Iron-binding peptide based on tuna blood mixed meat and application of iron-binding peptide

Abstract

The invention provides iron binding peptides based on tuna blood combined meat. The amino acid sequences of some polypeptides are SEQ ID NO: 1-6. The polypeptide provided by the invention can be used for preparing a product for treating iron-deficiency anemia. According to the tuna red meat hematopoietin provided by the invention, an organic iron compound with high bioavailability is formed through the chelation of specific polypeptide iron ions in preparation of a blood replenishing product, so that the absorption efficiency of intestinal tracts on iron is remarkably improved. A carrier formed by the polypeptide can protect iron ions from being damaged by gastric acid, and the iron ions are delivered to an absorption part in a targeted mode, so that the hemoglobin synthesis efficiency is improved by 40% or above. The natural polypeptide ligand disclosed by the invention avoids gastrointestinal irritation of a traditional iron supplementing agent, and is not interfered by absorption of dietary factors such as phytic acid and tannin. The tuna processing by-product is used as the raw material to prepare the functional blood-enriching peptide, so that high-value utilization of fishery resources is realized, and an eco-friendly new iron-enriching source is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of active polypeptide preparation, and particularly relates to an iron-binding peptide based on tuna blood and flesh and an application thereof. Background Art

[0002] Iron deficiency anemia (IDA) is a global public health problem caused by insufficient iron intake or increased metabolic demand, leading to depletion of stored iron and impaired hemoglobin synthesis. According to the World Health Organization (WHO), IDA ranks among the top five most common diseases worldwide and is particularly prevalent in developing countries and economically disadvantaged regions.

[0003] Currently, the clinical treatment of IDA mainly relies on iron supplements, including inorganic salts (such as ferrous sulfate) and organic acid salts (such as ferrous gluconate). However, these traditional iron supplements have the following limitations: 1) Inorganic salts have low bioavailability and are highly irritating to the gastrointestinal tract, easily causing adverse reactions such as nausea and vomiting; 2) The bioavailability of iron supplements based on organic acid salts has been improved, but they are still susceptible to interference from dietary components such as tannins and phytic acid, which affects the efficiency of iron absorption.

[0004] In recent years, bioactive peptide-iron complexes have become a hot topic in iron supplement research due to their high bioavailability, low toxicity, and excellent stability. Bioactive peptides (BPs) are small peptide fragments (typically with a molecular weight <6 kDa) obtained by protease hydrolysis of proteins. They possess various physiological regulatory functions, such as immunomodulation, antioxidant activity, and metal ion chelation.

[0005] Tuna blood and flesh (also known as red meat, dark meat, or dark meat) are often discarded due to their rough texture, but their crude protein content, as high as 25%, makes them ideal raw materials for developing bioactive peptides. However, current technologies for the development of tuna resources are still in their early stages. Summary of the Invention

[0006] The purpose of the present invention is to provide an iron-binding peptide based on tuna blood and flesh and its application, so as to make up for the shortcomings of the prior art.

[0007] The present invention first provides an iron ion binding peptide, which is separated and prepared from tuna blood; One of the polypeptides has an amino acid sequence of AEELKKEQDTSAH (SEQ ID NO: 1), Furthermore, the amino acid sequence of the polypeptide is one or more of the following: LDKENALDRAEQA (SEQ ID NO:2), VEEELDRAQER (SEQ ID NO:3), SISEELDHA (SEQ ID NO: 4), AISEELDHA (SEQ ID NO: 5), DLQHRLDEAEA (SEQ ID NO:6).

[0008] The present invention also provides a use of the polypeptide, which is use in preparing a product for treating iron deficiency anemia.

[0009] The present invention also provides a product for treating iron deficiency anemia, wherein the product contains the above polypeptide at a pharmacologically effective concentration.

[0010] Furthermore, the preparation is a composite peptide preparation, wherein the VEE polypeptide of SEQ ID NO: 3 is used as the main component, Furthermore, the composite peptide preparation is further added with a polypeptide of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 6.

[0011] As described in one embodiment, vitamins and / or microcrystalline cellulose are further added to the composite peptide preparation.

[0012] The tuna red meat blood peptide provided by the present invention has the following outstanding advantages in preparing blood-enriching products: Highly efficient iron absorption: Through the chelation of specific polypeptide iron ions, a highly bioavailable organic iron complex is formed, significantly improving the intestinal absorption efficiency of iron; Optimizes blood-enriching effect: The polypeptide carrier can protect iron ions from being destroyed by gastric acid and deliver them to the absorption site in a targeted manner, thereby increasing the efficiency of hemoglobin synthesis by more than 40%; Excellent biocompatibility: Natural peptide ligands avoid the gastrointestinal irritation of traditional iron supplements and are not affected by absorption interference from dietary factors such as phytic acid and tannins; Sustainable resource utilization: Functional blood-enriching peptides are prepared using tuna processing by-products as raw materials, which not only achieves high-value utilization of fishery resources (cost reduction by 60%), but also provides an eco-friendly new source of iron supplementation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Total ion current; Figure 2 Technical roadmap for animal experimental research on the efficacy verification of tuna blood peptide; Figure 3 Effects of different drug administration groups on the body weight growth of IDA rats at 50 days; Figure 4Effects of different drug administration groups on hepcidin content in the liver of IDA rats; Figure 5 Effects of different drug administration groups on the iron content in liver tissue of IDA rats; Figure 6 Effects of different drug administration groups on serum total iron binding capacity in IDA rats; different letters (a, b) represent significant differences between different drug administration groups ( p < 0.05); Figure 7 Effects of different drug administration groups on superoxide dismutase (TSOD), an oxidative stress indicator in IDA rats; Figure 8 Effects of different drug administration groups on the total antioxidant capacity of IDA rats; different letters (a, b) represent significant differences between different drug administration groups ( p < 0.05); Figure 9 Schematic diagram of molecular docking simulation. DETAILED DESCRIPTION

[0014] The present invention isolated and identified a group of novel iron-binding peptides from tuna red meat, named blood-binding peptides (to distinguish them from the blood peptides in the control group, the iron-binding peptides screened in the accompanying figure are named red meat peptides), and confirmed that they have significant effects in the treatment of iron deficiency anemia IDA.

[0015] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0016] Example 1: Identification and characterization of blood-binding peptides Liquid chromatography-mass spectrometry (LCMS / MS) was used to analyze the F4 active component of the tuna red meat enzymatic hydrolysate, and the total ion current (TIC) was obtained. Figure 1 shown.

[0017] The steps for separating the F4 component of the tuna red meat enzymatic hydrolysate are as follows: 1. Enzymatic hydrolysis optimization: Orthogonal experiments determined the optimal conditions to be a solid-liquid ratio of 1:4 g / mL, a temperature of 50°C, a time of 6 h, and a compound enzyme (trypsin: papain = 1:1) addition amount of 1.2% (w / w); 2. Stepwise enzymatic hydrolysis: Trypsin (0.6%, 50℃, 3 h) and papain (0.6%, 50℃, 3 h) were used in sequence. The enzymes were inactivated in a boiling water bath, followed by centrifugation, ultrafiltration, and spray drying. 3. Purification: Separation by Sephadex G15 gel column (collecting the fourth group of components), combined with cation exchange chromatography and reversed-phase HPLC purification, the purity is ≥95%.

[0018] Total ion chromatogram (TIC) data were compared with databases (setting parameters: protein card value ≥ 1 unique peptide, peptide card value 101 gP ≥ 20). A total of 60 peptide sequences with potential bioactivity were identified in the F4 active fraction. Among them, the following six peptides (Table 1) exhibited significant iron-binding properties and are hereby designated as AEE, LDK, VEE, SIS, AIS, and DLQ.

[0019] Table 1: Sequences and characteristics of iron-binding peptides from tuna red meat Example 2: Verification of tuna iron-binding peptide activity 1. Steps and grouping of animal experiments Experimental groups and treatment plans 1.1 Experimental Animal Grouping After the rat model was successfully established, they were randomly divided into 11 groups, with 8 rats in each group, as follows: Normal control group (NC): gavage with normal saline; Iron deficiency model group (ID): gavage with normal saline; Positive control group (PC, salmon blood peptide group): oral administration of salmon blood peptide (12 mg / kg); Ferrous sulfate group (FeSO4): intragastric administration of ferrous sulfate (2 mg / kg, calculated as iron ion concentration); Tuna red meat blood peptide group (THP): gavage of tuna red meat blood peptide (12 mg / kg).

[0020] Synthetic peptide single component grouping: AEE group: oral administration of synthetic peptide AEE (12 mg / kg); LDK group: oral administration of synthetic peptide LDK (12 mg / kg); VEE group: oral administration of synthetic polypeptide VEE (12 mg / kg); SIS group: oral administration of synthetic peptide SIS (12 mg / kg); AIS group: oral administration of synthetic peptide AIS (12 mg / kg); DLQ group: oral administration of synthetic peptide DLQ (12 mg / kg); Compound peptide group (THPMix): AEE, LDK, VEE, SIS, AIS, and DLQ mixed in equal proportions were administered orally (2 mg of each peptide, total dose of 12 mg / kg).

[0021] 1.2 Experimental treatment Dosage: daily gavage for 20 consecutive days; Solution preparation: All peptide groups (PC, THP, AEE, LDK, VEE, SIS, AIS, DLQ, THPMix) were dissolved in normal saline and adjusted to the same volume (5 mL / kg); Detection indicators: On the day of drug withdrawal, 200 μL of tail vein blood was collected to measure peripheral blood count (Hb, RBC, HCT); 2 Experimental Results 2.1 Body mass and organ index After drug withdrawal, the body weights of rats in each treatment group were measured. The body weight of rats in the iron deficiency anemia model group was 229.40 g. Compared with the iron deficiency anemia model group, the body weights of rats in other groups increased significantly ( p < 0.05), and basically returned to normal levels ( Figure 3 ).

[0022] The organ index refers to the ratio of the mass of an animal's organ to its body weight before dissection. Under normal circumstances, the organ index value is relatively stable. When an organ is damaged, the organ index will change accordingly. Therefore, the organ index reflects the pathological status of an animal's internal organs. As shown in Table 2, in this experiment, by comparing the organ index data of the normal group and the model group, it was found that the liver weight of the model group was significantly higher than that of the normal group ( p < 0.05), and kidney weight also increased significantly ( p < 0.05), suggesting that modeling has an impact on the liver and kidneys. Compared with the model group, there was no significant difference in liver weight in the blood peptide group, blood peptide group, AEE group, LDK group, VEE group, AIS group, DLQ group, and ferrous sulfate group ( p > 0.05), indicating that these interventions did not significantly increase the burden on the liver; the kidney weights of the blood peptide group and the blood peptide group were significantly lower than those of the model group ( p < 0.05), showing a certain renal protective potential. The spleen and heart weights of the experimental groups were not significantly different from those of the normal group and the model group ( p > 0.05), indicating that the experimental intervention had little effect on the spleen and heart. Overall, most interventions had a certain degree of safety in terms of their effects on the liver, kidneys, spleen, and heart, and some interventions showed positive effects on the kidneys.

[0023] Table 2: Effects of different drug administration groups on organ indexes of IDA rats Grouping liver spleen kidney heart Normal group <![CDATA[3.80 b ]]> 0.17 0.73 0.31 Model Group <![CDATA[4.37 a ]]> 0.18 <![CDATA[0.85 a ]]> 0.35 Blood peptide group <![CDATA[3.99 b ]]> 0.16 <![CDATA[0.80 a ]]> 0.31 Blood peptide group <![CDATA[4.04 b ]]> 0.18 <![CDATA[0.81 a ]]> 0.32 AEE group <![CDATA[3.97 b ]]> 0.17 0.74 0.31 LDK group <![CDATA[3.85 b ]]> 0.16 0.77 0.32 VEE group <![CDATA[4.12 b ]]> 0.17 0.74 0.32 SIS Group <![CDATA[4.67 b ]]> 0.16 0.72 0.31 AIS group <![CDATA[3.89 b ]]> 0.17 0.73 0.34 DLQ group <![CDATA[4.17 b ]]> 0.18 0.71 0.32 Ferrous sulfate group <![CDATA[3.67 b ]]> 0.16 0.75 0.31 Note: Different letters (a, b) represent significant differences among different drug groups ( p < 0.05).

[0024] 2.2 Routine peripheral blood indicators Routine peripheral blood tests are commonly used to diagnose iron-deficiency anemia. By measuring the levels of various blood markers, the severity and type of anemia can be determined with high accuracy. Iron-deficiency anemia is often accompanied by a decrease in hemoglobin, red blood cell count, and mean corpuscular volume (MCV). Hematocrit refers to the volume ratio of red blood cells to a given volume of whole blood and indirectly reflects the number and volume of red blood cells. Chronic hypoxia caused by long-term severe anemia can lead to an abnormal increase in reactive oxygen species (ROS), which in turn induces platelet apoptosis and an abnormal decrease in platelet count. Experimental data showed that compared with the normal group, the HGB, RBC, and HCT values ​​in the model group were significantly decreased, indicating that the modeling resulted in hematological changes similar to anemia. Among the treatment groups, the ferrous sulfate group, used as a positive control, showed significantly increased HGB, RBC, and HCT values ​​compared with the model group (p < 0.05), confirming its anemia-modifying effect. The HGB and RBC levels of the hemopeptide group, AEE group, LDK group, VEE group, AIS group and DLQ group were significantly higher than those of the model group (p < 0.05). Among them, the HGB and RBC values ​​of the hemopeptide group even exceeded those of the normal group, showing a stronger hematopoietic function. At the same time, the HCT of these groups also increased significantly, indicating that it has a positive regulatory effect on the blood concentration state.

[0025] In terms of platelet leukemia (PLL), the PLT levels in the hemopeptide, AEE, LDK, VEE, SIS, AIS, and DLQ groups were significantly higher than those in the model group (p < 0.05). The hemopeptide group showed a significant increase in PLT, suggesting a mechanism promoting platelet production or release. However, there were no significant differences in MCV between most of the treatment groups and the normal or model groups, indicating that the interventions had minimal effects on mean red blood cell volume. Overall, the hemopeptide group and other peptide-administered groups performed exceptionally well in improving anemia-related hematological indicators.

[0026] Table 3: Effects of different drug administration groups on peripheral blood routine indicators of IDA rats Drug administration group HGB (g / L) <![CDATA[RBC(10 12 / L)]]> HCT (%) MCV (fL) <![CDATA[PLT(10 9 / L)]]> Normal group 125.75±9.00 4.33±0.22 20.95±1.18 48.35±1.61 202.50±94.22 Model Group 100.50±11.26 3.02±0.34 15.38±1.88 47.90±1.24 159.00±113.80 Ferrous sulfate group 112.67±19.51* 3.71±0.82* 18.23±4.13* 49.10±1.36 245.50±120.26 Blood peptide group 123.83±13.39 4.20±0.60 20.55±2.49 49.08±1.74 172.50±119.63 Blood peptide group 131.83±9.85* 4.53±0.22* 22.05±1.71* 48.63±1.57 417.33±165.42* AEE group 127.71±8.65* 4.98±0.75* 17.90±1.24* 48.35±1.24 365.47±95.42* LDK group 139.83±4.07* 4.23±0.69 17.33±1.42* 48.90±1.34 401.53±105.47* VEE group 130.46±9.81* 4.85±0.51* 17.50±1.63* 47.78±1.74 387.47±152.81* SIS Group 124.91±4.82 3.43±0.93* 18.69±1.13* 49.18±1.67 393.58±142.75* AIS group 137.23±7.96* 3.93±0.49* 20.50±1.26 48.49±1.51 369.89±145.50* DLQ group 129.41±6.15* 4.15±0.53* 17.88±2.27* 49.35±1.19 398.31±125.21* Note: "*" indicates that there are significant differences between different drug groups. p < 0.05, indicating p < 0.01 2.3 Iron metabolism indicators SI and TIBC together reflect the metabolic state of iron in the body's blood circulation. The liver is the central regulator of iron homeostasis. The hepcidin it secretes is a cysteine-rich antimicrobial peptide that is closely related to the production of red blood cells. It participates in regulating the dynamic balance of iron, maintaining iron homeostasis and normal physiological functions. The levels of iron metabolism indicators in rats in each group can be seen from the data in Figure 4. Different drug administration groups have different effects on the hepcidin content in the liver of rats with iron deficiency anemia (IDA). Compared with the anemia group, each experimental group (hemopeptide, ferrous sulfate, composite peptide group, etc.) regulated the hepcidin level in the liver to a certain extent. There are also differences between the polypeptide groups, which means that different polypeptides have different regulatory effects on the hepcidin content in the liver. Hemopeptide and composite peptide affect hepcidin content through unique mechanisms of action, which is related to their polypeptide structure and metabolic process in the body, indicating that they have potential unique advantages in regulating iron metabolism. As Figure 5 、 Figure 6 As shown. The SI and TIBC levels of the normal group were high, and the modeling successfully reduced the SI level of rats. In terms of SI indicators, some drug-administered groups such as the blood peptide group and the AEE group were significantly higher than the blood peptide group and the ferrous sulfate group, showing a better effect in improving serum iron. For the TIBC index, the levels of drug-administered groups such as the blood peptide group were significantly higher than those of the blood peptide group, the ferrous sulfate group, and the composite peptide group, indicating a stronger regulatory effect on the ability of transferrin to bind iron. Overall, multiple drug-administered groups such as the blood peptide group were superior to the blood peptide group and others in improving the iron metabolism indicators of IDA rats. Different interventions had significant differences in the effects of iron metabolism, and some drug-administered groups had great potential in correcting iron metabolism disorders. The results showed that tuna blood peptides have good comprehensive effects and safety in improving the three iron metabolism indicators of rats.

[0027] 2.4 Oxidative stress indicators When the body is in a state of iron deficiency for a long time, the body's free radical metabolism is disordered and the antioxidant capacity decreases, which will induce severe oxidative stress reactions, causing tissue damage and cell apoptosis. The total antioxidant capacity can reflect the overall function of the body's antioxidant system and measure the body's ability to eliminate reactive oxygen species and resist oxidative stress reactions. Superoxide dismutase (SOD) is a key antioxidant metalloenzyme in the body that maintains the dynamic balance of free radicals by eliminating various reactive oxygen species. Figure 7It can be seen that the T-SOD activity of each drug-treated group showed different degrees of change compared with the normal group. The T-SOD activity of some drug-treated groups, such as the blood peptide group, AEE group, LDK group, VEE group, and SIS group, was significantly higher than that of the blood peptide group, ferrous sulfate group, and composite peptide group. This shows that these drug-treated groups have enhanced the body's ability to scavenge superoxide anion free radicals, thereby increasing the activity of T-SOD, and have a positive effect on improving the oxidative stress state of IDA rats. The positive regulation of T-SOD activity by the above-mentioned drug-treated groups helps to reduce oxidative damage and promote the body's recovery. Figure 8 The data show that compared with the blood peptide group, the total antioxidant capacity of the blood peptide group, AEE group, LDK group, VEE group, SIS group, AIS group, DLQ group and normal group was significantly higher, while the ferrous sulfate group and the composite peptide group were also higher than the blood peptide group, but the difference was not significant and the degree was slightly weaker. This shows that some drug-administered groups such as the blood peptide group have outstanding effects in improving the total antioxidant capacity of IDA rats. Oxidative stress plays a negative role in the pathological process of iron deficiency anemia, and insufficient antioxidant capacity of the body will aggravate the damage. These drug-administered groups with better effects enhance the body's ability to scavenge free radicals by regulating the activity of antioxidant enzymes, increasing the content of antioxidant substances, and other means, thereby improving the total antioxidant capacity. As a common iron supplement, the ferrous sulfate group has relatively limited performance in improving the total antioxidant capacity.

[0028] Example 3: Molecular mechanism of action of tuna iron-binding peptide 1 Molecular docking and binding mechanism analysis Binding properties of single peptides to transferrin receptor (TfR) Through molecular docking simulation (AutoDock Vina), the binding energies and key interactions of the six peptides with TfR are shown in Table 4.

[0029] Table 4: Binding energy and key interactions between 6 peptides and TfR

[0030] VEE (Val-Glu-Glu) has the lowest binding energy (-8.5 kcal / mol), and its histidine-mediated iron chelation is the core mechanism for enhancing iron absorption.

[0031] AEE and DLQ stabilize TfR binding through hydrogen bonds / salt bridges, assisting iron transport.

[0032] LDK and AIS rely on hydrophobic interactions (Phe315 π-π stacking) to enhance the stability of the complex.

[0033] Example 4: Safety Evaluation of Tuna Iron-Binding Peptide All peptides were evaluated by the ToxinPred online tool (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) and showed no toxicity and possessed good water solubility (logP < 0), high intestinal absorption rate (> 80%), and low allergenic risk.

[0034] The results of in vitro (Caco-2 cells) and in vivo (rat acute toxicity test) evaluations are shown in Table 5 .

[0035] Table 5: Safety evaluation results of tuna iron-binding peptides

[0036] Safety ranking: VEE ≈ SIS > AEE ≈ LDK > DLQ > AIS Optimal options: VEE and SIS offer both high safety (non-cytotoxic, non-gastric irritation) and strong iron-binding capacity. Limiting factors: DLQ and AIS require dose control (<500 mg / kg).

[0037] Based on binding capacity and safety data, the following ratio is used in blood-enriching preparations: Core polypeptide: VEE (50%, dominant in iron chelation and transport); auxiliary polypeptide: AEE+DLQ (30%, enhancing TfR binding stability); safety regulation: SIS (20%, reducing potential irritation risks).

[0038] Specific formula: Active ingredients: VEE (25 mg) + AEE (10 mg) + DLQ (10 mg) + SIS (5 mg); Excipients: Vitamin C (20 mg) + microcrystalline cellulose The peptides in this complex peptide formulation form stable chelates with iron ions (Fe²⁺ / Fe³⁺) through their carboxyl (Glu / Asp) and amino (Lys / Gln) groups, enhancing iron bioavailability (approximately 35% higher than ferrous sulfate). The VEE and AEE in the complex peptides target the iron transporter (DMT1) in intestinal epithelial cells, promoting iron absorption.

[0039] Vitamin C can maintain the reduced state of iron ions (Fe²⁺), prevent oxidative failure, and enhance the solubility of the polypeptide-iron complex.

[0040] In the iron deficiency anemia rat model, after 20 days of intervention with the compound peptide preparation, hemoglobin (Hb) increased to 98.5% of the normal group (vs. 92.3% in the ferrous sulfate group), and serum ferritin levels increased by 2.1 times compared with the model group ( p < 0.01).

[0041] The above results indicate that the polypeptide prepared by the present invention can be used to prepare blood-enriching products for iron-deficiency anemia.

Claims

1. An iron ion binding peptide, characterized in that The iron ion binding peptide is separated and prepared from tuna blood meat.

2. The iron ion binding peptide according to claim 1, wherein The amino acid sequence of the iron ion binding peptide is SEQ ID NO:

1.

3. The iron ion binding peptide according to claim 1, wherein The amino acid sequence of the iron ion binding peptide is any one of SEQ ID NOs: 2-6.

4. Use of the iron ion binding peptide according to any one of claims 1 to 3 in the preparation of a product for treating iron deficiency anemia.

5. A product for treating iron deficiency anemia, characterized in that: The product contains the iron ion binding peptide according to any one of claims 1 to 3 at a pharmacologically effective concentration.

6. The product according to claim 5, wherein The product is a composite peptide preparation, which contains a polypeptide with a sequence of SEQ ID NO:

3.

7. The product according to claim 6, wherein The product is further added with a polypeptide having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO:

6.

8. The product according to claim 6 or 7, characterized in that Vitamins and / or microcrystalline cellulose are also added into the product.

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