Lentinan-ferrous complex for improving intestinal flora and oxidative stress and preparation method thereof

Abstract

The invention discloses a lentinan-ferrous complex for improving intestinal flora and oxidative stress as well as a preparation method and application of the lentinan-ferrous complex, and belongs to the technical field of functional foods and medicines. The preparation method comprises the following steps: dissolving lentinan and trisodium citrate in water in proportion, heating to 50-55 DEG C under the protection of nitrogen, slowly dropwise adding a FeSO4 solution, simultaneously maintaining the pH value to be 8-9 by using a NaOH solution, reacting, centrifuging the reaction solution, precipitating with alcohol, washing, and drying to obtain the lentinan. The prepared complex can significantly improve the stability of ferrous ions, and the bioavailability of the complex in a rat body is superior to that of ferrous sulfate and ferrous glycinate. More importantly, the complex has multiple effects of supplementing iron, regulating intestinal flora (such as reducing F / B value), increasing the yield of short-chain fatty acid and relieving oxidative stress and inflammatory response, and can be used for developing a novel iron supplementing preparation with an intestinal microenvironment improving function.

Description

Technical Field

[0001] This invention relates to the preparation technology of an iron supplement containing lentinan-ferrous complex that can improve gut microbiota and oxidative stress. Background Technology

[0002] Iron deficiency anemia (IDA) is one of the four major nutritional deficiency diseases recognized by the World Health Organization (WHO). According to WHO statistics, approximately 2 billion people worldwide suffer from varying degrees of anemia, with about 50% attributable to iron deficiency. IDA not only affects an individual's growth, development, and cognitive abilities but can also lead to a series of serious health problems. Iron is an essential trace element for the human body, primarily involved in the synthesis of hemoglobin. Iron deficiency leads to a reduction in hemoglobin, thereby affecting oxygen supply to various tissues and organs, resulting in anemia symptoms. The 2002 my country National Nutrition and Health Survey showed that the average prevalence of anemia among Chinese residents was 15.2%, with higher prevalence rates in infants under 2 years old, the elderly over 60 years old, and women of childbearing age. Iron deficiency not only causes anemia but also leads to a series of health problems such as decreased physical strength, weakened immunity, and cognitive impairment.

[0003] The global distribution of iron deficiency anemia (IDA) shows significant regional differences. According to a UNICEF report, approximately 50% of children in developing countries suffer from varying degrees of iron deficiency, while this figure is around 10% in developed countries. Certain physiological stages, such as infants, adolescents, pregnant women, and women of childbearing age, are at high risk for IDA. The iron content in newborns is directly related to their birth weight; low birth weight infants (<2.5 kg) have insufficient iron reserves and are more prone to IDA during rapid growth. Pregnant women require 2-3 times more iron than usual due to increased blood volume and fetal needs; failure to supplement iron can easily lead to anemia. Insufficient dietary iron intake is a major cause of IDA in developing countries. Dietary iron is divided into heme iron and non-heme iron. Heme iron, mainly found in animal foods, is a divalent ferrous ion and is closely related to hematopoiesis and the pathogenesis of IDA, with an absorption rate of 15%-35%. Non-heme iron, mainly found in plant foods, is a trivalent ferric ion, with an absorption rate of only 2%-20%, and is unrelated to hematopoiesis and the pathogenesis of IDA.

[0004] IDA prevention requires supplementation with divalent heme iron (II). The traditional clinical iron supplement is ferrous sulfate, which has the advantages of being inexpensive and having a clear iron-supplementing effect. However, it has disadvantages such as: a strong metallic taste, affecting palatability and making long-term use difficult; potential tooth discoloration with prolonged use; significant gastrointestinal irritation (nausea, vomiting, constipation); low iron absorption rate, leading to low iron supplementation efficiency; and a tendency to trigger oxidative stress. Intravenous iron supplements can rapidly increase iron reserves and are suitable for people with severe iron deficiency or oral intolerance, but there is a risk of allergic reactions, requiring close monitoring. Traditional iron supplements have complex interactions with other medications; for example, co-administration with antacids can significantly reduce efficacy.

[0005] In addition, there are reports of using ferrous ions as a nutritional fortifier in the development of functional foods with iron-supplementing effects. However, during food processing and storage, ferrous ions are easily oxidized to trivalent non-heme iron. This oxidation process reduces the bioavailability of iron and can also cause undesirable sensory changes in food, such as darkening of color, the development of a pronounced metallic taste, and a reduction in the nutritional quality of the product. Constructing complexes with ferrous ions using natural macromolecules is currently the most promising method. Common methods include constructing amino acid chelated iron, peptide chelated iron, dextran iron, and synthesizing polysaccharide iron complexes.

[0006] Many studies favor constructing complexes with peptides, amino acids, and ferrous ions because peptides and amino acids have relatively high absorption efficiency in the intestines, which helps reduce the probability of ferrous ions being oxidized. However, peptides and amino acids themselves do not have strong antioxidant capabilities, and ferrous ions are still easily oxidized to ferric ions during complex preparation. Polysaccharides naturally possess antioxidant properties, which can improve the stability of ferrous ions in food systems. Furthermore, polysaccharides have the advantage of improving the intestinal microenvironment. Using polysaccharides as ligands to prepare ferrous complexes can achieve iron supplementation while simultaneously improving the function of the intestinal flora. In terms of preparation methods, the direct chelation method is generally used, which involves mixing ferrous salts (e.g., FeSO4) with ligands under suitable pH conditions and promoting the reaction through heating or stirring.

[0007] Ferrous ions (Fe) 2+ Ferrous ions are easily oxidized in food systems, primarily due to their reactive chemical properties, readily reacting with oxygen, free radicals, and other oxidizing agents. In the food environment, dissolved oxygen directly reacts with ferrous ions, oxidizing them to ferric (Fe3+) ions. 3+ At the same time, superoxide radicals (•O2) are generated. -This leads to a chain oxidation reaction, accelerating the degradation of lipids, vitamins, and other sensitive components in food. Furthermore, the pH, water activity, temperature, and coexisting components (such as ascorbic acid, polyphenols, and transition metal ions) of the food system significantly affect the stability of ferrous ions. For example, under weakly acidic to neutral conditions (pH 5–7), the oxidation rate of ferrous ions is high, and while ascorbic acid itself has reducing properties, it can promote the oxidation of ferrous ions through electron transfer in certain situations. Light, heating, and metal ions (such as Cu) also contribute to the oxidation. + The presence of ferrous ions further catalyzes this process, leading to deterioration in the color, flavor, and nutritional value of food. Therefore, chelating agents, antioxidants, or physical encapsulation techniques are often used during food processing and storage to slow down the oxidation of ferrous ions and maintain food quality.

[0008] In recent years, the interaction between metal ions and polysaccharides has attracted widespread attention. Active groups such as hydroxyl and carboxyl groups in polysaccharide molecules can coordinate with metal ions (such as iron, zinc, and calcium) to form stable polysaccharide-metal ion complexes. Polysaccharide-iron complexes are a novel oral iron supplement. With their unique molecular structure and excellent bioavailability, they have become an important choice for iron supplementation in modern medicine. Compared with traditional iron supplements, polysaccharide-iron complexes have several significant advantages. Their stable molecular structure makes them less susceptible to destruction by gastric acid, allowing for the slow release of iron ions in the intestines, reducing gastrointestinal irritation and significantly lowering the incidence of adverse reactions such as nausea, vomiting, and constipation. Polysaccharide-iron complexes are efficiently absorbed and utilized by the intestines, with a bioavailability far exceeding that of ordinary iron supplements, meaning patients can achieve iron supplementation effects in a shorter time. Polysaccharide-iron complexes also exhibit good tolerability, and even long-term use is unlikely to induce drug resistance, making them suitable for patients requiring long-term iron supplementation, such as those with iron deficiency anemia and pregnancy anemia. Polysaccharide-iron complexes, with their high efficiency, safety, and good tolerability, have become a new option for iron supplementation in modern medicine. They offer patients a more convenient and safer way to supplement iron. Summary of the Invention

[0009] The purpose of this invention is to provide a lentinan-iron(II) complex, and the lentinan-iron(II) complex can be used to prepare iron supplements that improve gut microbiota and oxidative stress.

[0010] The technical solution adopted in this invention is:

[0011] A method for preparing a lentinan-ferrous complex that improves gut microbiota and oxidative stress, the method comprising:

[0012] Lentinan and trisodium citrate were dissolved in water and heated to 50-55°C under nitrogen protection. FeSO4 solution was slowly added dropwise while the pH was adjusted to 8-9 with NaOH solution. The reaction was maintained at this temperature for 1-2 hours. After the reaction was completed, the solid and liquid were separated, and the supernatant was precipitated with alcohol to obtain the lentinan-ferrous complex.

[0013] This invention uses a co-heating method to prepare polysaccharide ferrous ion complexes. To prevent the ferrous iron from being oxidized by air during the experiment, nitrogen gas is introduced throughout the experiment.

[0014] The mass ratio of lentinan to trisodium citrate is 3-4:1, preferably 3:1.

[0015] The concentration of the FeSO4 solution is preferably 0.5~1 mol / L; the concentration of the NaOH solution is preferably 1~3%, more preferably 2%.

[0016] After the reaction is completed, solid and liquid are separated, which is generally done by centrifugation. Centrifugation can be carried out at 4℃ and 8000 r / min for 10 min, and the supernatant is collected.

[0017] The supernatant was precipitated by adding 3 times the volume of 90% ethanol to the supernatant and precipitating at -18 to -20°C for 8 to 15 hours. Then, it was centrifuged at 4°C and 8000 r / min for 10 min. The precipitate was washed with anhydrous ethanol and freeze-dried under vacuum to obtain the lentinan-ferrous complex.

[0018] The lentinan generally needs to be pre-dried to remove moisture. Preferably, the lentinan is dried at 60°C for 2 hours, and the sample is placed in a sealed bag and placed in a desiccator.

[0019] This invention uses a co-heating method to prepare lentinan-ferrous ions (Fe2+). 2+ The mechanism of this complex is mainly based on the coordination ability of polysaccharides and the properties of ferrous ions, with heating promoting their binding. Lentinan molecules (such as β-glucan) contain abundant oxygen-containing functional groups such as hydroxyl (-OH) and carboxyl (-COOH), which can act as electron donors to bind with ferrous ions (Fe²⁺). 2 + Coordination bonds are formed. Increasing the temperature increases the flexibility of the polysaccharide chains and the diffusion rate of ferrous ions, promoting their contact. Upon heating, the polysaccharide chains partially unfold, exposing more hidden coordinating groups. Heating may remove the hydration layer on the polysaccharide surface, reducing steric hindrance and allowing Fe... 2+ It is easier to access the active site. Fe 2+ Easily oxidized to Fe 3+ However, it can be partially stabilized in the coordination environment of polysaccharides. The hydrophobic microenvironment of polysaccharides may reduce Fe 2+Contact with dissolved oxygen in water. Operation must be performed in an inert gas environment (such as nitrogen), or with the addition of reducing agents such as ascorbic acid to prevent Fe... 2+ Oxidation. Avoid excessively high temperatures that could lead to polysaccharide degradation or Fe oxidation. 2+ Oxidation is accelerated. Through a co-heating method, lentinan and ferrous ions form a stable complex, the core of which is the synergistic effect of coordination chemistry and thermodynamics.

[0020] The present invention also provides a lentinan-ferrous complex prepared by the above method.

[0021] This invention provides the application of lentinan-ferrous complex in the preparation of iron supplements.

[0022] Lentinan-ferrous complex can effectively improve the stability of ferrous ions during storage, and its effect is better than that of commercially available glycine iron products.

[0023] Furthermore, the lentinan-ferrous complex can improve the bioavailability of ferrous ions in rats, and its iron supplementation effect is superior to that of ferrous sulfate and commercially available glycine iron products.

[0024] The present invention also provides the role of lentinan-ferrous complex in the preparation of formulations that improve intestinal flora imbalance.

[0025] Furthermore, the lentinan-ferrous complex can be used to prepare formulations that reduce the relative abundance of Firmicutes, Proteobacteria, and Actinobacteria in the gut, increase the relative abundance of Bacteroidetes and Verrucous, and decrease the Firmicutes / Bacteroidetes ratio. Its effects are superior to ferrous sulfate and commercially available ferric glycine products.

[0026] Furthermore, the present invention also provides the role of lentinan-ferrous complex in the preparation of formulations that improve inflammatory responses caused by intestinal flora imbalance.

[0027] Lentinan-ferrous complex can be used to prepare formulations that reduce the levels of lipopolysaccharide (LPS), lipopolysaccharide-binding protein (LBP), Toll-like receptor 4 (TLR4), nuclear factor κ-B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) in plasma and liver, while increasing the level of interleukin-10 (IL-10).

[0028] The present invention also provides the role of lentinan-ferrous complex in the preparation of formulations that increase the level of intestinal short-chain fatty acids (SCFAs).

[0029] Lentinan-ferrous complex can be used to prepare formulations that increase the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid in the intestines.

[0030] This invention also provides the application of lentinan-ferrous complex in the preparation of formulations that improve oxidative stress in the body.

[0031] The beneficial effects of this invention are:

[0032] This invention constructs a complex by combining lentinan with ferrous ions, thereby protecting the stability of ferrous ions in food systems and improving their bioavailability in rats. It also improves gut microbiota dysbiosis in mice induced by a high-fat diet, reducing the relative abundance of Firmicutes, Proteobacteria, and Actinobacteria while increasing the relative abundance of Bacteroidetes and Verrucous Microbes, lowering the Firmicutes / Bacteroidetes ratio, increasing the production of short-chain fatty acids, and improving oxidative stress and inflammatory responses. Furthermore, the above-mentioned effects of the lentinan-ferrous complex obtained by this invention are superior to those of ferrous sulfate and commercially available glycine iron products. The lentinan-ferrous complex of this invention has significant market potential as an iron supplement that improves the intestinal microenvironment. Attached Figure Description

[0033] Figure 1 The Fourier transform infrared (FTIR) spectra of lentinan and lentinan-iron(II) are shown in Figure A. Figure B shows the FTIR spectra of lentinan and lentinan-iron(II).

[0034] Figure 2 The effects of FeSO4, glycine iron(II) (Gly-Fe(II)), and lentinan-iron(II) on Fe during simulated storage. 2+ The loss rate. Figure A shows the Fe loss rate of three samples before and after 3 days of storage. 2+ The mass fraction of Fe in the three samples before and after storage is shown in Figure B. 2+ The loss rate.

[0035] Figure 3 The mass fraction of iron in the blood of rats administered FeSO4, glycine iron (II) (Gly-Fe(II)), and lentinan-iron (II) by gavage at various time points was determined.

[0036] Figure 4Figures show the effects of each group of samples on the mouse gut microbiota. Figure A shows the Chao1 analysis results of mouse gut microbiota diversity; Figure B shows the Shannon analysis results of mouse gut microbiota diversity; Figure C shows the Simpson analysis results of mouse gut microbiota diversity; Figure D shows the relative abundance of Firmicutes in the mouse gut; Figure E shows the relative abundance of Bacteroidota in the mouse gut; Figure F shows the Firmicutes / Bacteroidota ratio in the mouse gut; Figure G shows the relative abundance of Proteobacteria in the mouse gut; Figure H shows the relative abundance of Actinobacteriota in the mouse gut; and Figure I shows the relative abundance of Verrucomicrobiota in the mouse gut. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0037] Figure 5 The following figures illustrate the effects of each group of samples on short-chain fatty acids (SCFA) in the mouse intestine. Figure A shows the bar chart of total SCFA levels in the mouse intestine; Figure B shows the bar chart of acetic acid levels in the mouse intestine; Figure C shows the bar chart of propionic acid levels in the mouse intestine; Figure D shows the bar chart of butyrate levels in the mouse intestine; Figure E shows the bar chart of isobutyrate levels in the mouse intestine; Figure F shows the bar chart of valerate levels in the mouse intestine; Figure G shows the bar chart of isovalerate levels in the mouse intestine; and Figure H shows the bar chart of hexanoate levels in the mouse intestine. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0038] Figure 6 The following figures illustrate the effects of redox status on mouse blood: Figure A shows the bar chart of total antioxidant capacity (T-AOC) in mouse blood; Figure B shows the bar chart of the ratio of reduced glutathione to oxidized glutathione (GSH / GSSG) in mouse blood; Figure C shows the bar chart of malondialdehyde (MDA) level in mouse blood; Figure D shows the bar chart of superoxide dismutase (SOD) activity in mouse blood; Figure E shows the bar chart of catalase (CAT) activity in mouse blood; and Figure F shows the bar chart of glutathione peroxidase (Gpx) activity in mouse blood. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0039] Figure 7The following figures illustrate the effects of these factors on the inflammatory response in mouse blood: Figure A shows the bar charts for mouse blood lipopolysaccharide (LPS) levels; Figure B shows the bar charts for mouse blood lipopolysaccharide receptor (LBP) levels; Figure C shows the bar charts for mouse blood interleukin-6 (IL-6) levels; Figure D shows the bar charts for mouse blood interleukin-8 (IL-8) levels; Figure E shows the bar charts for mouse blood interleukin-10 (IL-10) levels; Figure F shows the bar charts for mouse blood Toll-like receptor 4 (TLR4) levels; Figure G shows the bar charts for mouse blood tumor necrosis factor-α (TNFα) levels; and Figure H shows the bar charts for mouse blood nuclear factor-κB (NFκB) levels. Significant differences were defined as follows: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group. Detailed Implementation

[0040] To make the objectives, technical solutions, and effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention. Unless otherwise specified, the experimental materials and reagents used are all commercially available consumables and reagents.

[0041] Example 1: Preparation of Lentinan-Iron(II) Complex by Heating Method

[0042] Principle: Preparation of lentinan-ferrous ions (Fe) by co-heating method 2+ The mechanism of this complex is mainly based on the coordination ability of polysaccharides and the properties of ferrous ions, with heating promoting their binding. Lentinan molecules (such as β-glucan) contain abundant oxygen-containing functional groups such as hydroxyl (-OH) and carboxyl (-COOH), which can act as electron donors to bind with ferrous ions (Fe²⁺). 2+ Coordination bonds are formed. Increasing the temperature increases the flexibility of the polysaccharide chains and the diffusion rate of ferrous ions, promoting their contact. Upon heating, the polysaccharide chains partially unfold, exposing more hidden coordinating groups. Heating may remove the hydration layer on the polysaccharide surface, reducing steric hindrance and allowing Fe... 2+ It is easier to access the active site. Fe 2+ Easily oxidized to Fe 3+ However, it can be partially stabilized in the coordination environment of polysaccharides. The hydrophobic microenvironment of polysaccharides may reduce Fe 2+ Contact with dissolved oxygen in water. Operation must be performed in an inert gas environment (such as nitrogen), or with the addition of reducing agents such as ascorbic acid to prevent Fe... 2+ Oxidation. Avoid excessively high temperatures that could lead to polysaccharide degradation or Fe oxidation. 2+ Oxidation is accelerated. Through a co-heating method, lentinan and ferrous ions form a stable complex, the core of which is the synergistic effect of coordination chemistry and thermodynamics.

[0043] Procedure: Weigh 0.75g of dried lentinan and add 0.25g of trisodium citrate, mixing them (mass ratio 3:1) in 250mL of water. The polysaccharide-ferrous ion complex is prepared using a co-heating method. To prevent the oxidation of ferrous iron by air during the experiment, nitrogen gas should be introduced throughout the process. Adjust the temperature of the electromagnetic stirrer to 50℃ to maintain the reaction temperature at 50℃. Pour the lentinan-trisodium citrate mixed solution into a beaker, introduce nitrogen gas into the beaker, and slowly add 0.5 mol / L FeSO4 solution until the final FeSO4 solution volume is approximately 4 mL. Simultaneously, adjust the pH with 2% NaOH solution to maintain it at around 8. After stirring with the electromagnetic stirrer for 1 hour, centrifuge at 4℃ and 8000 r / min for 10 min, and collect the supernatant. Approximately three volumes of 90% ethanol were added to the supernatant, and the mixture was incubated overnight at -20°C for alcohol precipitation. The precipitate was then centrifuged at 8000 r / min for 10 min at 4°C. The precipitate was collected, washed three times with anhydrous ethanol, and then freeze-dried under vacuum. 0.78 g of the lentinan-iron(II) complex was obtained, with an iron mass fraction of 3.91%.

[0044] Example 2: Fourier transform infrared spectroscopy analysis of lentinan and lentinan-iron(II)

[0045] In recent years, Fourier transform infrared spectroscopy (FTIR) has been widely applied and is often used as a key technology for monitoring the functional group composition of organic / inorganic structures. This invention, based on infrared spectroscopy, analyzes the infrared absorption peaks of lentinan and its ferrous ion complex samples to explore their functional group composition and structural changes. The characteristic peaks of the infrared spectra are labeled and analyzed as follows: Figure 1 As shown.

[0046] FTIR results of lentinan (shiitake mushroom polysaccharide) Figure 1 In Figure A), 3410 cm -1 The absorption peak at 2927 cm⁻¹ is mainly due to the stretching vibration mode of the polysaccharide OH group. The peak shifts due to varying degrees of association of the hydroxyl groups, resulting in a broad peak shape. -1 The absorption peak at 1654 cm⁻¹ is attributed to the methylene CH stretching vibration mode of the polysaccharide. -1 The absorption peak at 1425 cm⁻¹ is a characteristic peak of the C=O stretching vibration in -COOH, and the appearance of this peak indicates that lentinan is an acidic polysaccharide; -1 The overlapping peaks at this location are relatively complex, mainly related to the symmetric stretching vibration mode of C=O in -COOH. Absorption peaks from OH deformation vibration and methylene bending vibration also appear in this region; in the 1155-1024 cm⁻¹ range... -1The absorption peak at that point is the stretching vibration peak of COC and C-OH in pyranose, indicating that the lentinan polysaccharide structure is extremely rich in pyranose monosaccharides.

[0047] The infrared characteristics of the lentinan-ferrous ion complex sample changed significantly, such as... Figure 1 As shown in Figure B, the sample is at 3414 cm. -1 An absorption peak for the OH stretching vibration of lentinan was observed at 2929 cm⁻¹. -1 An absorption peak for the methylene CH stretching vibration appears at 1630 cm⁻¹; -1 An absorption peak for C=O stretching vibration appears at 1155-1024 cm⁻¹. -1 The absorption peak at that location corresponds to the stretching vibrations of COC and C-OH in pyranose. The relative intensity of these absorption peaks is significantly lower than that of lentinan, indicating a lower proportion of polysaccharide components in both the lentinan and its ferrous ion complex samples. These characteristics demonstrate the successful construction of the lentinan-ferrous ion complex.

[0048] Example 3: Stability Study of Iron(II) in the Composite

[0049] In this part of the examples, FeSO4, commercially available glycine iron(II), and lentinan-iron(II) were selected to compare the stability of three forms of ferrous ions during simulated food storage. The samples were prepared into 10 μg / mL solutions and stored in a 37°C incubator for 3 days. The ferrous ion content was measured once before the start of the storage experiment and once after the end of the experiment, and the loss rate of ferrous ions was calculated.

[0050] Detection method: Determination of iron (Fe) by o-phenanthroline colorimetric method (o-phenanthroline spectrophotometric method) 2+ Mass fraction. Fe 2+ It forms a complex with o-phenanthroline, and has a maximum absorption peak at 510 nm.

[0051] Reagent Preparation: 0.1% o-phenanthroline solution: Weigh 0.1 g o-phenanthroline, dissolve in 50 mL distilled water, and gently heat to 60℃ to aid dissolution. Cool before use and bring to a final volume of 100 mL. 10% hydroxylamine hydrochloride solution: Weigh 10 g hydroxylamine hydrochloride and dissolve in 100 mL distilled water. Sodium acetate buffer: Weigh 8.2 g CH3COONa·3H2O, dissolve in 50 mL water, add 5.7 mL glacial acetic acid, adjust to pH 4.5 using a pH meter, and bring to a final volume of 100 mL. Iron standard solution (100 μg / mL): Accurately weigh 0.0702 g ferrous ammonium sulfate ((NH4)2Fe(SO4)2·6H2O), dissolve in 50 mL 0.1 mol / L H2SO4 solution (to prevent oxidation of ferrous iron), and bring to a final volume of 100 mL. Take 10 mL of the iron standard solution and bring to a final volume of 100 mL.

[0052] Standard curve preparation: Take six 25 mL colorimetric tubes and add 0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of iron standard solution (10 μg / mL), corresponding to iron contents of 0, 5, 10, 20, 30, and 40 μg, respectively. Add distilled water to each tube to approximately 10 mL. Then, add 1 mL of hydroxylamine hydrochloride solution sequentially, mix well, and let stand for 2 minutes (to reduce Fe). 3+ →Fe 2+ Add 2 mL of o-phenanthroline solution and 5 mL of sodium acetate buffer. Adjust the volume to 25 mL, shake well, and let stand in the dark for 15 minutes. Measure the absorbance of each tube at 510 nm. Plot a standard curve with iron content (μg) on ​​the x-axis and absorbance on the y-axis. Fit the regression equation. Take 10 mL of the treated sample solution, add reagents according to the standard curve procedure, develop the color, and measure the absorbance. Calculate the Fe content of the sample. 2+ Quality score.

[0053] Figure 2 The effects of FeSO4, glycine iron(II) (Gly-Fe(II)), and lentinan-iron(II) (LNT-Fe(II)) on Fe before and after storage. 2+ mass fraction (A) and Fe 2+ The loss rate. It can be seen that, because the molecular weight of polysaccharides is much larger than that of glycine, the Fe in glycine iron(II) is... 2+ The mass fraction was relatively high. After 3 days of simulated storage, FeSO4 was stored without any protection, and Fe... 2+ All were oxidized to Fe 3+ The loss rate reached 100%. Fe in glycine iron(II) 2+ The loss rate was approximately 50%, while the Fe content in lentinan-iron(II) was... 2+ The loss rate was only 20%. This indicates that the lentinan-iron(II) complex provided by this invention effectively protects Fe...2+ It has better stability.

[0054] Example 4: Bioavailability of Lentinan-Iron(II) in Animals

[0055] Blood drug concentration-time assay: Eighteen healthy male Wistar rats weighing approximately 200 g were randomly divided into three groups of six each: FeSO4 group, glycine iron (II) group, and lentinan-iron (II) group. The mass fraction of iron in rat blood was determined using atomic absorption spectrophotometry. Baseline iron levels in the blood of each group were measured before gavage. Rats were then administered a dose of 5 mg Fe / kg BW via gavage. Blood samples were collected from the tail of the rats at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours after administration to determine the mass fraction of iron in the blood.

[0056] according to Figure 3 As shown, by detecting the iron content in the blood of rats at various time points, it was found that under the same iron gavage conditions, the concentration of iron in the blood of rats gavaged with lentinan-iron(II) was higher. This indicates that constructing lentinan-iron(II) helps to improve the bioavailability of iron, and this effect is better than that of glycine-iron(II).

[0057] Example 5: Effects of Lentinan-Iron(II) on High-Fat-Induced Gut Microbiota in Mice

[0058] (1) Animal model construction and grouping

[0059] Specific pathogen-free (SPF) male C57BL / 6 mice (4 weeks old, weighing 18±2 g) were housed in an environment with an ambient temperature of 20-24 ℃, relative humidity of 40-60%, and a 12-hour / 12-hour light-dark cycle. All mice used in this experiment had free access to food and purified water throughout the experiment. After 7 days of acclimatization, the mice were fed a normal diet (fat energy ratio: 20%, energy: 3,530 kcal / kg, 8 mice) and a high-fat diet (HFD, fat energy ratio: 60%, energy: 5,127 kcal / kg, 32 mice) for 6 weeks to induce gut microbiota dysbiosis.

[0060] After 3 weeks of feeding, the mice were randomly divided into 5 groups. Control group (CON, n=8): normal diet, daily intragastric injection of physiological saline (0.9% NaCl); HFD group (n=8): HFD feeding, daily intragastric injection of physiological saline; FeSO4 group (n=8): HFD feeding, oral gavage of FeSO4 solution, gavage dose calculated based on iron content: 5 mg Fe / kg BW; Gly-Fe(II) group (n=8): HFD feeding, oral gavage of glycine iron(II) solution, gavage dose calculated based on iron content: 5 mg Fe / kg BW; LNT-Fe(II) group (n=8): HFD feeding, oral gavage of lentinan-iron(II) solution, gavage dose calculated based on iron content: 5 mg Fe / kg BW.

[0061] (2) Effects of Lentinan-Iron(II) on gut microbiota and SCFAs in mice induced by high-fat diet

[0062] The structural characteristics of the gut microbiota were analyzed by 16S rDNA sequencing. Total DNA was eluted with 50 µL of elution buffer and analyzed by PCR.

[0063] Each sample was labeled with a specific barcode at the 5' end of the primers and sequenced using universal primers. PCR amplification was performed in a 25 μL reaction mixture containing 25 ng template DNA, 12.5 μL of 2×PCR premix, 1 μL of each primer, and PCR-grade water to adjust the volume. PCR conditions for amplifying the prokaryotic 16S fragment included: initial denaturation at 98 °C for 30 s; 32 cycles of denaturation at 98 °C for 15 s, annealing at 54 °C for 20 s, and extension at 72 °C for 20 s; followed by a final extension at 72 °C for 5 min. PCR products were confirmed by 2% agarose gel electrophoresis. Amplicon libraries were prepared for sequencing, and the size and number of amplicon libraries were evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA) and an Illumina Library Quantification Kit (Kapa Biosciences, Woburn, MA, USA). Libraries were sequenced on a NovaSeq PE250 platform.

[0064] Data Analysis

[0065] Following manufacturer recommendations from LC Bio, samples were sequenced on the Illumina NovaSeq platform. After de-replication using DADA2, we obtained a feature table and feature sequences. Feature abundance was then normalized using the relative abundance of each sample according to the SILVA (version 132) classifier. α-diversity analysis was applied to assess the species diversity complexity of the samples using three indices: Chao1, Shannon, and Simpson, and all these indices were calculated using QIME2. Feature sequences for each representative sequence were annotated using the SILVA database. These graphs were implemented using the R package (v3.5.2). SCFA content analysis was performed using a GC-MS system.

[0066] Figure 4 Figures show the effects of each group of samples on the mouse gut microbiota. Figure A shows the Chao1 analysis results of mouse gut microbiota diversity; Figure B shows the Shannon analysis results of mouse gut microbiota diversity; Figure C shows the Simpson analysis results of mouse gut microbiota diversity; Figure D shows the relative abundance of Firmicutes in the mouse gut; Figure E shows the relative abundance of Bacteroidota in the mouse gut; Figure F shows the Firmicutes / Bacteroidota ratio in the mouse gut; Figure G shows the relative abundance of Proteobacteria in the mouse gut; Figure H shows the relative abundance of Actinobacteriota in the mouse gut; and Figure I shows the relative abundance of Verrucomicrobiota in the mouse gut. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0067] Figure 5 The following figures illustrate the effects of each group of samples on short-chain fatty acids (SCFA) in the mouse intestine. Figure A shows the bar chart of total SCFA levels in the mouse intestine; Figure B shows the bar chart of acetic acid levels in the mouse intestine; Figure C shows the bar chart of propionic acid levels in the mouse intestine; Figure D shows the bar chart of butyrate levels in the mouse intestine; Figure E shows the bar chart of isobutyrate levels in the mouse intestine; Figure F shows the bar chart of valerate levels in the mouse intestine; Figure G shows the bar chart of isovalerate levels in the mouse intestine; and Figure H shows the bar chart of hexanoate levels in the mouse intestine. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0068] Figure 4 and Figure 5 The results show that:

[0069] Lentinan-iron(II) significantly alleviated the effects of HFD on the gut microbiota structure in mice, while FeSO4 and glycine iron(II) had no significant effect on the gut microbiota. The Chao1 model was used to determine bacterial richness, thereby assessing the α-diversity of the gut microbiota. The Shannon and Simpson models were used to assess bacterial diversity. The results showed that bacterial diversity in the HFD group was significantly lower than that in the CON group, and there was a significant difference in bacterial diversity between the HFD group and the lentinan-iron(II) treatment group, indicating that lentinan-iron(II) can effectively alleviate the effects of HFD on gut microbiota diversity.

[0070] The gut microbiota of mice in the HFD group differed significantly from that in the CON group. HFD increased the relative abundance of Firmicutes, Proteobacteria, and Actinobacteria, decreased the relative abundance of Bacteroidetes and Verrucous Microbes, and increased the Firmicutes / Bacteroidetes ratio. Lentinan-iron(II) treatment was found to alleviate the effects of HFD on the gut microbiota in mice, while FeSO4 and glycine iron(II) had no significant effect on the gut microbiota.

[0071] Compared with the CON group mice, the HFD group mice showed significantly reduced production of total SCFA and some fatty acids (including acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, propionic acid, isobutyric acid, and hexanoic acid). The total SCFA and fatty acid production of the lentinan-iron(II) treatment group mice were significantly higher than those of the HFD group mice, while the effects of FeSO4 and glycine iron(II) were not significant.

[0072] Example 6: Effects of Lentinan-Iron(II) on high-fat induced oxidative stress and inflammatory response in mice

[0073] The experimental steps are as follows:

[0074] Various biochemical parameters were measured using appropriate kits. These parameters included: activity of antioxidant enzymes (SOD, CAT, Gpx), MDA content, T-AOC level, GSH content, GSSG content, TNF-α, IL-6, IL-8, IL-10, NFκB, TLR4, and total protein content, which were determined using appropriate kits.

[0075] Figure 6The following figures illustrate the effects of redox status on mouse blood: Figure A shows the bar chart of total antioxidant capacity (T-AOC) in mouse blood; Figure B shows the bar chart of the ratio of reduced glutathione to oxidized glutathione (GSH / GSSG) in mouse blood; Figure C shows the bar chart of malondialdehyde (MDA) level in mouse blood; Figure D shows the bar chart of superoxide dismutase (SOD) activity in mouse blood; Figure E shows the bar chart of catalase (CAT) activity in mouse blood; and Figure F shows the bar chart of glutathione peroxidase (Gpx) activity in mouse blood. Significant differences: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0076] Figure 7 The following figures illustrate the effects of these factors on the inflammatory response in mouse blood: Figure A shows the bar charts for mouse blood lipopolysaccharide (LPS) levels; Figure B shows the bar charts for mouse blood lipopolysaccharide receptor (LBP) levels; Figure C shows the bar charts for mouse blood interleukin-6 (IL-6) levels; Figure D shows the bar charts for mouse blood interleukin-8 (IL-8) levels; Figure E shows the bar charts for mouse blood interleukin-10 (IL-10) levels; Figure F shows the bar charts for mouse blood Toll-like receptor 4 (TLR4) levels; Figure G shows the bar charts for mouse blood tumor necrosis factor-α (TNFα) levels; and Figure H shows the bar charts for mouse blood nuclear factor-κB (NFκB) levels. Significant differences were defined as follows: ***p<0.001 compared to the CON group; ###p<0.001 compared to the HFD group.

[0077] Figure 6 The study revealed the levels of oxidative stress markers and antioxidant capacity indicators. A decreased plasma GSH / GSSG ratio and increased MDA levels were observed in HFD-fed mice, indicating that HFD feeding induced oxidative stress. The plasma T-AOC and antioxidant enzyme activities (including SOD, CAT, and Gpx) in HFD-treated mice were significantly lower than those in the CON group, indicating a decrease in antioxidant capacity in HFD-fed mice. Exposure to lentinan-iron(II) significantly alleviated the effects of HFD on oxidative stress and antioxidant activity in mice, while FeSO4 and glycine iron(II) had no significant effects.

[0078] Figure 7 The results showed that, compared with CON mice, the levels of IL-6, IL-8, LPS, LBP, TLR4, TNFα, and NFκB were significantly increased in the HFD group mice, but the level of IL-10 was decreased. Lentinan-iron(II) significantly alleviated the inflammatory response induced by HFD feeding, while FeSO4 and glycine iron(II) had no significant effect.

Claims

1. A method for preparing a Lentinan-ferrous complex, characterized in that, The method comprises the following steps: The lentinan and trisodium citrate are dissolved in water, and under the protection of inert gas, heated to 50-55℃, slowly drop FeSO4 solution, while adjusting and maintaining pH to 8-9 with NaOH solution, incubate for 1-2h; after the reaction, solid-liquid separation is carried out, take the supernatant for alcohol precipitation, collect the precipitate and dry, the lentinan-ferrous complex is obtained.

2. The method of claim 1, wherein: The mass ratio of the lentinan to trisodium citrate is 3-4:

1.

3. The method of claim 1, wherein: The alcohol precipitation step is: adding 2-4 times volume of 85%-95% ethanol to the supernatant, standing at-15--25℃ for 8-15h, then centrifugal collection of the precipitate.

4. The method of claim 1, wherein: The drying is vacuum freeze-drying.

5. A lentinan-ferrous complex, characterized by: Prepared by the method of any one of claims 1-4.

6. The lentinan-ferrous complex of claim 5 for use in the preparation of an iron supplement.

7. The lentinan-ferrous complex of claim 5 for use in the preparation of a preparation for improving intestinal flora disorder.

8. The lentinan-ferrous complex of claim 5 for use in the preparation of a preparation for improving intestinal short-chain fatty acid level.

9. The lentinan-ferrous complex of claim 5 for use in the preparation of a preparation for improving body oxidative stress.

10. The lentinan-ferrous complex of claim 5 for use in the preparation of a preparation for improving body inflammatory response.

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