A regulator of body iron homeostasis and uses thereof

Abstract

The present application relates to the application of legume gum or its protein component as a body iron homeostasis regulator to promote the absorption of iron nutrients by humans and animals. The legume gum or its protein component disclosed in the present application can activate the hypoxia-inducible factor signaling pathway, thereby up-regulating the expression of intestinal epithelial iron absorption transport proteins, inhibiting the overexpression of hepcidin in the liver, and promoting the production of erythropoietin in the liver and kidney, thereby achieving the regulation of the body iron homeostasis of humans and animals, and can be applied in feed, food, health food and medicine for preventing and treating iron deficiency and related diseases. The body iron homeostasis regulator used in the present application is of food origin, and therefore has the advantage of no toxic side effects compared with conventional chemically synthesized drugs. The application described in the present application can be used as a complementary or alternative therapy for iron nutritional intervention, and can make up for the shortcomings of iron nutritional intervention in safety and effectiveness, and is particularly effective for functional iron deficiency caused by inflammation or kidney damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of legume gum or its protein components as iron homeostasis regulators to promote the absorption of iron nutrients in humans and animals. Background Technology

[0002] Iron is the most abundant trace element in the human body, essential for many physiological and cellular processes, and a leading cause of disability among women in 35 countries. Iron deficiency often leads to a variety of symptoms, such as chronic fatigue, poor concentration, impaired motor skills, and reduced quality of life. Iron deficiency is associated with premature birth, lower birth weight, and increased mortality in fetuses, and also increases the incidence of neonatal infections and heart failure. Currently, many oral iron-fortified products in various dosages and formulations are cost-effective methods for controlling the iron deficiency epidemic; however, meta-analyses have found that oral iron supplements are ineffective in more than 15% of children and significantly increase the risk of infections and gastrointestinal diseases in children. Therefore, increasing iron intake is insufficient to address the current reality of iron deficiency. Thus, alternative interventions for iron deficiency are crucial.

[0003] Iron homeostasis is closely related to oxygen supply in various tissues. The hypoxia-inducible factor (HIF) signaling axis is a core component of the cellular oxygen sensing pathway. Proline hydroxylase catalyzes the hydroxylation of the HIF-α subunit, which is subsequently degraded by ubiquitination. Once this process is inhibited, HIF-α is stably expressed in cells and can bind to hypoxia response elements (HREs), subsequently inducing p300 / CREB binding proteins to activate downstream transcription. HRE elements have been found in upstream genes of iron absorption and transport-related proteins in the brush border membrane and basolateral aspect of the intestine. Therefore, regulating iron homeostasis through the HIF signaling pathway provides a new approach to addressing iron deficiency. Recent research reports that hydroxyproline-containing dipeptides / tripeptides in collagen protein hydrolysates can regulate iron homeostasis through the HIF-2α signaling pathway, providing a new direction for the discovery of novel iron homeostasis regulators. Leguminous gums are viscous fluids exuded from the seeds or wounds of leguminous plants. After drying, they form gelatinous masses with a certain color and irregular shape. They have wide applications in traditional Chinese medicine and other fields, and contain abundant hydroxyproline-rich glycoproteins, showing potential as novel plant-derived iron homeostasis regulators. No reports have been found in the literature regarding the regulation of iron homeostasis in organisms by leguminous gums.

[0004] Our experimental data show that the hydroxyproline-rich glycoprotein component in legume gums can produce hydroxyproline-containing dipeptides / tripeptides after digestion and absorption. These peptides activate the HIF-2α signaling pathway in the intestine, liver, and kidney tissues by inhibiting prolyl hydroxylase. This upregulates the expression of iron absorption transport proteins such as divalent metal ion carrier 1, duodenal cytochrome b, membrane iron transporter, and ferrooxidase in intestinal epithelial cells, inhibits the overexpression of hepcidin in the liver, and promotes the expression of erythropoietin in the liver and kidneys. In this way, they regulate the body's iron homeostasis and alleviate nutritional and functional iron deficiency. Summary of the Invention

[0005] The purpose of this invention is to use legume gum or its protein components as a regulator of iron homeostasis in the body for the prevention and treatment of iron deficiency and related diseases in humans and animals.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: An iron homeostasis regulator, wherein the regulator is a legume gum or its protein component, and the iron homeostasis regulation is any one or more of the following: regulation of the expression of iron absorption transport proteins in the human and animal intestines, regulation of the expression of hepcidin in the liver, and regulation of the expression of erythropoietin in the liver and kidneys.

[0007] The legume gum mentioned is derived from legumes (scientific name: Fabaceae, alternative names: Leguminosae, Papilionaceae) and contains hydroxyproline-rich glycoprotein (HRGP).

[0008] The protein component is a protein separation or degradation product of legume gum.

[0009] The protein separation is performed by one or more of the following processes: alcohol precipitation, column chromatography, and membrane separation. The protein degradation is performed by one or more of the following processes: alkaline hydrolysis, acid hydrolysis, or protease hydrolysis.

[0010] The alcohol used in the alcohol precipitation process is methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, or a combination thereof.

[0011] The gel used in the column chromatography process is any one or more of the following: Sephadex G50 / G75 / G100 / G150 dextran gel and Sepharose 4B / 6B / CL-2B / CL-4B / CL-6B agarose gel.

[0012] The membrane used in the membrane separation process is an ultrafiltration membrane with a molecular weight cutoff range of 3-100 kDa.

[0013] The alkali used in the alkaline hydrolysis process is one or more of sodium hydroxide, potassium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, and sodium bicarbonate.

[0014] The acid used in the acid hydrolysis process is any one or more of hydrochloric acid, acetic acid, citric acid, lactic acid, and sulfuric acid.

[0015] The protease used in the protease hydrolysis process includes any one or more of pepsin, trypsin, alkaline protease, papain, ginger protease, bromelain, fig protease, and neutral protease.

[0016] The aforementioned iron homeostasis regulator is used in feed and medicines for the prevention and treatment of iron deficiency and related diseases in humans and animals.

[0017] The feed and medicine use the legume gum and its protein components as active ingredients and can be used in combination with iron supplements.

[0018] The iron agent is selected from one or more of the following: ferrous sulfate, ferrous lactate, ferrous gluconate, ferrous fumarate, ferric dextran, ferrous succinate, polysaccharide iron complex, ferric ammonium citrate, ferric citrate, ferric heme chloride, ferric pyrophosphate, ferric porphyrin, ferrous glycinate, reduced iron, ferric sodium EDTA, carbonyl iron powder, ferrous carbonate, ferrous citrate, ferrous fumarate, heme iron, and electrolytic iron.

[0019] The iron deficiency and related diseases mentioned are one or more of the following: iron deficiency syndrome, iron deficiency anemia, renal anemia, exercise anemia, chronic disease anemia, tumor-related anemia, and blood loss anemia.

[0020] The drug can be administered orally or parenterally.

[0021] The method of parenteral administration is one of the following: intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and local injection.

[0022] Compared with the prior art, the technical advantages of the present invention are as follows: 1) The use of legume gum or its protein components as a regulator of iron homeostasis is a first of its kind in this invention. Legume gum is often used as a food additive. Therefore, compared with conventional chemically synthesized regulators of iron homeostasis such as roxadustat (FG-4592), the regulator of iron homeostasis described in this invention is not expected to have toxic side effects. 2) Experimental data show that legume gums or their protein components can act as a regulator of iron homeostasis and stimulate the body's own absorption of iron nutrients. Compared with traditional exogenous iron nutrient supplementation or food fortification, it has significant advantages in addressing functional iron deficiency caused by inflammation or kidney damage. 3) While many iron-absorbing dietary factors are derived from animal sources, plant-based dietary components such as polyphenols, phytic acid, and dietary fiber often hinder iron absorption. This invention takes a different approach, extracting an iron homeostasis regulator from legume gums. Compared to traditional animal-derived iron-absorbing dietary factors, the regulator described in this invention aligns with the needs of green, low-carbon, and sustainable development, and expands the applicable population for iron-absorbing dietary factors, making it particularly suitable for vegetarians. Attached Figure Description

[0023] The specific embodiments and beneficial effects of the present invention will be further described in detail below with reference to the accompanying drawings.

[0024] Figure 1 The promoting effect of tragacanth gum, ebony gum and gum arabic on intestinal iron absorption.

[0025] Figure 2 The promoting effect of gum arabic (GA) on intestinal iron absorption: (a) kinetics of iron absorption in Caco-2 monolayer cells; (b) concentration of ferrous iron accumulated in the supernatant culture medium; (c) concentration of iron transported to the lower chamber via monolayer cells in the Transwell iron transporter.

[0026] Figure 3 Effects of gum arabic (GA) on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) iron transporter (FPN); (d) ferrous oxidase (HEPH).

[0027] Figure 4 Effects of gum arabic (GA) and its protein and polysaccharide components on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) iron transporter (FPN); (d) ferrous oxidase (HEPH); (e) Ser-Hyp-Hyp-Hyp; (f) Ser-Hyp-Ser-Hyp.

[0028] Figure 5 The relative abundance of hydroxyproline-containing components in cell lysates after incubation with Caco-2 following alkaline hydrolysis of gum arabic: (a) free hydroxyproline, (b) Hyp-Hyp, (c) Pro-Hyp, (d) Ser-Hyp.

[0029] Figure 6 Effects of gum arabic (GA) and its protein and polysaccharide components on intestinal hypoxia-inducible factors: (a) (b) Western blotting images and density analysis of HIF-2α; (c) determination of hypoxia response element (HRE) activity by dual-luciferase reporter gene assay; (d) determination of proline hydroxylase (PHD) activity by dual-luciferase reporter gene assay.

[0030] Figure 7 Acute effects of protein components of gum arabic (GA) on the expression levels of iron transporter mRNA in rat duodenum: (a) divalent metal transporter 1 (DMT1) and duodenal cytochrome b (Dcytb); (b) iron transporter (FPN) and ferrous oxidase (HEPH); (c) Western blotting images of HIF-2α; (d) density analysis.

[0031] Figure 8 The effects of gum arabic (GA) and its protein and polysaccharide components on renal anemia: (a) kidney index; (b) urea nitrogen content in urine; (c) hemoglobin content in blood; (d) number of red blood cells in blood.

[0032] Figure 9 The regulatory effects of gum arabic and its protein and polysaccharide components on iron homeostasis-related proteins in a mouse model of renal anemia: (a) EPO and HIF-2α expression levels in the kidney; (b) Hepcidin and HIF-2α expression levels in the kidney; (c) EPO and HIF-2α expression levels in the liver; (d) Hepcidin and HIF-2α expression levels in the liver.

[0033] Figure 10 Peptide sequence identification of protein components separated by alkaline hydrolysis of gum arabic. a The underscore represents hydroxyproline; heo.MH + This represents the theoretical molecular weight. All matched peptide library data were obtained from the protein analysis software Proteome Iscoverer 2.2. Detailed Implementation

[0034] Example 1 (I) The promoting effect of three legume gums on intestinal iron absorption in an in vitro Caco-2 intestinal cell model. The specific experimental procedures and conclusions are as follows: Figure 1 The promoting effects of gum arabic, tragacanth gum, and ebony gum on intestinal iron absorption. This experiment used Caco-2 colon cells, which have a structure and function similar to small intestinal epithelial cells. The cell line used was purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphoteric acid B solution at a depth of 25 cm. 2 Cells were cultured in flasks placed in a cell culture incubator at 37°C and 5% CO2 humidity. The culture medium was changed every 2 days. When the cell confluence reached 80%, the cells were plated according to the experimental requirements. To further verify its promoting effect on iron absorption, the kinetics of cell iron absorption were measured using the calcein fluorescence method. HBSS buffer at pH 5.5 was used to simulate the intestinal fluid conditions of the proximal small intestine, and the cells were cultured at a concentration of 5 × 10⁻⁶ cells / mL. 4 Caco-2 cells were seeded at a density of cells / well in 24-well plates. After complete differentiation, the cells were incubated for 48 h in a medium containing 1 mg / mL of gum arabic, tragali gum, and erythrina gum. Then, calcein-AM was added and the cells were incubated at 37°C for 30 min. Finally, 970 μL of HBSS solution at pH 5.5 and 30 μL of a solution containing 1 mM aminotriacetic acid-Fe3+ were added. 3+ The sample solution was incubated at 37°C for 30 minutes in a SpectraMax i3x fluorescence microplate reader, with calcein fluorescence (485 nm excitation, 530 nm emission) recorded every 3 minutes. Calcein-AM is a membrane-permeable non-fluorescent molecule that emits fluorescence after being cleaved by cytoplasmic esterases within cells, and can label the content of divalent metal ions in the weakly bound iron pool of cells. In iron absorption experiments ( Figure 1 (a) It can be seen that after incubation with the three legume gums, the quenching rate of calcein fluorescence in cells was significantly enhanced in a dose-dependent manner (P < 0.05), indicating that the three legume gums can promote the rate of iron absorption by Caco-2 cells.

[0035] Example 2 (II) The regulatory effect of gum arabic on intestinal iron homeostasis in an in vitro Caco-2 intestinal cell model. The specific experimental procedures and conclusions are as follows: Figure 2 The promoting effect of gum arabic (GA) on intestinal iron absorption: (a) kinetics of iron absorption in Caco-2 monolayer cells; (b) concentration of ferrous iron accumulated in the culture supernatant; (c) concentration of iron transported to the lower chamber via the cell monolayer in the Transwell iron transporter. Figure 3Effects of gum arabic (GA) on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) iron transporter (FPN); (d) ferrooxidase (HEPH). This experiment was the same as in Example 1. To further verify the promoting effect of gum arabic on iron absorption, the cellular iron absorption kinetics were determined using the calcein fluorescence method. HBSS buffer at pH 5.5 was used to simulate the intestinal fluid conditions of the proximal small intestine, with 5 × 10⁻⁶ cells / mL. 4 Caco-2 cells were seeded at a density of cells / well in 24-well plates. After complete cell differentiation, the medium was replaced with different concentrations of gum arabic medium. Calcein-AM was then added and incubated at 37°C for 30 minutes. Finally, 970 μL of HBSS solution and 30 μL of medium containing 1 mM aminotriacetic acid-Fe2+ were added. 3+ The sample solution was incubated at 37 °C for 30 minutes in a SpectraMax i3x fluorescence microplate reader, with calcein fluorescence (485 nm excitation, 530 nm emission) recorded every 3 minutes. In the iron absorption experiment ( Figure 2 a) It can be seen that the quenching rate of calcein fluorescence in cells after GA incubation was significantly enhanced in a dose-dependent manner (P < 0.05), indicating that the rate of iron uptake by cells was enhanced. To study the cells' ability to reduce ferrous iron, sodium phenanthrene disulfonate (BPDS) was used, which can rapidly bind Fe. 2+ A pink complex is formed, and the absorbance is used to determine the cell's response to Fe. 3+ Its restorative ability. From Figure 2 As shown in b, the content of complexes formed by cells incubated with GA increased significantly (P<0.05), indicating that the cells' ability to reduce ferrous iron was enhanced. Further verification of the iron content transported from the basal side of the cell was conducted using a Transwell iron transport model. It was found that after incubation with different doses of GA, the iron content transported to the lower chamber by Caco-2 increased to varying degrees, indicating that the cells' ability to absorb and transport iron was enhanced. Figure 2 c). The above results indicate that Caco-2 cells incubated with GA showed significantly enhanced abilities in iron absorption, reduction, and transport (P < 0.05).

[0036] Further, Caco-2 cells were added at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells / well in 6-well cell culture plates. The culture medium was changed every 2 days. After approximately 10 days, when the cells had fully differentiated into a monolayer, different concentrations of GA were added for incubation. A control group without GA was also included. After culturing the cells in an incubator for 48 hours, Western blotting was used to investigate the effect of GA on the expression of intestinal iron absorption-related proteins. Figure 3 Western blotting results from a-3d showed that after 48 h of cell incubation, GA significantly promoted the expression of divalent metal ion transporter 1 (DMT-1) responsible for iron transport in the intestinal lumen and duodenal cytochrome C (Dcytb) responsible for iron reduction, as well as the expression of basal side iron transporter (FPN) and ferrous oxidase (HEPH) in a dose-dependent manner (P < 0.05), thereby further promoting the absorption, reduction and transport of iron nutrients in the intestine.

[0037] (III) The specific molecular mechanism of the regulatory effect of gum arabic on intestinal iron homeostasis was studied. The specific experimental procedures and conclusions are as follows: See Figure 4-6 , 10.

[0038] Figure 4 The relative abundance of hydroxyproline-containing components in cell lysates after incubation with Caco-2 following alkaline hydrolysis of gum arabic: (a) free hydroxyproline, (b) Hyp-Hyp, (c) Pro-Hyp, (d) Ser-Hyp, (e) Ser-Hyp-Hyp-Hyp, (f) Ser-Hyp-Ser-Hyp.

[0039] Figure 5 Effects of gum arabic (GA) and its protein and polysaccharide components on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) iron transporter (FPN); (d) ferrooxidase (HEPH). Figure 6 Effects of gum arabic (GA) and its protein and polysaccharide components on intestinal hypoxia-inducible factors: (a) (b) Western blotting images and density analysis of HIF-2α; (c) determination of hypoxia response element (HRE) activity by dual-luciferase reporter gene assay; (d) determination of proline hydroxylase (PHD) activity by dual-luciferase reporter gene assay.

[0040] Figure 10 Peptide sequence identification of protein components separated by alkaline hydrolysis of gum arabic.

[0041] To further investigate the specific molecular mechanism of GA's regulatory effect on intestinal iron homeostasis, polysaccharide and protein components of gum arabic were separated by alkaline hydrolysis with 1 M NaOH and 2 M NaBH4 at 100℃ for 6 h, followed by precipitation with 95% ethanol at 5 times their volume. Table 1 shows the peptide sequence identification of the protein components after alkaline hydrolysis of gum arabic, indicating that they are hydroxyproline-rich peptides containing repeating X-(Pro / Hyp)n. Caco-2 cells were incubated with 2 mg / mL of the protein component at 37℃ for 30 min. The protein solution was then washed away with fresh HBSS. Caco-2 cells were collected at 0, 0.5, 1, 2, and 4 h throughout the experiment and lysed with RIPA lysis buffer. After centrifugation at 12,000 g for 10 min at 4℃, the supernatant was obtained, the protein was precipitated with acetonitrile, and desalted with Ziptip C18. The samples were analyzed by UPLC-MS / MS. Figure 4 As shown, several hydroxyproline-containing peptides, including Hyp-Hyp, Pro-Hyp, Ser-Hyp, Ser-Hyp-Hyp-Hyp and Ser-Hyp-Ser-Hyp, were detected in high abundance in the lysates of polarized Caco-2 cell monolayers by LC-MS / MS. Figure 4 In fact, collagen-derived hydroxyproline-containing dipeptides and tripeptides have recently been discovered to be PHD inhibitors, capable of activating the HIF2α signaling pathway and inducing the expression of iron transporter genes in intestinal cells. To further investigate the regulation of intestinal iron homeostasis by different components of GA, the protein and polysaccharide components were incubated with the GA component separately for 48 h. Figure 5 As shown in a-5d, Western blotting analysis and subsequent density measurements revealed that the GA protein component significantly upregulated the expression of non-heme iron absorption-related proteins (DMT1, Dcytb, FPN, HEPH) in Caco-2 cells, with a similar promoting effect to GA (P<0.05). However, the GA-derived polysaccharide component did not exhibit the corresponding promoting effect. Based on these results, GA stimulates the absorption of non-heme iron by intestinal cells by upregulating iron absorption-related proteins, and iron absorption primarily depends on its protein component rather than its polysaccharide component.

[0042] HIF-2α is a key regulator of hypoxia response, occupying a central position in oxygen homeostasis in various organisms. Furthermore, HIF-2α plays a close role in regulating iron homeostasis and oxygen metabolism. Studies have shown that HIF-2α can directly or indirectly activate the expression of iron transporter genes in intestinal cells. Figure 6As shown in a and 6b, GA and GA-derived protein components significantly increased the expression level of HIF-2α in polarized Caco-2 cell monolayers (P<0.05), while GA-derived polysaccharide components had no significant effect on HIF-2α protein expression. HIF-2α can activate the transcription of iron transport-related protein genes by binding to specific locations in the promoter or enhancer regions of target genes, namely hypoxia response elements (HREs). Therefore, we used the pGL4.42 [luc2P / HRE / Hygro] luciferase reporter vector and applied a dual-luciferase reporter gene assay to evaluate the transcriptional induction activity of HIF-2α. Figure 6 c). The transcriptional activation activity of HIF-2α increased twofold under the influence of 100 μM CoCl2, GA, and GA-derived protein components, indicating that GA and its protein components can effectively upregulate the transcriptional induction activity of HIF-2α (P<0.05). However, the GA-derived polysaccharide components did not promote the transcriptional activity of HIF-2α (P>0.05). Based on the above results, GA can activate intestinal HIF-2α signaling. Considering that HIF-2α is an upstream regulator of duodenal protein levels involved in iron uptake and translocation, this activation process may stimulate the absorption of non-heme iron by intestinal cells through a hypoxia-induced pathway.

[0043] Under normal oxygen levels, proline hydroxylase (PHD) hydroxylates the proline subunit of HIF-2a, leading to the ubiquitination and degradation of HIF-2a. To investigate the effect of GA incubation in polarized Caco-2 cell monolayers on PHD activity, we used the HIF2α-ODD-luciferase reporter gene system, constructed by fusing luciferase into the hydroxylation domain of a typical PHD. Figure 6 As shown in Figure d, treatment with GA and its protein components significantly increased the activity of luciferase in Caco-2 cells (P<0.05), a slightly lower increase than that of CoCl2, a chemical inhibitor of PHD. These results indicate that the GA-derived protein components can inhibit PHD activity and activate HIF-2α signaling in intestinal cells.

[0044] Example 3 Gum arabic (GA) demonstrated a regulatory effect on intestinal iron homeostasis in rats during an acute experiment. The specific experimental procedure and conclusions are as follows: Figure 7 Acute effects of oral administration of gum arabic (GA) on the expression levels of iron transporter mRNA in rat duodenum: (a) divalent metal transporter 1 (DMT1) and duodenal cytochrome b (Dcytb); (b) iron transporter (FPN) and ferrous oxidase (HEPH); (c) Western blotting images of HIF-2α; (d) density analysis. To verify whether gum arabic can stimulate the absorption of non-heme iron in the intestine in vivo, 21 ± 3-day-old female Sprague-Dawley rats were housed individually in cages. The temperature in the animal room was controlled at 25 ± 2℃, and the humidity at 55 ± 15%. The animal room was subjected to 12 / 12-hour light / dark cycles. Throughout the study, the animals had free access to food and ultrapure water. After one week of acclimatization, the rats were randomly divided into 5 groups. The rats were first fasted for 12 hours (with normal supply of ultrapure water) to eliminate dietary interference. Then, they were administered GA-derived protein components by gavage at high doses (1.2 g / kg) and low doses (0.6 g / kg). Two hours after gavage, the rats were anesthetized, and the duodenum and jejunum were harvested for mRNA and protein extraction. The transcriptional levels of the duodenal genes Dcytb, DMT1, FPN, and HEPH were measured. Figure 7 As shown in a and 7b, the protein components of GA induced dose-dependent upregulation of the gene transcription levels of Dcytb, DMT1, FPN, and HEPH in the rat duodenum, confirming the direct stimulatory effect of GA on the absorption of non-heme iron in the intestine in vivo. Based on Western blotting results ( Figure 7 (c and 7d) The protein components of GA increased the protein level of HIF-2α in the duodenum of rats in a dose-dependent manner, verifying the activation of the HIF-2α signaling pathway by GA in vivo.

[0045] Example 4 The effects of gum arabic (GA) and its protein and polysaccharide components on the regulation of iron homeostasis in patients with renal anemia were evaluated. The specific experimental procedures and conclusions are as follows: Figure 8 The effects of gum arabic and its protein and polysaccharide components on renal anemia: (a) kidney index; (b) urea nitrogen content in urine; (c) hemoglobin content in blood; (d) number of red blood cells in blood. Figure 9 The regulatory effects of gum arabic and its protein and polysaccharide components on iron homeostasis-related proteins in a mouse model of renal anemia: (a) Erythropoietin (EPO) and HIF-2α expression levels in the kidney; (b) Hepcidin and HIF-2α expression levels in the liver; (c) EPO and HIF-2α expression levels in the liver; (d) Hepcidin and HIF-2α expression levels in the liver.

[0046] Six-week-old C57BL / 6 mice, eight per group, were used in the animal room at a temperature of 25 ± 2 ℃ and a humidity of 55 ± 15%, with a 12 / 12 h light cycle (lighting from 8 am to 8 pm). Animals had free access to food and ultrapure water throughout the study. The study was approved by the Animal Experiment Ethics Committee of Ocean University of China (Approval No.: SPXY20221110).

[0047] After one week of acclimatization, mice in each group were administered 100 μL of adenine (50 mg / kg body weight in 0.5% sodium carboxymethyl cellulose solution) daily by gavage to induce adenine model. Simultaneously, according to the groupings, the GA group received 200 μL of GA (0.5% sodium carboxymethyl cellulose solution) daily by gavage, with a high dose of 2 g / kg and a low dose of 0.5 g / kg; the GA-protein group received 200 μL of GA-derived protein fraction (0.5% sodium carboxymethyl cellulose solution) daily by gavage, with a high dose of 600 mg / kg and a low dose of 150 mg / kg; the GA-polysaccharide group received 200 μL of GA-derived polysaccharide fraction (0.5% sodium carboxymethyl cellulose solution) daily by gavage, with a high dose of 1.9 g / kg and a low dose of 0.4 g / kg; the Roxadustat group received 200 μL of 25 mg / kg (0.5% sodium carboxymethyl cellulose solution); and the control group received 200 μL of PBS (0.5% sodium carboxymethyl cellulose solution). The model was established and treated in sodium carboxymethyl cellulose solution for a total of four weeks.

[0048] like Figure 8 As shown in Figure a, the PBS gavage group, after prolonged adenine-induced kidney damage, exhibited significant renal enlargement and a high renal index. Gavage administration of GA significantly alleviated this renal enlargement (P < 0.05), with the GA-derived protein component playing a major role. The GA-derived polysaccharide group showed a poorer effect. The presence of adenine leads to renal function impairment, resulting in a large amount of urea nitrogen failing to be excreted through urine. Therefore, the urea nitrogen level in the urine of the PBS gavage group was lower than that of other groups (P < 0.05). Figure 8 (b) Oral administration of GA and GA-derived protein components increased urea nitrogen levels. These results all indicate that GA has a protective effect on renal function, with the protein components playing a major role. Simultaneously, GA can increase hemoglobin and red blood cell count in the blood (b). Figure 8 GA (adenosine monophosphate) improved anemia caused by adenine-induced kidney injury, showing effects consistent with the positive control drug Roxadustat. Therefore, GA can effectively alleviate kidney damage, and is particularly effective in improving functional iron deficiency caused by kidney injury.

[0049] Figure 9This study demonstrated the effects of GA and its protein components on hypoxia levels in the liver and kidneys of mice with renal anemia, as well as the expression of hepcidin in the liver and erythropoietin (EPO) in the kidneys. EPO is a circulating hematopoietic hormone primarily produced in the kidneys in adults, and EPO deficiency is a major cause of anemia in patients with renal anemia. Figure 9 As shown in Figure a, Western blot analysis revealed that treatment with GA and its protein components significantly increased EPO production in the kidneys of mice with renal anemia (P<0.05), while the GA-derived polysaccharide component did not play a related role. These results indicate that GA and its protein components can promote EPO production in mice with renal anemia. Hepcidin is a circulating hormone derived from hepatocytes that regulates iron homeostasis through its interaction with ferroportin, the only known mammalian membrane iron transporter. Excessive Hepcidin production due to systemic inflammation is thought to cause renal anemia by disrupting systemic iron homeostasis. The level of Hepcidin produced in the liver of adenine-controlled mice was significantly higher than that in the normal control group. Treatment with GA and its protein components significantly inhibited the increase in adenine-induced hepcidin production in hepatocytes (P<0.05), while the GA polysaccharide component did not have a related effect. These results indicate that GA and its protein components can effectively reduce excessive Hepcidin production in mice with renal anemia. HIF-2α is a key regulator of systemic iron homeostasis by mediating hypoxic EPO induction and Hepcidin inhibition. The levels of HIF-2α protein in the liver and kidneys of GA and GA-derived protein groups were higher than those in the adenine control group. P <0.05). Therefore, GA and its protein components regulate iron homeostasis in functional iron-deficient organisms by activating HIF-2α-dependent hypoxia signaling in the liver and kidneys, thereby regulating the expression of EPO and Hepcidin.

[0050] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention and are not intended to limit the scope of the invention in any way. Various changes and modifications can be made to the invention without departing from its scope, and all such changes and modifications fall within the scope of the claims.

Claims

1. The application of legume gum or legume gum protein components in the preparation of feed or medicines for the prevention and treatment of iron deficiency in humans and animals, characterized in that, The legume gum is either tragacanth gum or ebony gum; The legume gum protein component is prepared by alkaline hydrolysis of gum arabic at 100°C for 6 hours using 1M NaOH and 2M NaBH4, followed by alcohol precipitation with 95% ethanol at 5 times its volume. The legume gum or legume gum protein component can promote iron absorption.

2. The application as described in claim 1, characterized in that, Legume gums are derived from legumes and contain hydroxyproline-rich glycoproteins.

3. The application as described in claim 1, characterized in that, The legume gum or legume gum protein component can be used in combination with iron supplements.

4. The application as described in claim 3, characterized in that, The iron agent is selected from one or more of the following: ferrous sulfate, ferrous lactate, ferrous gluconate, ferrous fumarate, ferric dextran, ferrous succinate, polysaccharide iron complex, ferric ammonium citrate, ferric citrate, ferric heme chloride, ferric pyrophosphate, ferric porphyrin, ferrous glycinate, reduced iron, ferric sodium ethylenediaminetetraacetate, carbonyl iron powder, ferrous carbonate, ferrous citrate, and electrolytic iron.

5. The application of legume gum protein components in the preparation of medicines for the prevention or treatment of renal anemia, characterized in that, The legume gum protein component is prepared by alkaline hydrolysis of gum arabic at 100°C for 6 hours using 1M NaOH and 2M NaBH4, followed by alcohol precipitation with 95% ethanol at 5 times its volume. The legume's gum protein component can promote iron absorption.

6. The application as described in claim 5, characterized in that, Legume gums are derived from legumes and contain hydroxyproline-rich glycoproteins.

7. The application as described in claim 5, characterized in that, The legume collagen component can be used in combination with iron supplements.

8. The application as described in claim 7, characterized in that, The iron agent is selected from one or more of the following: ferrous sulfate, ferrous lactate, ferrous gluconate, ferrous fumarate, ferric dextran, ferrous succinate, polysaccharide iron complex, ferric ammonium citrate, ferric citrate, ferric heme chloride, ferric pyrophosphate, ferric porphyrin, ferrous glycinate, reduced iron, ferric sodium ethylenediaminetetraacetate, carbonyl iron powder, ferrous carbonate, ferrous citrate, and electrolytic iron.

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