phenanthroline spectrophotometry and recorded in Table 1. The in vitro simulated gastrointestinal iron release of Sargassum fusiformis polysaccharide-iron complex is as follows Figure 1 shown.

[0116] Table 1

[0117] Group Iron content (%) Example 1 60.34 Example 2 57.68 Example 3 58.87 Example 4 56.45 Example 5 56.95 Example 6 57.32 Example 7 57.36 Example 8 57.64 Example 9 57.86 Comparative Example 1 47.63 Comparative Example 2 47.78 Comparative Example 3 46.51 Comparative Example 4 46.57 Comparative Example 5 46.45 Comparative Example 6 46.47 Comparative Example 7 46.13 Comparative Example 8 46.34 .

[0118] From the data analysis in Table 1, it can be seen that the iron ion content in the Sargassum polysaccharide iron complex prepared by the preparation method proposed in the present invention generally reaches more than 46%, indicating that the Sargassum polysaccharide iron complex prepared by the preparation method proposed in the present invention has a significant advantage in iron loading, and due to the easy absorbability of Sargassum polysaccharides and their own pharmacological effects, the Sargassum polysaccharide iron complex as an iron supplement not only has high bioavailability, but also has additional positive effects. At the same time, the iron content of each comparative example is generally lower than that of the embodiment, which also shows that the Sargassum polysaccharide iron complex prepared by the preparation method of the present invention after the optimization of each parameter has a higher iron content.

[0119] The iron release was measured in artificial gastric juice (pH = 2.0) and artificial intestinal juice (pH = 7.0) for 120 min and 180 min, respectively. Figure 1As shown in the figure, the Sargassum polysaccharide-iron complex was gradually released within 120 minutes, and the release amount gradually increased with time. In the simulated intestinal fluid, the amount of free iron in the solution decreased significantly and stabilized after 200 minutes. This is due to the re-adsorption of iron ions by Sargassum polysaccharides under alkaline conditions. Studies have shown that free iron can be absorbed by the duodenum near the stomach. The results show that the Sargassum polysaccharide-iron complex has a good release rate in the human body.

[0120] [Test on the hydrolysis degree of Sargassum polysaccharide-iron complex at different pH values]

[0121] The SFIC was prepared according to the preparation method of Example 1. The SFIC and FeCl3 solutions were prepared at a concentration of 1 mg / mL, 20 mL were taken respectively, and titrated with 0.02 mol / L sodium hydroxide. The pH change of the reaction solution was measured with a pH acidity meter, and the turbidity of the solution was observed. Figure 2 The sodium hydroxide titration curve of Sargassum fusiformis polysaccharide iron complex (SFIC) and FeCl3 is shown in Figure 3 shown.

[0122] The hydrolysis of two iron preparations, Sargassum fusiformis polysaccharide iron complex (SFIC) and FeCl3, under different pH conditions was compared. Figure 2 and Figure 3 It can be seen that the pH when FeCl3 begins to become turbid is 2.79, and the pH when a large amount of turbidity appears is 12.24, while the SFIC has no precipitation between pH 7.92 and 12.82 and remains clear and transparent. The results show that the SFIC can be soluble in water and exist stably between pH 7.92 and 12.82.

[0123]

Antioxidant activity test

[0124] The Sargassum polysaccharide iron complex was prepared according to the preparation method of Example 1. Sargassum polysaccharide was prepared according to the preparation method of Comparative Example 9. Sargassum polysaccharide, Sargassum polysaccharide iron complex and ascorbic acid solution were prepared with deionized water at concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg / mL, respectively, and the activity of scavenging DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anion free radicals was detected according to the following method.

[0125] The DPPH free radical scavenging activity was carried out according to the method of Liu Tong (Liu Tong. Preparation, structural characterization and anti-iron deficiency anemia activity of black fungus polysaccharide iron complex [D]. Jilin Agricultural University, 2019.).

[0126] The ABTS free radical scavenging activity was carried out according to the method of Wang et al. (Zichao Wang, Shutong Jia, et al. Antioxidant activity of a polysaccharide produced by Chaetomium globosum CGMCC 6882 [J]. International Journal of Biological Macromolecules, 2019, 233 (141): 248-257.).

[0127] The hydroxyl radical scavenging activity was carried out according to the method of Jhonatas et al. (Jhonatas Rodrigues Barbosa, Maurício Madson S. Freitas, et al. Obtaining extracts rich in antioxidant polysaccharides from the edible mushroom Pleurotus ostreatus using binary system with hotwater and supercritical CO2[J]. Food Chemistry, 2020, 330(10): 127-137.).

[0128] The superoxide anion radical scavenging activity was carried out according to the method of Chen et al. (Juncheng Chen, Li Lin, et al. Preliminary characterization and antioxidant and hypoglycemic activities in vivo of polysaccharides from Huidouba [J]. Food & function, 2018, 9 (12): 6337-6348.).

[0129] All data are expressed as mean ± standard error. Figure 4 The activities of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide iron complex and ascorbic acid in scavenging ABTS free radicals are shown in Figure 5The scavenging activities of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide-iron complex and ascorbic acid in scavenging hydroxyl free radicals are shown in Figure 6 The activities of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide iron complex and ascorbic acid in scavenging superoxide anion free radicals are shown in Figure 7 shown.

[0130] The experiment measured the scavenging rates of DPPH, ABTS, hydroxyl, and superoxide anion free radicals of Sargassum fusiformis polysaccharide, Sargassum fusiformis polysaccharide iron complex, and ascorbic acid. Figures 4 to 7 Data analysis showed that, with ascorbic acid as the positive control, both Sargassum polysaccharide and Sargassum polysaccharide-iron complex had certain antioxidant activity. The antioxidant activity of Sargassum polysaccharide was generally higher than that of Sargassum polysaccharide-iron complex. The scavenging rates of Sargassum polysaccharide and Sargassum polysaccharide-iron complex on DPPH free radicals were low, and the antioxidant activity on ABTS and hydroxyl free radicals was better. In the determination of superoxide anion free radical scavenging rate, the scavenging rate of Sargassum polysaccharide-iron complex decreased with increasing concentration.

[0131] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a Sargassum polysaccharide-iron complex, characterized in that: The following steps are involved: (S.1) Extraction of Sargassum fusiformis polysaccharides by hot water extraction; (S.2) dissolving the Sargassum fusiformis polysaccharide and the chelating agent obtained in step (S.1) in water, stirring and heating in a water bath, slowly dripping in a sodium hydroxide solution and adjusting the pH value of the solution, then dropwise adding an aqueous solution of a trivalent iron compound and maintaining the pH value of the solution constant, and stopping the addition of samples when reddish-brown insoluble matter appears in the solution, continuing heating in a water bath, and centrifuging and filtering the supernatant while the reaction is completed to obtain a mixed solution, adding anhydrous ethanol for alcohol precipitation, and obtaining a Sargassum fusiformis polysaccharide-iron complex after centrifugation, filtering, washing and drying; the chelating agent is any one or more combinations of trisodium citrate, sodium alginate and sodium tartrate; the mass ratio of the Sargassum fusiformis polysaccharide to the chelating agent is 1 to 8:

1.

2. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The trivalent iron compound is ferric chloride.

3. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The molar concentration of the aqueous solution of the trivalent iron compound is 0.1 to 10 mol / L.

4. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The reaction temperature of the water bath heating in the step (S.2) is 50-90°C.

5. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The specific steps of adjusting the pH of the solution in the step (S.2) are as follows: Slowly drip a sodium hydroxide solution with a mass ratio of 10-30% and adjust the solution pH to 7-12.

6. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The specific steps of the alcohol precipitation treatment in step (S.2) are as follows: Anhydrous ethanol is added until the anhydrous ethanol accounts for 50-90% of the total volume of the mixed solution, and then placed at 0-16°C for alcohol precipitation overnight.

7. The method for preparing a Sargassum polysaccharide-iron complex according to claim 1, characterized in that: The specific steps of hot water extraction of Sargassum fusiformis polysaccharide in step (S.1) are as follows: The Sargassum fusiformis is homogenized by adding deionized water at a solid-liquid ratio of 1-2:1-20, the homogenate is heat-insulated and soaked, and then filtered. The filter residue is heat-insulated and soaked in hot water at a solid-liquid ratio of 0.5-1:1-5, and the operation is repeated twice. The filtrate is collected, concentrated, and dried to powder to obtain Sargassum fusiformis polysaccharide.

8. Use of the preparation method of the Sargassum fusiformis polysaccharide-iron complex according to any one of claims 1 to 7 in iron-supplementing health products for preventing iron-deficiency anemia.

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