Composition based on synergistic effect of sod-lf covalent conjugate and neuromodulation and application thereof in preparation of medicine for relieving anxiety insomnia and improving sleep quality

Abstract

The application discloses a composition based on synergistic effect of SOD-LF covalent conjugate and nerve regulation and application thereof in preparation of medicine for relieving anxiety insomnia and improving sleep quality, and belongs to the technical field of biotechnology.The present application prepares SOD-LF covalent conjugate by using lactoferrin and SOD; the SOD-LF covalent conjugate is uniformly dispersed in a sodium alginate solution, then sprayed into a calcium chloride solution, and then placed, filtered to obtain SOD-LF covalent conjugate coated with sodium alginate; the SOD-LF covalent conjugate coated with sodium alginate, fucoidan and sodium tripolyphosphate are dispersed in a chitosan solution, stirred, reacted, centrifuged and collected to obtain the composition based on synergistic effect of SOD-LF covalent conjugate and nerve regulation.The composition can enhance the blood-brain barrier permeability through SOD-LF covalent conjugate, regulate the intestinal flora-gut-brain axis in combination with fucoidan, and synergistically inhibit nerve inflammation and oxidative stress, so as to improve anxiety insomnia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to compositions based on the synergistic effect of SOD-LF covalent conjugates and neuromodulation, and their application in the preparation of drugs for relieving anxiety and insomnia and improving sleep quality. Background Technology

[0002] Insomnia refers to a group of diseases characterized by dissatisfaction with sleep quality and duration despite adequate sleep conditions, accompanied by daytime social dysfunction. With increasing societal pressures, insomnia has become a prevalent and common clinical condition. Over 20% of adults suffer from chronic insomnia. Many factors contributing to chronic insomnia, such as shift work, irregular work hours, jet lag, and stress, are related to modern lifestyles. Insufficient sleep can lead to memory loss, irritability, depression, poor concentration, and fatigue. In addition to cognitive function, sleep disorders are also associated with metabolic syndrome, such as obesity, inflammation, diabetes, and cardiovascular disease. Although many medications are available for treating insomnia, including monoamine oxidase inhibitors, tricyclic antidepressants, tetracyclic antidepressants, and serotonin reuptake inhibitors, most are hormone and neurotransmitter modulators. Existing medications have certain toxic side effects, leading to the development of various traditional Chinese medicine combinations to alleviate anxiety and improve sleep; however, their effects are not significant and require long-term use. Summary of the Invention

[0003] In view of this, the present invention aims to provide a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation, and its application in the preparation of drugs for relieving anxiety and insomnia and improving sleep quality. The composition of the present invention enhances the blood-brain barrier permeability through SOD-LF covalent conjugate, regulates the gut microbiota-gut-brain axis in combination with fucoidan, and synergistically inhibits neuroinflammation and oxidative stress, thereby improving anxiety and insomnia.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a composition based on the synergistic effect of SOD-LF covalent conjugate and neural regulation, characterized by comprising the following preparation steps:

[0006] S1. Lactoferrin was dissolved in PBS solution, and then a thiol reducing agent (TCEP) was added. The mixture was then incubated, allowed to stand, and purified to obtain reduced lactoferrin.

[0007] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC), stir and react, then add glycine to terminate the reaction, and finally purify to obtain activated SOD.

[0008] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then obtained after purification.

[0009] S4. The SOD-LF covalently coupled compound was uniformly dispersed in a sodium alginate solution and then sprayed into a calcium chloride solution. After standing and filtration, the sodium alginate-coated SOD-LF covalently coupled compound was obtained.

[0010] S5. Sodium alginate-coated SOD-LF covalently coupled with fucoidan was dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0011] Preferably, the molar ratio of lactoferrin to TCEP in S1 is 1:(5-10); the ratio of lactoferrin to PBS solution is (2-5) mg:1 mL.

[0012] Preferably, the incubation time in S1 is 0.5 to 1.5 hours, and the temperature is 35 to 37°C.

[0013] Preferably, the settling time in step S1 is 30-50 minutes and the temperature is 2-5°C.

[0014] Preferably, the molar ratio of SOD to Sulfo-SMCC in S2 is (10-20):1;

[0015] The ratio of SOD to PBS solution is (4-8) mg / mL.

[0016] Preferably, the temperature of the stirring reaction in S2 is 20-25°C, and the time is 1-3 hours.

[0017] Preferably, the molar ratio of reduced lactoferrin to activated SOD in S3 is 1:(1-3).

[0018] Preferably, the ratio of SOD-LF covalent conjugate to sodium alginate solution in S4 is 1g:(10-30)mL;

[0019] The concentration of the sodium alginate solution is 1–3 wt%.

[0020] The concentration of the calcium chloride solution is 0.1–0.2 mol / L.

[0021] Preferably, the particle size of the SOD-LF covalently coupled with sodium alginate in S4 is 10-20 μm.

[0022] Preferably, the ratio of sodium alginate-coated SOD-LF covalently coupled compound, fucoidan, and chitosan solution in S5 is (1-3) mg: (1-3) mg: (10-20) mL.

[0023] The concentration of the chitosan solution is 2–5 mg / mL.

[0024] Preferably, the amount of sodium tripolyphosphate used in S5 is 0.1 to 0.3 wt%.

[0025] Secondly, the present invention provides the use of the above-described composition in the preparation of a medicament for relieving anxiety and insomnia and improving sleep quality.

[0026] It contains at least the following beneficial technical effects:

[0027] The composition prepared by this invention has a significant effect on relieving anxiety and insomnia and improving sleep quality, while having no side effects and using widely available raw materials. Detailed Implementation

[0028] This invention provides a composition based on the synergistic effect of SOD-LF covalent conjugate and neural regulation, characterized by comprising the following preparation steps:

[0029] S1. Lactoferrin was dissolved in PBS solution (pH 7.4), then TCEP was added and incubated, allowed to stand, and purified to obtain reduced lactoferrin.

[0030] S2. Dissolve SOD in PBS solution, then add Sulfo-SMCC and stir to react. Then add glycine to terminate the reaction and finally purify to obtain activated SOD.

[0031] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then obtained after purification.

[0032] S4. The SOD-LF covalently coupled compound was uniformly dispersed in a sodium alginate solution and then sprayed into a calcium chloride solution. After standing and filtration, the sodium alginate-coated SOD-LF covalently coupled compound was obtained.

[0033] S5. Sodium alginate-coated SOD-LF covalently coupled with fucoidan was dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0034] This invention involves dissolving lactoferrin in PBS solution, then adding TCEP for incubation, standing, and purification to obtain reduced lactoferrin.

[0035] The present invention exposes the free thiol groups of LF through TCEP using the above-described technical solution.

[0036] In this invention, SOD is dissolved in PBS solution, then Sulfo-SMCC is added and stirred to react, then glycine is added to terminate the reaction, and finally purification is performed to obtain activated SOD.

[0037] The present invention uses the above technical solution to covalently link the NHS ester end of Sulfo-SMCC with the primary amino group (-NH2) of SOD.

[0038] This invention involves uniformly dispersing SOD-LF covalently coupled compounds in a sodium alginate solution, then spraying them into a calcium chloride solution, allowing them to stand, and filtering to obtain sodium alginate-coated SOD-LF covalently coupled compounds.

[0039] This invention employs the above-described technical solution to link activated SOD (containing maleimide groups) with reduced LF (containing free thiol groups). LF binds to the low-density lipoprotein receptor (LRP1) on the surface of brain endothelial cells, triggering receptor-mediated endocytosis and transporting SOD across the blood-brain barrier. The binding of LF to SOD reduces renal clearance and improves bioavailability.

[0040] In this invention, SOD-LF covalently coupled compounds are uniformly dispersed in sodium alginate solution, then sprayed into calcium chloride solution, allowed to stand, and filtered to obtain sodium alginate-coated SOD-LF covalently coupled compounds.

[0041] This invention employs the above-mentioned technical solution to encapsulate SOD-LF covalently coupled compounds with sodium alginate. Sodium alginate has good environmental tolerance and cross-links with calcium ions to form a gel, which is then released by bacterial enzymatic decomposition in the colon.

[0042] This invention involves dispersing sodium alginate-coated SOD-LF covalently coupled with fucoidan in a chitosan solution, adding sodium tripolyphosphate, stirring and reacting, and centrifuging to collect the mixture, thereby obtaining a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0043] This invention employs the above-mentioned technical solution to encapsulate both sodium alginate-coated SOD-LF covalently and fucoidan with chitosan. The SOD-LF covalently is a double-layer coating, while the fucoidan is a single-layer coating. Chitosan allows the encapsulated material to pass through gastric juices and release both substances in the alkaline intestinal environment. Fucoidan is released first and exerts its effect. Fucoidan is a sulfated polysaccharide extracted from brown algae (such as kelp and wakame), containing a large amount of fucose and sulfate groups. It cannot be directly digested by the human body, but it can serve as "food" (prebiotic) for specific intestinal flora, especially butyrate-producing bacteria, and activate metabolic pathways related to butyrate synthesis in the flora (such as the butyrate kinase pathway), increasing butyrate production. Then, the SOD-LF covalently coupled compound is slowly released and synergistically interacts with butyrate. Butyrate stimulates intestinal endocrine cells (such as enterochromaffin cells) to release serotonin (5-HT). 5-HT activates vagal nerve afferent fibers, transmitting signals to the nucleus tractus solitarius (NTS) in the brainstem, inhibiting the hypothalamus-pituitary-adrenal (HPA) axis, reducing the release of stress hormones (cortisol), and alleviating anxiety. Butyrate can enter the bloodstream via passive diffusion or the monocarboxylic acid transporter (MCT1), partially crossing the blood-brain barrier, and directly acting on the central nervous system, reducing the release of pro-inflammatory factors and lowering neuroinflammation. Butyrate enhances mitochondrial antioxidant capacity and reduces neuronal apoptosis by upregulating SOD. By enhancing endogenous SOD expression through the gut microbiota-gut-brain axis, it synergistically inhibits oxidative stress in conjunction with SOD-LF (exogenous antioxidant) and fucoidan (endogenous pro-antioxidant), blocking the vicious cycle of inflammation and oxidation.

[0044] This composition enhances blood-brain barrier permeability through SOD-LF covalent coupling, and, in conjunction with fucoidan, regulates the gut microbiota-gut-brain axis, synergistically inhibiting neuroinflammation and oxidative stress, thereby improving anxiety and insomnia.

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0051] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0052] Example 1

[0053] This embodiment provides a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation:

[0054] S1. Dissolve lactoferrin in PBS solution and then add thiol reducing agent (TCEP). Incubate at 36°C for 1 h, then let stand at 4°C for 40 min. Purify by desalting column to obtain reduced lactoferrin.

[0055] The molar ratio of lactoferrin to TCEP is 1:8; the ratio of lactoferrin to PBS solution is 4 mg: 1 mL.

[0056] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC), stir at 25°C for 2 h, then add glycine to terminate the reaction, and finally purify by desalting column to obtain activated SOD.

[0057] The molar ratio of SOD to Sulfo-SMCC is 15:1; the ratio of SOD to PBS solution is 6 mg / mL.

[0058] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then purified using a Superdex 200 column.

[0059] The molar ratio of reduced lactoferrin to activated SOD is 1:2.

[0060] S4. The SOD-LF covalent conjugate was uniformly dispersed in sodium alginate solution and then sprayed into calcium chloride solution. After standing and filtration, sodium alginate-coated SOD-LF covalent conjugate with a particle size of 15 μm was obtained.

[0061] The ratio of SOD-LF covalent conjugate to sodium alginate solution was 1g:20mL;

[0062] The concentration of the sodium alginate solution is 2 wt%.

[0063] The concentration of the calcium chloride solution is 0.2 mol / L.

[0064] S5. Sodium alginate-coated SOD-LF covalently coupled with fucoidan was dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0065] The ratio of sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution was 2 mg: 2 mg: 15 mL.

[0066] The chitosan solution concentration was 3 mg / mL, and the amount of sodium tripolyphosphate used was 0.2 wt%.

[0067] Example 2

[0068] This embodiment provides a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation:

[0069] S1. Dissolve lactoferrin in PBS solution and then add thiol reducing agent (TCEP). Incubate at 35°C for 0.5 h, then let stand at 2°C for 30 min. Purify by desalting column to obtain reduced lactoferrin.

[0070] The molar ratio of lactoferrin to TCEP is 1:5; the ratio of lactoferrin to PBS solution is 2 mg: 1 mL.

[0071] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC), stir at 20℃ for 1 h, then add glycine to terminate the reaction, and finally purify by desalting column to obtain activated SOD.

[0072] The molar ratio of SOD to Sulfo-SMCC is 10:1; the ratio of SOD to PBS solution is 4 mg / mL.

[0073] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then purified using a Superdex 200 column.

[0074] The molar ratio of reduced lactoferrin to activated SOD is 1:1.

[0075] S4. The SOD-LF covalent conjugate was uniformly dispersed in sodium alginate solution and then sprayed into calcium chloride solution. After standing and filtration, sodium alginate-coated SOD-LF covalent conjugate with a particle size of 10 μm was obtained.

[0076] The ratio of SOD-LF covalent conjugate to sodium alginate solution was 1g:10-mL;

[0077] The concentration of the sodium alginate solution is 1 wt%.

[0078] The concentration of the calcium chloride solution is 0.1 mol / L.

[0079] S5. Sodium alginate-coated SOD-LF covalently coupled with fucoidan was dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0080] The ratio of sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution was 1 mg: 1 mg: 10 mL.

[0081] The chitosan solution concentration was 2 mg / mL, and the amount of sodium tripolyphosphate used was 0.1 wt%.

[0082] Example 3

[0083] This embodiment provides a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation:

[0084] S1. Dissolve lactoferrin in PBS solution and then add thiol reducing agent (TCEP). Incubate at 37°C for 1.5 h, then let stand at 5°C for 50 min. Purify by desalting column to obtain reduced lactoferrin.

[0085] The molar ratio of lactoferrin to TCEP is 1:10; the ratio of lactoferrin to PBS solution is 5 mg: 1 mL.

[0086] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC), stir at 25°C for 3 h, then add glycine to terminate the reaction, and finally purify by desalting column to obtain activated SOD.

[0087] The molar ratio of SOD to Sulfo-SMCC is 20:1; the ratio of SOD to PBS solution is 8 mg / mL.

[0088] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then purified using a Superdex 200 column.

[0089] The molar ratio of reduced lactoferrin to activated SOD is 1:3.

[0090] S4. The SOD-LF covalent coupling compound was uniformly dispersed in sodium alginate solution and then sprayed into calcium chloride solution. After standing and filtration, sodium alginate-coated SOD-LF covalent coupling compound with a particle size of 20 μm was obtained.

[0091] The ratio of SOD-LF covalent conjugate to sodium alginate solution was 1g:30mL;

[0092] The concentration of the sodium alginate solution is 3 wt%.

[0093] The concentration of the calcium chloride solution is 0.2 mol / L.

[0094] S5. Sodium alginate-coated SOD-LF covalently coupled with fucoidan was dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled with neural regulation.

[0095] The ratio of sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution was 3 mg: 3 mg: 20 mL.

[0096] The concentration of the chitosan solution is 5 mg / mL.

[0097] The amount of sodium tripolyphosphate used in S5 is 0.3 wt%.

[0098] Comparative Example 1

[0099] This comparative example does not couple SOD with LF (the amount used is the same as in Example 1). The specific preparation method is as follows:

[0100] SOD and LF were uniformly dispersed in sodium alginate solution and then sprayed into calcium chloride solution. After standing and filtration, SOD-LF covalently coupled with sodium alginate with a particle size of 15 μm was obtained.

[0101] The ratio of total SOD and LF to sodium alginate solution was 1g:20mL;

[0102] The concentration of the sodium alginate solution is 2 wt%.

[0103] The concentration of the calcium chloride solution is 0.2 mol / L.

[0104] S5. Sodium alginate-coated SOD, LF and fucoidan were dispersed in a chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and centrifuged to collect the mixture to obtain a composition based on the synergistic effect of SOD-LF covalent coupling and neuromodulation.

[0105] The ratio of sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution was 2 mg: 2 mg: 15 mL.

[0106] The chitosan solution concentration was 3 mg / mL, and the amount of sodium tripolyphosphate used was 0.2 wt%.

[0107] Comparative Example 2

[0108] The preparation method of this comparative example is the same as that of Example 1, except that fucoidan is not added in step S5.

[0109] Comparative Example 3

[0110] All comparative examples used a single-layer coating method, as follows:

[0111] S1. Dissolve lactoferrin in PBS solution and then add thiol reducing agent (TCEP). Incubate at 36°C for 1 h, then let stand at 4°C for 40 min. Purify by desalting column to obtain reduced lactoferrin.

[0112] The molar ratio of lactoferrin to TCEP is 1:8; the ratio of lactoferrin to PBS solution is 4 mg: 1 mL.

[0113] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC), stir at 25°C for 2 h, then add glycine to terminate the reaction, and finally purify by desalting column to obtain activated SOD.

[0114] The molar ratio of SOD to Sulfo-SMCC is 15:1; the ratio of SOD to PBS solution is 6 mg / mL.

[0115] S3. Reduced lactoferrin and activated SOD were mixed in PBS solution and stirred. After stirring, cysteine ​​was added to terminate the reaction. The SOD-LF covalent conjugate was then purified using a Superdex 200 column.

[0116] The molar ratio of reduced lactoferrin to activated SOD is 1:2.

[0117] S4. SOD-LF covalently coupled compound and fucoidan were dispersed in chitosan solution, sodium tripolyphosphate was added, the mixture was stirred and reacted, and the mixture was collected by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalently coupled compound and neuromodulation.

[0118] The ratio of SOD-LF covalent conjugate, fucoidan, and chitosan solution was 2 mg: 2 mg: 15 mL.

[0119] The chitosan solution concentration was 3 mg / mL, and the amount of sodium tripolyphosphate used was 0.2 wt%.

[0120] Experimental Example 1

[0121] Experimental animals: SPF-grade healthy male BALB / c mice, weighing 15-20g, were purchased from Beijing Vital River Technology Co., Ltd.; 70 mice were selected and randomly divided into 7 groups after one week of acclimatization.

[0122] Drug preparation: The compositions obtained in Examples 1-3 and Comparative Examples 1-3 were prepared with distilled water to form a 10 mg / mL solution.

[0123] Gavage dosage: Each group was given 25 mL / kg of the drug solution used in the examples or comparative examples, while the blank group was given an equal volume of distilled water.

[0124] Experimental method: Each mouse was given the corresponding drug solution twice daily by gavage for 7 consecutive days. After that, a light-dark box test was conducted (50cm*50cm, half of which was covered as the dark box and the other half was uncovered as the light box). The mice were placed in the open box and the test was started. Each test was timed for 10 minutes and repeated 3 times. The total time the mice stayed in the light box was recorded.

[0125] The test results are shown in Table 1.

[0126] Table 1

[0127] Group Average dwell time in the lightbox / s Example 1 219 Example 2 211 Example 3 195 Comparative Example 1 151 Comparative Example 2 149 Comparative Example 3 168 Blank group 127

[0128] Experiment Example 2

[0129] Experimental animals: SPF-grade healthy male BALB / c mice, weighing 15-20g, were purchased from Beijing Vital River Technology Co., Ltd.; 70 mice were selected and randomly divided into 7 groups after one week of acclimatization.

[0130] Forced swimming experiment: Mice were placed in a bucket that could not reach the bottom and were kept for 5 minutes twice a day for 7 consecutive days to establish a mouse depression model.

[0131] Drug preparation: The compositions obtained in Examples 1-3 and Comparative Examples 1-3 were prepared with distilled water to form a 10 mg / mL solution.

[0132] Gavage dosage: Each group was gavaged with 25 mL / kg of the drug solution used in the examples or comparative examples, while the blank group was gavaged with an equal volume of distilled water. Each mouse was gavaged twice with the corresponding drug solution. Three days later, a forced swimming experiment was conducted and the swimming time was recorded. See Table 2.

[0133] Table 2

[0134]

[0135]

[0136] Experimental Example 3

[0137] Mouse insomnia experiment

[0138] Experimental animals: SPF-grade healthy male BALB / c mice, weighing 15-20g, were purchased from Beijing Vital River Technology Co., Ltd.; 70 mice were selected and randomly divided into 7 groups after one week of acclimatization.

[0139] The model was successfully established when mice were injected intraperitoneally with 400 mg / kg PCPA at a dose of 0.1 mL / 10 g for 3 consecutive days. The mice exhibited loss of circadian rhythm, increased aggression, disheveled fur, and weight loss.

[0140] Drug preparation: The compositions obtained in Examples 1-3 and Comparative Examples 1-3 were prepared with distilled water to form a 10 mg / mL solution.

[0141] Gavage dosage: Each group was given 25 mL / kg of the drug solution used in the examples or comparative examples, while the blank group was given an equal volume of distilled water. The administration was carried out for 3 consecutive days, and the time to fall asleep was recorded. See Table 3.

[0142] Table 3

[0143] Group Sleep onset time / min Example 1 47.9 Example 2 43.0 Example 3 44.7 Comparative Example 1 34.8 Comparative Example 2 31.5 Comparative Example 3 38,4 Blank group 13.4

[0144] As can be seen from the above experimental examples, the composition prepared by this invention has a significant effect in relieving anxiety and insomnia and improving sleep quality. Comparative Example 1 shows that the coupling of SOD and LF significantly enhances the effect of SOD; Comparative Examples 2-3 show that the combined effect of SOD-LF covalently coupled with fucoidan significantly enhances the effect. However, in Comparative Example 3, because both SOD-LF covalently coupled and fucoidan are released simultaneously, the SOD-LF covalently coupled is absorbed before the fucoidan can exert its effect, making it difficult to achieve a truly simultaneous effect.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition based on the synergistic effect of SOD-LF covalent conjugate with neuroregulation, characterized in that, The composition is prepared by the following preparation method: S1. Lactoferrin is dissolved in a PBS solution, then a thiol reducing agent TCEP is added, and incubation, standing, and purification are sequentially performed to obtain reduced lactoferrin; S2. SOD is dissolved in a PBS solution, then Sulfo-SMCC is added to perform stirring reaction, then glycine is added to terminate the reaction, and finally purification is performed to obtain activated SOD; S3. The reduced lactoferrin and the activated SOD are mixed in a PBS solution to perform stirring reaction, then cysteine is added to terminate the reaction, and finally purification is performed to obtain an SOD-LF covalent conjugate; S4. The SOD-LF covalent conjugate is uniformly dispersed in a sodium alginate solution, then is sprayed into a calcium chloride solution, and standing and filtration are performed to obtain a sodium alginate-coated SOD-LF covalent conjugate; S5. The sodium alginate-coated SOD-LF covalent conjugate and fucoidan are dispersed in a chitosan solution, then sodium tripolyphosphate is added to perform stirring reaction, and centrifugal collection is performed to obtain a composition based on the synergistic effect of SOD-LF covalent conjugate and nerve regulation.

2. The composition of claim 1, wherein, In S1, the molar ratio of lactoferrin to the thiol reducing agent TCEP is 1: (5-10); and the ratio of lactoferrin to the PBS solution is (2-5) mg: 1 mL.

3. The composition of claim 1, wherein, In S2, the molar ratio of SOD to Sulfo-SMCC is (10-20):

1. The ratio of SOD to the PBS solution is (4-8) mg: 1 mL.

4. The composition of claim 1, wherein, In S2, the stirring reaction is performed at a temperature of 20-25°C for 1-3 h.

5. The composition of claim 1, wherein, In S3, the molar ratio of reduced lactoferrin to activated SOD is 1: (1-3).

6. The composition of claim 1, wherein, In S4, the ratio of the SOD-LF covalent conjugate to the sodium alginate solution is 1 g: (10-30) mL; The concentration of the sodium alginate solution is 1-3 wt%; The concentration of the calcium chloride solution is 0.1-0.2 mol / L.

7. The composition of claim 1, wherein In S4, the particle size of the sodium alginate-coated SOD-LF covalent conjugate is 10-20 μm.

8. The composition of claim 1, wherein, In S5, the ratio of the sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution is (1-3) mg: (1-3) mg: (10-20) mL; The concentration of the chitosan solution is 2-5 mg / mL.

9. The composition of claim 1, wherein, In S5, the amount of sodium tripolyphosphate used is 0.1-0.3 wt%.

10. Use of the composition of any one of claims 1-9 in the preparation of a medicament for relieving anxiety.

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