Peptide-zinc complex with improved efficacy for improving anxious mood and use thereof

Abstract

This invention discloses a method for preparing and applying a peptide-zinc complex with anxiety-relieving effects. The peptide-zinc complex comprises an IE peptide and zinc, wherein the amino acid sequence of the IE peptide is IEELEEELEEAER, and the molar ratio of the IE peptide to zinc is 1:2 to 1:3. This invention verifies the efficacy of the peptide-zinc complex in relieving anxiety through behavioral changes, tissue morphological changes, and changes in the expression of neurotrophic and inflammatory factors in a mouse anxiety model. This invention establishes an evaluation system for animal models, providing an efficient approach for the application of the peptide-zinc complex IE-Zn.

Description

Technical Field

[0001] This invention relates to the fields of food science and biotechnology, and in particular to the preparation of a peptide-zinc complex with the effect of improving anxiety. Background Technology

[0002] In recent years, anxiety has become an "epidemic" in modern society. According to data from the World Health Organization (WHO) and authoritative organizations at home and abroad, approximately 264 million people worldwide suffer from anxiety disorders. Epidemiological surveys in China in recent years have also shown that the prevalence of anxiety disorders is on the rise, with a lifetime prevalence of approximately 7.6% among adults. If undiagnosed subclinical states and anxiety are included, the prevalence would be even higher.

[0003] The relationship between zinc supplementation and anxiety has garnered significant scientific attention in recent years. Zinc, as a structural factor in proteins and a participant in certain physiological responses, plays a crucial role in the biosynthesis and metabolism of various enzymes. A growing body of clinical and basic research indicates that zinc plays a vital role in regulating anxiety, and it is considered a natural "mood regulator." Observational studies have shown that patients with anxiety disorders (especially those with comorbid depression) typically have significantly lower blood zinc levels than healthy individuals. In clinical studies, zinc supplementation in addition to routine anti-anxiety / antidepressant medication significantly improves treatment efficacy and accelerates its onset.

[0004] However, anxiety involves multiple physiological and psychological factors and is not simply caused by zinc deficiency. Simply supplementing with zinc (such as inorganic zinc) has limited effect on improving anxiety. Although zinc peptide chelates have been shown to have high zinc absorption efficiency, it is unknown whether other structures of zinc peptide chelates, in addition to improving zinc absorption efficiency, can help improve anxiety.

[0005] Therefore, developing a peptide-zinc complex that can improve anxiety has significant scientific research value and practical application implications. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a peptide-zinc complex with the effect of improving anxiety and its application. The peptide-zinc complex is prepared by combining peptides with zinc using specific amino acid sequences, so as to significantly improve anxiety.

[0007] To achieve the above objectives, the present invention first provides a peptide-zinc complex that improves anxiety, wherein the amino acid sequence of the peptide in the peptide-zinc complex is IEELEEELEAER, denoted as IE peptide, and the molar ratio of IE peptide to zinc is 1:2 to 1:3.

[0008] The present invention also provides a method for preparing the above-mentioned peptide zinc complex, the specific steps of which are as follows: S1. After mixing IE peptide, zinc salt and deionized water evenly, adjust the pH of the mixed solution to 7 and carry out the chelation reaction under water bath conditions. S2. The reaction product of S1 was dialyzed using a dialysis bag with a molecular weight <100 Da to remove free zinc ions. The solution was then freeze-dried to obtain the peptide zinc complex IE-Zn.

[0009] In one embodiment of the present invention, the molar ratio of IE peptide in S1 to zinc ions in zinc salt is 1:2, wherein the zinc salt is selected from sulfate or zinc chloride.

[0010] In one embodiment of the present invention, in step S1, the mass ratio of the mixture of IE peptide and zinc salt to deionized water is 1:5~20, the water bath temperature is 30~50℃, and the chelation time is 30~50 min.

[0011] In one embodiment of the present invention, dialysis is performed at 4°C for 24-48 hours. The dialyzed solution is then freeze-dried to obtain IE-Zn.

[0012] The present invention also provides the use of the above-mentioned peptide zinc complex in improving anxiety.

[0013] In one embodiment of the invention, the use includes improving anxiety behavior induced by compound stress.

[0014] In one embodiment of the invention, the combined stress includes a combination of daily fixed noise, restraint, crowded environment, and irregular swimming exhaustion and irregular diet.

[0015] In one embodiment of the present invention, the peptide zinc complex can alleviate stress-induced weight loss.

[0016] In one embodiment of the present invention, the peptide zinc complex can alleviate pathological damage to hippocampal neurons, the pathological damage including at least one of nuclear condensation, staining intensification, and intercellular vacuolization.

[0017] In one embodiment of the present invention, the peptide zinc complex can regulate the expression of neuroplasticity-related factors and / or inflammatory factors in the hippocampus.

[0018] In one embodiment of the present invention, the neuroplasticity-related factor is selected from at least one of FY8, P300, PSD95, GluN2B, GluA2, and NGF; preferably, the zinc peptide chelate can significantly upregulate the expression of FY8 and / or P300.

[0019] In one embodiment of the present invention, the inflammatory factor is selected from at least one of TNF-α, IL-18, and ANXA2; preferably, the zinc peptide chelate can significantly inhibit the release of the inflammatory factor.

[0020] In one embodiment of the present invention, the dosage of the zinc peptide chelate is 1-50 mg / kg body weight / day (based on the total amount of the zinc peptide complex), preferably 3-20 mg / kg body weight / day.

[0021] The present invention also provides an application of the above-mentioned peptide zinc complex in the preparation of products that improve anxiety.

[0022] In one embodiment of the invention, the product includes gummies or solid beverages.

[0023] Beneficial effects: 1. This invention uses a dodecapeptide as a raw material to obtain a peptide-zinc complex that improves anxiety by chelating it with zinc ions. Through complex stress models and multi-dimensional indicators (behavioral, pathological, and molecular biological), this invention is the first to discover and confirm the significant effect of the chelate of IE peptide and zinc in improving anxiety.

[0024] 2. Based on experimental results of behavioral changes, hippocampal morphological changes, and neurotransmitter expression changes, this invention confirms that the chelate of IE peptide and zinc significantly improves anxiety in mice.

[0025] 3. The peptide-zinc complex of this invention is safe, healthy, and non-toxic, and does not have the side effects of first- or second-generation zinc supplements. It is highly effective in supplementing zinc for consumers with zinc deficiency and anxiety, and can also improve nerve cell damage by promoting zinc absorption, promoting neurotransmitter recovery, and inhibiting the expression of inflammatory factors, thereby alleviating anxiety. For normal consumers, IE-Zn does not produce side effects; on the contrary, it can effectively supplement zinc and reduce the risk of zinc deficiency and mood disorders. Attached Figure Description

[0026] Figure 1 The diagram shows a mouse anxiety model and the results of weight change. In the diagram, A is a mouse anxiety model and B is the results of weight change. Figure 2 For the behavioral evaluation of a mouse anxiety model, A is the walking route of the mouse in the open field experiment, B is the walking distance of the mouse in the open field experiment, and C is the change in the immobility time of the mouse in the forced swimming experiment. Figure 3 Results of morphological changes in hippocampal tissue in a mouse anxiety model; Figure 4 The results show the expression of neural factors in the hippocampus tissue of a mouse anxiety model; Figure 5The results show the expression of inflammatory factors in the hippocampus tissue of a mouse anxiety model. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] The IE peptide used in the following examples was purchased from Suzhou Qiangyao Biotechnology Co., Ltd., and its amino acid sequence is IEELEEELEAER.

[0029] Statistical analyses involved in the following examples: all experiments were performed at least three times. GraphpadPrism 10.0.3 software was used for data analysis.

[0030] Example 1: Preparation of the peptide-zinc complex IE-Zn A method for preparing a peptide-zinc complex IE-Zn with the effect of improving anxiety, the specific steps of which are as follows: Weigh out IE peptide and zinc sulfate separately according to a 1:2 molar ratio with zinc ions. Add deionized water and a rotor at a material-to-liquid ratio of 1:10. Mix thoroughly on a magnetic stirrer, and adjust the pH to 7 with 1 mol / L NaOH or HCl solution. Chelate the solution in a water bath at 40°C for 40 min.

[0031] The solution after chelation was dialyzed to remove free zinc ions. Dialysis bags with a molecular weight <100 Da were selected and activated by boiling in deionized water. The solution was added to the activated dialysis bag, and dialyzed at 4°C for 24 hours, changing the water every 8 hours. The dialyzed solution was then pre-frozen in a refrigerator. Finally, the pre-frozen sample was lyophilized using a lyophilizer. The lyophilized IE-Zn can be used for subsequent experiments.

[0032] Example 2: Evaluation of the anxiety-improving function of the peptide-zinc complex IE-Zn This study aims to evaluate the functional efficacy of IE-Zn at the level of an animal model that more closely resembles the physiological environment.

[0033] 1. Establishment of a mouse anxiety model Ninety male C57 mice (8 weeks old) were acclimatized for one week. They were then randomly divided into five groups of 16 mice each: normal control group (NC Group), model group (MC Group), inorganic zinc group (Zn Group), low-dose IE-Zn group (IEL Group), and high-dose IE-Zn group (IEH Group).

[0034] Model establishment method: Except for the normal group, the other four groups were placed in a noisy environment from 8:00 AM to 12:00 PM, rested from 12:00 PM to 2:00 PM, placed in a restrained environment from 2:00 PM to 6:00 PM, rested from 6:00 PM to 7:00 PM, and then swam to exhaustion from 7:00 PM to 8:00 PM. After 8:00 PM, they rested and were placed in a crowded environment. The swimming exhaustion experiment was conducted irregularly, 2-3 times per week. The feed content was also reduced periodically to simulate irregular eating habits. A schematic diagram of the model establishment is shown below. Figure 1 As shown in Figure A.

[0035] Swimming exhaustion test method: Place mice in a swimming tank with a water depth of 50×50×40 cm and a water depth of ≥30 cm at a temperature of 25-30 ℃, ensuring that the mice cannot touch the bottom of the tank. Gently hold the mouse by the tail and put it into the water. If the mouse sinks into the water for more than 3 seconds and cannot float to the surface, immediately remove it. After drying and keeping the mouse warm, return it to the cage.

[0036] Based on the recommended daily zinc intake for humans, the daily zinc intake of mice was calculated to be approximately 1.5 mg / kg according to body surface area index. Two weeks after model establishment, gavage administration began in the third week and continued for four weeks. The normal control group and model group were administered deionized water by gavage, the inorganic zinc group was administered ZnSO4 (3.7 mg / kg), the low-dose IE-Zn group was administered IE-Zn (3.8 mg / kg), and the high-dose IE-Zn group was administered IE-Zn (18.8 mg / kg). The mice's condition and weight were recorded daily.

[0037] The results are as follows Figure 1 As shown, during the first two weeks of modeling, the weight of each group did not change significantly before and after gavage administration. Significant differences in weight emerged after gavage administration. The results indicated that the model group mice exhibited marked anxiety-like behaviors (such as irritability and reduced activity) and had significantly lower weights than the normal group, suggesting that prolonged combined stress successfully induced an anxiety model and affected physiological state. At the end of the sixth week, the weight of the inorganic zinc group mice was not significantly different from the model group, indicating that zinc supplementation alone failed to improve stress-induced weight loss. However, after administration of the peptide zinc chelate (IE-Zn) of this invention, although the weight of the low-dose group mice was still lower than the normal group, it was significantly higher than the model group; the weight of the high-dose group mice further recovered and was significantly higher than both the model and normal groups. These results demonstrate that the peptide zinc chelate of this invention can effectively alleviate systemic physiological damage caused by stress, and its effect is superior to that of inorganic zinc at the same zinc dose, suggesting that its mechanism of action is not solely dependent on zinc supplementation.

[0038] 2. Evaluation of changes in mouse behavior Behavioral experiments on mice were conducted after the sixth week.

[0039] Open field experiment: Mice were brought to the laboratory 1 hour in advance to acclimatize to noise and light, reducing transport stress. Each mouse was placed gently in a square open box (approximately 50cm x 50cm x 40cm) with its head facing the wall in one corner. Uniform, bright, but not glaring light was provided above the box. The interior of the box was white, creating high contrast with the color of the C57BL / 6 mice. Video recording was performed for 10 minutes, tracking the mice's movement paths and analyzing dwell time and distance traveled. After each mouse was tested, the bottom of the box was cleaned and wiped to remove any fecal or urine odors left by the previous mouse.

[0040] Forced swimming experiment: Mice were placed in a transparent cylindrical container, approximately 30 cm high and 15 cm in diameter. Water at approximately 25°C and a depth of 20 cm was poured into the cylinder. Mice were placed into the cylinder one by one, and the immobility time over 6 minutes was recorded and analyzed. Immobility time was defined as a floating state where only the head was above water and the limbs remained largely still. After the experiment, the mice were quickly placed in a drying oven to dry and then returned to their cages.

[0041] The results are as follows Figure 2 As shown, in the open field experiment, compared to the model group, mice that ingested zinc ions, especially those given the peptide zinc complex, exhibited significantly increased dwell time and movement distance in the central region. This indicates that the mice's anxiety was alleviated, and they resumed exploring the central region. In the forced swimming experiment, mice given the peptide zinc complex showed significantly shorter immobility time, indicating a change in survival motivation, and the effect was superior to the inorganic zinc group. This suggests that the peptide zinc complex, through the intake of IE peptides, can effectively alleviate anxiety in mice.

[0042] 3. Morphological changes in mouse hippocampus tissue After mouse sacrifice, brain tissue was collected and fixed in 4% paraformaldehyde solution for 24 h, followed by rinsing with running water for 24 h. The tissue was then trimmed smooth in a fume hood and placed in a labeled dehydration box. A gradient of ethanol was applied for dehydration and paraffin impregnation: 75% ethanol for 1.5 h, 85% ethanol for 1.5 h, 90% ethanol for 1.5 h, 95% ethanol for 1.5 h, anhydrous ethanol I for 1.5 h, anhydrous ethanol II for 1.5 h, benzene for 5-10 min, xylene I for 10-20 min, xylene II for 10-20 min, molten paraffin I at 65°C for 0.5 h, molten paraffin II at 65°C for 1 h, and molten paraffin III at 65°C for 1.5 h (where I, II, and III refer to the same reagent). Next, the tissue was embedded: the paraffin-impregnated tissue was placed in an embedding machine for embedding. First, place the melted wax into the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it into the embedding frame according to the required embedding surface, then cover with the corresponding label. Cool on a -20°C freezing stage. After the wax solidifies, remove the wax block from the embedding frame and trim it. For tissue sectioning after embedding: Place the trimmed wax block into a paraffin microtome and section it to a thickness of 4 μm. Float the sections on a 40°C warm water spreader to flatten the tissue. Lift the tissue onto a glass slide and bake it in a 60°C oven. After the water dries and the wax melts, remove it and store it at room temperature for later use. Finally, perform mounting: Immerse the baked paraffin sections in the molten paraffin in a sealing machine for a few seconds, remove and cool to dry, ensuring the entire slide and tissue are covered with a paraffin film. Place the sections in a 67°C oven and bake until the wax film melts.

[0043] H&E staining method: First, dewax the paraffin sections to water. Then, sequentially immerse the sections in environmentally friendly dewaxing solution I for 15 min, environmentally friendly dewaxing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, followed by rinsing with tap water. Next, stain with hematoxylin for 3 min, rinse with tap water, and then stain with eosin for 15 s. Afterward, dehydrate using a concentration gradient. Then, sequentially immerse the sections in anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, anhydrous ethanol III for 1 min, n-butanol I for 1 min, n-butanol II for 1 min, xylene I for 1 min, and xylene II for 1 min (where I, II, and III refer to the same reagent). Finally, mount the sections with neutral resin. Observe the morphological changes of the hippocampus tissue under a microscope and acquire images.

[0044] The results are as follows Figure 3 As shown, the hippocampus tissue of mice in the model group exhibited reduced nuclear volume, darker staining, and vacuolation between tissue cells. No significant damage was observed in the hippocampus tissue of mice in both groups treated with the peptide zinc complex. This indicates that the peptide zinc complex can improve tissue damage caused by anxiety.

[0045] 4. Evaluation of changes in the expression of neurotrophic and inflammatory factors in mice Real-time quantitative PCR (qRT-PCR) experiment: Mouse hippocampal tissue was collected, and tissue RNA was extracted using the TRNzol method. 0.4 mL of TRNzol lysis buffer was added to 50 mg of hippocampal tissue, and the tissue was homogenized using a cryo-mortar. After standing at room temperature, it was centrifuged at 4°C (10000 rpm). The supernatant was collected, and 1 / 5 volume of chloroform was added. After mixing, it was allowed to stand for 3 min. After centrifugation, the upper aqueous phase was collected, and an equal volume of isopropanol was added. After mixing, it was allowed to stand for 10 min. After centrifugation at 4°C, the supernatant was discarded, and the precipitate was washed with 75% ethanol (prepared with RNase-free water). After centrifugation, the liquid was poured off, dried, and dissolved in 50 μL of DEPC water to obtain total RNA. The RNA concentration was measured using a micro spectrophotometer, ensuring that A260 / 280 and A260 / 230 were within the normal range. The total RNA was transcribed into cDNA according to the reverse transcription kit instructions. Real-time quantitative PCR analysis was performed using the cDNA as a template. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 15 min, followed by 40 cycles (95°C for 10 s, 60°C for 30 s), with melting curve conditions set sequentially at 95°C, 60°C, and 95°C for 15 s each. The mRNA expression levels of neurocytokines GluN2B, PSD95, P300, FY8, GluA2, NGF, and inflammatory factors ANXA2, TNF-α, and IL-18 were detected. mRNA levels were normalized using GADPH mRNA assays and analyzed using a 2... -ΔΔCt The method is used to calculate the results. Three parallel experiments are set up for each sample test group.

[0046] Changes in anxiety are directly related to the expression of various factors in nerve cells. Factors such as GluN2B, PSD95, P300, FY8, GluA2, and NGF directly affect the expression of two important neurotransmitters, NMDA and AMPA. Simultaneously, abnormalities in neurotransmitters lead to oxidative stress and inflammation in cells.

[0047] The test results of neurotrophic factors are as follows Figure 4 As shown, compared with the normal group, the expression levels of GluN2B, PSD95, GluA2, NGF, FY8, and P300 in the hippocampus of the model group mice were significantly downregulated, suggesting that stress inhibited neural synaptic plasticity.

[0048] The hippocampus is a core brain region regulating emotion, learning, memory, and anxious behavior. The normal expression of neurotrophic factors and receptors within the hippocampus is crucial for maintaining neurotransmitter homeostasis and synaptic structure integrity. The occurrence and development of anxiety are closely related to the abnormal expression of key neurotrophic factors such as GluN2B, PSD95, P300, GluA2, and NGF in the hippocampus. These factors directly regulate the function of two important excitatory neurotransmitters, NMDA and AMPA. Disorders in their expression can further induce neurotransmitter imbalance, leading to secondary oxidative stress damage and inflammatory responses, ultimately exacerbating anxiety-like behaviors. Results showed that compared to the inorganic zinc group, the peptide zinc complex significantly restored the expression of neurotrophic factors FY8 and P300 in the hippocampus. Its promoting effect on the recovery of GluA2 and GluN2B was comparable to that of inorganic zinc, while inorganic zinc only significantly promoted the recovery of NGF. Overall, the peptide zinc complex more effectively promoted the recovery of neurotrophic factor expression in the hippocampus. This indicates that the zinc peptide complex can fundamentally improve the abnormal state of the NMDA and AMPA neurotransmitter systems, alleviating the pathological changes of downregulated neurotrophic factor expression in the anxiety model. By repairing neurotrophic factor expression, the zinc peptide complex helps stabilize synaptic structure and function, reduce oxidative stress and inflammatory damage mediated by neurotransmitter disorders, thereby protecting hippocampal neurons and ultimately improving and regulating anxiety.

[0049] At the same time, combined Figure 5 The changes in neurotrophic factor levels shown indicate that the peptide zinc complex can further inhibit the release of inflammatory factors. Compared with the inorganic zinc group, the intake of the peptide zinc complex has better efficacy, which may be due to the synergistic effect of peptide intake and zinc ion formation. This demonstrates the efficacy of the peptide zinc complex in improving anxiety.

[0050] In summary, this invention has discovered that the peptide-zinc complex formed by the chelation of the dodecapeptide with the sequence IEELEEELEAER and zinc ions has good activity in promoting zinc absorption and improving anxiety.

[0051] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A peptide-zinc complex that improves anxiety, characterized in that, The peptide-zinc complex is a complex of IE peptide and zinc, the amino acid sequence of the IE peptide is IEELEEELEAER, and the molar ratio of the IE peptide to zinc is 1:2 to 1:

3.

2. The peptide-zinc complex according to claim 1, characterized in that, Its preparation method includes the following steps: S1. After mixing IE peptide, zinc salt and deionized water evenly, adjust the pH of the mixed solution to 7 and carry out the chelation reaction under water bath conditions. S2. The reaction product of S1 was dialyzed using a dialysis bag with a molecular weight <100 Da to remove free zinc ions. The solution was then freeze-dried to obtain the peptide zinc complex IE-Zn.

3. The peptide-zinc complex according to claim 2, characterized in that, The molar ratio of IE peptide in S1 to zinc ions in zinc salt is 1:2, and the zinc salt is selected from sulfate or zinc chloride.

4. The peptide zinc complex according to claim 2, characterized in that, In step S1, the mass ratio of the mixture of IE peptide and zinc salt to deionized water is 1:5~20, the water bath temperature is 30~50℃, and the chelation time is 30~50 min.

5. The peptide zinc complex according to claim 2, characterized in that, Dialysis was performed at 4°C for 24–48 h. The dialyzed solution was then freeze-dried to obtain IE-Zn.

6. Use of the peptide zinc complex according to any one of claims 1 to 5 in improving anxiety.

7. The use according to claim 6, characterized in that, The intended use includes improving anxiety-induced behavior caused by combined stress, which includes a combination of daily fixed noise, restraint, crowded environments, and irregular swimming exhaustion and eating habits.

8. The use according to claim 7, characterized in that, The peptide-zinc complex can alleviate stress-induced weight loss, reduce pathological damage to hippocampal neurons, and regulate the expression of neuroplasticity-related factors and / or inflammatory factors in the hippocampus. The pathological damage includes at least one of nuclear pyknosis, intensified staining, and intercellular vacuolization. The neuroplasticity-related factors are selected from at least one of FY8, P300, PSD95, GluN2B, GluA2, and NGF. The inflammatory factors are selected from at least one of TNF-α, IL-18, and ANXA2. The peptide-zinc chelate can significantly inhibit the release of the inflammatory factors.

9. The use of the peptide zinc complex according to any one of claims 1 to 5 in the preparation of products that improve anxiety.

10. The application according to claim 9, characterized in that, The product includes food or medicine, and the food includes candy or solid beverage.

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