Iron-binding peptide based on tuna blood meat and application thereof

Abstract

The application provides a kind of iron-binding peptide based on tuna blood meat, wherein the amino acid sequence of some polypeptides is SEQ ID NO:1-6.The polypeptide provided by the application can be used to prepare a preparation for treating iron deficiency anemia.The tuna red meat blood peptide provided by the application can be used to prepare a blood supplement preparation, and through specific polypeptide iron ion chelation, a high-bioavailability organic iron complex is formed, which significantly improves the absorption efficiency of iron in the intestinal tract.The carrier formed by the polypeptide can protect iron ions from being destroyed by gastric acid and target delivery to the absorption site, so that the hemoglobin synthesis efficiency is increased by more than 40%.The natural polypeptide ligand of the application avoids the gastrointestinal irritation of traditional iron supplements and is not affected by dietary factors such as phytic acid and tannin absorption interference.Functional blood supplement peptides are prepared from tuna processing by-products, which not only realizes the high-value utilization of fishery resources, but also provides an ecological-friendly new source of iron supplement.

Description

Technical Field

[0001] This invention belongs to the field of active peptide preparation technology, specifically relating to an iron-binding peptide based on tuna blood and meat and its application. Background Technology

[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 diseases with the highest global incidence, and is particularly prevalent in developing countries and economically underdeveloped regions.

[0003] Currently, clinical treatment for IDA primarily 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:

[0004] 1) Inorganic salts have low bioavailability, are highly irritating to the gastrointestinal tract, and can easily cause adverse reactions such as nausea and vomiting;

[0005] 2) The bioavailability of iron supplements containing organic acid salts has improved, but they are still easily affected by components such as tannins and phytic acid in the diet, which can affect the absorption efficiency of iron.

[0006] In recent years, bioactive peptide iron complexes have become a hot topic in iron supplement research due to their high bioavailability, low toxicity, and good stability. Bioactive peptides (BPs) are small molecule peptides (usually <6 kDa) obtained by proteolytic hydrolysis of proteins and have various physiological regulatory functions, such as immune regulation, antioxidation, and metal ion chelation.

[0007] Tuna blood and flesh (also known as red meat, akami, or dark meat) are often discarded due to their coarse texture, but their crude protein content is as high as 25%, making them an ideal raw material for developing bioactive peptides. However, current technologies for the resource development of tuna are still in their early stages. Summary of the Invention

[0008] The purpose of this invention is to provide an iron-binding peptide based on tuna blood and meat and its application, thereby overcoming the shortcomings of the prior art.

[0009] The present invention first provides an iron-binding peptide, which is isolated and prepared from tuna blood and flesh;

[0010] One of the polypeptides has the amino acid sequence AEELKKEQDTSAH (SEQ ID NO:1).

[0011] Furthermore, the polypeptide has an amino acid sequence of one or more of the following:

[0012] LDKENALDRAEQA (SEQ ID NO:2),

[0013] VEEELDRAQER (SEQ ID NO:3),

[0014] SISEELDHA (SEQ ID NO:4)

[0015] AISEELDHA (SEQ ID NO:5)

[0016] DLQHRLDEAEA (SEQ ID NO:6).

[0017] The present invention also provides an application of the aforementioned polypeptide in the preparation of products for treating iron deficiency anemia.

[0018] The present invention also provides a product for treating iron deficiency anemia, wherein the product contains the above-mentioned polypeptide at a pharmacologically effective concentration.

[0019] Furthermore, the product is a complex peptide formulation, wherein the VEE polypeptide of SEQ ID NO:3 is used as the main component.

[0020] Furthermore, the compound peptide formulation also contains polypeptides of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6.

[0021] As one example, the compound peptide preparation also contains vitamins and / or microcrystalline cellulose.

[0022] The tuna red meat blood peptide provided by this invention has the following outstanding advantages in the preparation of blood-enriching products:

[0023] Highly efficient iron absorption: Through the chelation of iron ions by specific polypeptides, a highly bioavailable organic iron complex is formed, which significantly improves the efficiency of iron absorption in the intestine.

[0024] Optimizes blood replenishment effect: The polypeptide carrier can protect iron ions from being destroyed by gastric acid and deliver them to the absorption site, increasing the efficiency of hemoglobin synthesis by more than 40%.

[0025] Excellent biocompatibility: Natural polypeptide ligands avoid the gastrointestinal irritation of traditional iron supplements and are not affected by the absorption of dietary factors such as phytic acid and tannins.

[0026] Sustainable resource utilization: Functional blood-enriching peptides prepared from tuna processing by-products not only realize the high-value utilization of fishery resources (reducing costs by 60%), but also provide an eco-friendly new source of iron supplementation. Attached Figure Description

[0027] Figure 1 Total ion chromatogram;

[0028] Figure 2 Technical roadmap for animal experiments to verify the efficacy of tuna hemolysin peptides;

[0029] Figure 3 The effect of different drug administration groups on the body weight gain of IDA rats over 50 days;

[0030] Figure 4 The effect of different drug administration groups on hepcidin content in the liver of IDA rats;

[0031] Figure 5 The effect of different drug administration groups on iron content in liver tissue of IDA rats;

[0032] Figure 6 The effect of different drug administration groups on serum total iron binding capacity in IDA rats; where different letters (a, b) represent significant differences between different drug administration groups. p < 0.05);

[0033] Figure 7 The effect of different drug administration groups on superoxide dismutase (TSOD), an oxidative stress marker in IDA rats;

[0034] Figure 8 The effect of different drug administration groups on the total antioxidant capacity of IDA rats; where different letters (a, b) represent significant differences between different drug administration groups. p < 0.05);

[0035] Figure 9 A schematic diagram of a molecular docking simulation. Detailed Implementation

[0036] This invention isolates and identifies a group of novel iron-binding peptides from red tuna meat, named blood peptides (to distinguish them from the control group blood peptides, the iron-binding peptides screened in the attached figure are named red meat peptides), and confirms their significant effect in the treatment of iron deficiency anemia (IDA).

[0037] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0038] Example 1: Identification and Characterization of Blood Synthesis Peptides

[0039] The F4 active component of the enzymatic hydrolysis product of tuna red meat was analyzed using liquid chromatography-mass spectrometry (LC-MS / MS), and the total ion chromatogram (TIC) was obtained as follows: Figure 1 As shown.

[0040] The steps for separating the F4 fraction of the tuna red meat enzymatic hydrolysate are as follows:

[0041] 1. Enzymatic hydrolysis optimization: The optimal conditions were determined through orthogonal experiments to be a material-to-liquid ratio of 1:4 g / mL, a temperature of 50℃, a time of 6h, and a compound enzyme (trypsin:papain = 1:1) addition of 1.2% (w / w).

[0042] 2. Stepwise enzymatic hydrolysis: Trypsin (0.6%, 50℃, 3 h) and papain (0.6%, 50℃, 3 h) were used sequentially. After inactivation of the enzymes by boiling water bath, the mixture was centrifuged, ultrafiltered, and spray-dried.

[0043] 3. Purification: The fractions were separated by Sephadex G15 gel column chromatography (collecting the fractions from group 4), and purified by cation exchange chromatography and reversed-phase HPLC to a purity of ≥95%.

[0044] Data obtained from total ion chromatograms (TIC) were compared with a database (parameters set: protein card value ≥ 1 unique peptide, peptide card value 10 l gP ≥ 20). A total of 60 potentially bioactive peptide sequences were identified from the F4 active components. Among them, the following six peptides (Table 1) exhibited significant iron-binding properties and are simply named AEE, LDK, VEE, SIS, AIS, and DLQ.

[0045] Table 1: Sequences and characteristics of iron-binding polypeptides derived from tuna red meat

[0046] Example 2: Validation of the activity of tuna iron-binding peptide

[0047] 1. Procedures and grouping in animal experiments

[0048] Experimental grouping and treatment scheme

[0049] 1.1 Grouping of experimental animals

[0050] After successful establishment of the rat model, the rats were randomly divided into 11 groups of 8 rats each, as follows:

[0051] Normal control group (NC): administered physiological saline by gavage;

[0052] Iron deficiency model group (ID): administered physiological saline by gavage;

[0053] Positive control group (PC, salmon blood peptide group): salmon blood peptide (12 mg / kg) was administered by gavage.

[0054] Ferrous sulfate group (FeSO4): Ferrous sulfate (2 mg / kg, based on iron ion concentration) was administered by gavage.

[0055] Tuna red meat blood peptide group (THP): Tuna red meat blood peptide (12 mg / kg) was administered by gavage.

[0056] Synthetic polypeptide single-group grouping:

[0057] AEE group: AEE (12 mg / kg) was administered via gavage.

[0058] LDK group: LDK (12 mg / kg) synthesized by gavage.

[0059] VEE group: administered synthetic peptide VEE (12 mg / kg) by gavage;

[0060] SIS group: Synthetic polypeptide SIS (12 mg / kg) was administered by gavage.

[0061] AIS group: AIS synthetic polypeptide (12 mg / kg) was administered by gavage.

[0062] DLQ group: DLQ (12 mg / kg) synthesized by gavage.

[0063] The complex peptide group (THPMix) consists of AEE, LDK, VEE, SIS, AIS, and DLQ mixed in equal proportions by gavage (2 mg of each peptide, total dose 12 mg / kg).

[0064] 1.2 Experimental Treatment

[0065] Administration method: Daily gavage for 20 consecutive days;

[0066] Solution preparation: All peptide groups (PC, THP, AEE, LDK, VEE, SIS, AIS, DLQ, THPMix) were dissolved in physiological saline and adjusted to the same volume (5 mL / kg).

[0067] Testing indicators: On the day of drug discontinuation, 200 μL of tail vein blood was collected to measure peripheral blood counts (Hb, RBC, HCT).

[0068] 2. Experimental Results

[0069] 2.1 Body weight and organ index

[0070] After drug withdrawal, the body weight of rats in each treatment group was measured. The body weight of rats in the iron deficiency anemia model group was 229.40 g. Compared with this, the body weight of rats in other groups was significantly increased. p < 0.05), and has basically returned to normal levels ( Figure 3 ).

[0071] The organ index refers to the ratio of the mass of a specific organ in an animal to its pre-dissection body weight. Under normal circumstances, the organ index value is relatively stable, but it changes when an organ is damaged. Therefore, the organ index reflects the lesion status of an animal's internal organs. Table 2 shows that in this experiment, comparing the organ index data between the normal group and the model group revealed that the liver weight in the model group was significantly higher than that in 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 were no significant differences in liver weight among 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 weight in the blood peptide group and the blood peptide group was significantly lower than that in the model group ( p < 0.05, indicating a certain potential for kidney protection. There were no significant differences in spleen and heart weight between the experimental groups and the normal and model groups. p The value > 0.05 indicates that the experimental intervention had a relatively small impact on the spleen and heart. Overall, most interventions showed some safety in their effects on the liver, kidneys, spleen, and heart, while some interventions showed positive effects on the kidneys.

[0072] Table 2: Effects of different drug administration groups on organ indices in IDA rats

[0073] 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

[0074] Note: Different letters (a, b) represent significant differences between different treatment groups. p < 0.05).

[0075] 2.2 Peripheral blood routine indicators

[0076] Peripheral blood routine tests are a common method for diagnosing iron deficiency anemia. By detecting the levels of various indicators in the blood, the degree and type of anemia can be determined with high accuracy. Iron deficiency anemia is generally accompanied by a decrease in hemoglobin, red blood cell count, and mean corpuscular volume (MCV). Hematocrit, which refers to the volume ratio of red blood cells in a given amount of whole blood, also 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 model group had significantly lower HGB, RBC, and HCT, indicating that modeling led to hematological changes similar to anemia. Among the drug-treated groups, the ferrous sulfate group, as a positive control, showed significantly higher HGB, RBC, and HCT than the model group (p < 0.05), which to some extent verified its effect in improving anemia. The levels of HGB and RBC in the blood peptide group, AEE group, LDK group, VEE group, AIS group, and DLQ group were significantly higher than those in the model group (p < 0.05). Among them, the HGB and RBC values ​​in the blood peptide group even exceeded those in the normal group, showing a strong hematopoietic function. At the same time, the HCT in these groups was also significantly increased, indicating that they have a positive regulatory effect on blood concentration.

[0077] From the perspective of platelet-to-thickness (PLT) levels, the PLT levels in the blood peptide group, AEE group, LDK group, VEE group, SIS group, AIS group, and DLQ group were significantly higher than those in the model group (p < 0.05), especially in the blood peptide group, where PLT levels were significantly elevated, suggesting a mechanism of promoting platelet production or release. However, the mean corpuscular volume (MCV) level showed no significant difference between most of the treatment groups and the normal and model groups, indicating that the interventions had a relatively small impact on mean corpuscular volume. Overall, the blood peptide group and other peptide-treated groups demonstrated outstanding performance in improving anemia-related hematological parameters.

[0078] Table 3: Effects of different drug administration groups on peripheral blood routine indicators in IDA rats

[0079] 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*

[0080] Note: "*" indicates that there are significant differences between different treatment groups. p < 0.05 indicates p < 0.01

[0081] 2.3 Iron metabolism indicators

[0082] 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, and its secreted hepcidin is a cysteine-rich antimicrobial peptide closely related to erythrocyte production, participating in the regulation of iron homeostasis and maintaining normal physiological functions. As shown in Figure 4, the levels of iron metabolism indicators in different groups of rats showed differences in the effects of different drug administration groups on hepcidin content in the liver of rats with iron deficiency anemia (IDA). Compared with the anemia group, all experimental groups (blood peptide, ferrous sulfate, complex peptide group, etc.) regulated hepatic hepcidin levels to some extent. Differences also existed among the various peptide groups, indicating that different peptides had different regulatory effects on hepatic hepcidin content. Blood peptide and complex peptide affect hepcidin content through unique mechanisms of action, which are related to their peptide structure and metabolic processes in vivo, suggesting their potential unique advantages in regulating iron metabolism. Figure 5 , Figure 6 As shown in the figure, the normal group had higher SI and TIBC levels, and the successful modeling reduced the SI level in rats. Regarding the SI index, some treatment 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 superior effect in increasing serum iron. Similarly, for the TIBC index, the levels in the blood peptide group and other treatment groups were significantly higher than those in the blood peptide group, the ferrous sulfate group, and the complex peptide group, indicating a stronger regulatory effect on the iron-binding capacity of transferrin. Overall, the blood peptide group and other treatment groups were superior to the blood peptide group in improving iron metabolism indicators in IDA rats, and the effects of different interventions on iron metabolism varied significantly. Some treatment groups showed considerable potential in correcting iron metabolism disorders. The results indicate that tuna blood peptide has a good comprehensive effect and safety in improving the three iron metabolism indicators in rats.

[0083] 2.4 Oxidative stress indicators

[0084] When the body is chronically iron deficient, free radical metabolism becomes disordered, antioxidant capacity decreases, and severe oxidative stress can be induced, leading to tissue damage and cell apoptosis. Total antioxidant capacity reflects the overall function of the body's antioxidant system and measures the body's ability to scavenge reactive oxygen species and resist oxidative stress. Superoxide dismutase (SOD) is a key antioxidant metalloenzyme in the body, maintaining a dynamic balance of free radicals by scavenging various reactive oxygen species. Figure 7It was found that T-SOD activity in each treatment group showed varying degrees of change compared to the normal group. Some treatment groups, such as the blood peptide group, AEE group, LDK group, VEE group, and SIS group, exhibited significantly higher T-SOD activity than the blood peptide group, ferrous sulfate group, and complex peptide group. This indicates that these treatment groups enhanced the body's ability to scavenge superoxide anion free radicals, thereby increasing T-SOD activity and playing a positive role in improving the oxidative stress state of IDA rats. The positive regulation of T-SOD activity by the aforementioned treatment groups helps to reduce oxidative damage and promote recovery. Figure 8 Data shows 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 complex peptide group also showed higher levels than the blood peptide group, the differences were not significant and the degree was slightly weaker. This indicates that some treatment 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 exacerbates the damage. These better-performing treatment groups enhance the body's ability to scavenge free radicals and thus improve total antioxidant capacity by regulating antioxidant enzyme activity and increasing the content of antioxidant substances. The ferrous sulfate group, as a common iron supplement, showed relatively limited performance in improving total antioxidant capacity.

[0085] Example 3: Mechanism of Action of Tuna Iron-Binding Peptides

[0086] 1. Analysis of molecular docking and binding mechanisms

[0087] Binding properties of a single polypeptide to the transferrin receptor (TfR)

[0088] The binding energies and key interactions between the six peptides and TfR were obtained through molecular docking simulation (AutoDock Vina), as shown in Table 4.

[0089] Table 4: Binding energies and key interactions between six peptides and TfR

[0090]

[0091] 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.

[0092] AEE and DLQ stabilize TfR binding through hydrogen bonds / salt bridges, facilitating iron transport.

[0093] LDK and AIS-dependent hydrophobic interactions (Phe315 π-π stacking) enhance the stability of the complex.

[0094] Example 4: Safety evaluation of tuna iron-binding peptides

[0095] Evaluation using the ToxinPred online tool (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) showed that all peptides were non-toxic and possessed the following characteristics: good water solubility (logP < 0), high intestinal absorption (> 80%), and low risk of sensitization.

[0096] The results of in vitro (Caco-2 cells) and in vivo (rat acute toxicity test) evaluations are shown in Table 5.

[0097] Table 5: Safety Evaluation Results of Tuna Iron-Binding Peptides

[0098]

[0099] Security ranking: VEE ≈ SIS > AEE ≈ LDK > DLQ > AIS

[0100] Optimal choices: VEE and SIS offer a combination of high safety (no cytotoxicity, no gastrointestinal irritation) and strong iron-binding capacity. Limitations: DLQ and AIS require controlled dosage (<500 mg / kg).

[0101] Based on binding capacity and safety data, the following formulation ratios are used in hematinic preparations:

[0102] Core peptide: VEE (50% content, mainly responsible for iron chelation and transport); Auxiliary peptide: AEE+DLQ (30%, enhances TfR binding stability); Safety regulation: SIS (20%, reduces potential stimulation risk).

[0103] Specific formulation: Active ingredients: VEE (25 mg) + AEE (10 mg) + DLQ (10 mg) + SIS (5 mg); Excipients: Vitamin C (20 mg) + Microcrystalline cellulose

[0104] In the aforementioned compound peptide formulation, the peptides form stable chelates with iron ions (Fe²⁺ / Fe³⁺) through carboxyl groups (Glu / Asp) and amino groups (Lys / Gln), thereby enhancing iron bioavailability (approximately 35% higher than ferrous sulfate). The VEE and AEE in the compound peptides can target the small intestinal epithelial cell iron transporter (DMT1), promoting iron absorption.

[0105] Vitamin C can maintain the reduced state of iron ions (Fe²⁺), prevent oxidative degradation, and enhance the solubility of peptide-iron complexes.

[0106] In a rat model of iron deficiency anemia, after 20 days of intervention with this compound peptide preparation: hemoglobin (Hb) increased to 98.5% of the normal group (vs. 92.3% of the ferrous sulfate group), and serum ferritin levels increased 2.1 times compared to the model group. p < 0.01).

[0107] The above results indicate that the polypeptide prepared by the present invention can be used to prepare iron-deficiency anemia supplements.

Claims

1. An iron-binding peptide, characterized in that, The amino acid sequence of the iron-binding peptide is SEQ ID NO:

1.

2. The use of the iron-binding peptide according to claim 1 in the preparation of products for treating iron deficiency anemia.

3. A product for treating iron deficiency anemia, characterized in that, The product contains a pharmacologically effective concentration of the iron-binding peptide of claim 1.

4. The article of claim 3, characterized in that, The product also contains vitamins and / or microcrystalline cellulose.

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