Composition based on synergistic effect of SOD-LF covalent conjugate and neuromodulation and application of composition in preparation of drugs for relieving anxiety and insomnia and improving sleep quality

Abstract

The invention discloses a composition based on the synergistic effect of an SOD-LF covalent conjugate and neuromodulation and application of the composition in preparation of drugs for relieving anxiety and insomnia and improving sleep quality, and belongs to the technical field of biology. The preparation method comprises the following steps: preparing an SOD-LF covalent conjugate from lactoferrin and SOD; uniformly dispersing the SOD-LF covalent conjugate in a sodium alginate solution, spraying into a calcium chloride solution, standing, and filtering to obtain a sodium alginate coated SOD-LF covalent conjugate; and dispersing the sodium alginate coated SOD-LF covalent conjugate and fucoidin in a chitosan solution, adding sodium tripolyphosphate, stirring for reaction, and centrifugally collecting to obtain the composition based on the synergistic effect of the SOD-LF covalent conjugate and nerve regulation. According to the composition disclosed by the invention, the blood brain barrier transmittance is enhanced through the SOD-LF covalent conjugate, and the fucoidin is combined to regulate the intestinal flora-enteric brain axis to synergistically inhibit neuroinflammation and oxidative stress, so that anxiety and insomnia are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to 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. Background Art

[0002] Insomnia refers to a disease in which the quality and duration of sleep are still unsatisfactory under suitable sleep conditions, accompanied by daytime social dysfunction. With the increasing social pressure, insomnia has become a common and frequently-occurring disease in clinical practice. More than 20% of adults suffer from chronic insomnia. Many factors leading to chronic insomnia, such as shift work, irregular working hours, jet lag and stress, are related to modern lifestyles. Lack of sleep can lead to memory loss, irritability, depression, inattention and fatigue. In addition to cognitive function, sleep disorders are also associated with metabolic syndrome, such as obesity, inflammation, diabetes and cardiovascular diseases. Although there are many drugs available for the treatment of insomnia, including monoamine oxidase inhibitors, tricyclic antidepressants, tetracyclic antidepressants, 5-hydroxytryptamine reuptake inhibitors, etc., most of them are drugs that regulate hormones and neurotransmitters. The drugs in the prior art have certain toxic and side effects, and thus a variety of traditional Chinese medicine compositions have been derived to relieve anxiety and improve sleep, but their effects are not obvious and the taking time is long. Summary of the Invention

[0003] In view of this, the purpose of the present invention is 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 the SOD-LF covalent conjugate, combines with fucoidan to regulate the gut microbiota-gut-brain axis, and synergistically inhibits neuroinflammation and oxidative stress, thereby improving anxiety and insomnia.

[0004] To achieve the above purpose, the present invention provides the following technical solutions:

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

[0006] S1. Dissolve lactoferrin in PBS solution, then add a thiol reducing agent (TCEP), incubate, stand still, and purify in sequence to obtain reduced lactoferrin;

[0007] S2. Dissolve SOD in PBS solution, then add sodium 4-(N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimide (Sulfo-SMCC) for stirring reaction, then add glycine to terminate the reaction, and finally purify to obtain activated SOD;

[0008] S3. Mix the reduced lactoferrin and activated SOD in PBS solution, stir and react, then add cysteine to terminate the reaction, and finally obtain the SOD-LF covalent conjugate through purification;

[0009] S4. Uniformly disperse the SOD-LF covalent conjugate in the sodium alginate solution, then spray it into the calcium chloride solution, let it stand, and filter to obtain the sodium alginate-coated SOD-LF covalent conjugate;

[0010] S5. Disperse the sodium alginate-coated SOD-LF covalent conjugate and fucoidan in the chitosan solution, add sodium tripolyphosphate, stir and react, and collect by centrifugation to obtain the composition based on the synergistic effect of the SOD-LF covalent conjugate and neuromodulation.

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

[0012] Preferably, in S1, the incubation time is 0.5 - 1.5 h; the temperature is 35 - 37 °C.

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

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

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

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

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

[0018] Preferably, in S4, the ratio of the SOD-LF covalent conjugate to the sodium alginate solution is 1 g:(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, in S4, the particle size of the sodium alginate-coated SOD-LF covalent conjugate is 10 - 20 μm.

[0022] Preferably, the ratio of the sodium alginate-coated SOD-LF conjugate, 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 dosage of sodium tripolyphosphate in S5 is 0.1-0.3 wt%.

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

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

[0027] The composition prepared by the present invention has an obvious effect of relieving anxiety and insomnia and improving sleep quality, and at the same time has no side effects and wide raw material sources. Detailed Embodiments

[0028] The present invention provides a composition based on the synergistic effect of SOD-LF conjugate and neuromodulation, which is characterized by including the following preparation steps:

[0029] S1. Dissolve lactoferrin in PBS solution (pH 7.4), then add TCEP, and incubate, stand, and purify in sequence to obtain reduced lactoferrin;

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

[0031] S3. Mix reduced lactoferrin and activated SOD in PBS solution, stir and react, then add cysteine to terminate the reaction, and then purify to obtain SOD-LF conjugate;

[0032] S4. Uniformly disperse the SOD-LF conjugate in sodium alginate solution, then spray it into calcium chloride solution, stand, and filter to obtain sodium alginate-coated SOD-LF conjugate;

[0033] S5. Disperse the sodium alginate-coated SOD-LF conjugate and fucoidan in chitosan solution, add sodium tripolyphosphate, stir and react, and centrifuge to collect the composition based on the synergistic effect of SOD-LF conjugate and neuromodulation.

[0034] The present invention dissolves lactoferrin in PBS solution, then adds TCEP, and incubates, stands, and purifies in sequence to obtain reduced lactoferrin.

[0035] Through the above technical solution, the present invention exposes the free sulfhydryl group of LF through TCEP.

[0036] The present invention dissolves SOD in PBS solution, then adds Sulfo-SMCC for stirring reaction, then adds glycine to terminate the reaction, and finally purifies to obtain activated SOD.

[0037] Through the above technical solution, the present invention covalently connects the NHS ester end of Sulfo-SMCC with the primary amino group (-NH2) of SOD.

[0038] The present invention uniformly disperses the SOD-LF conjugate in sodium alginate solution, then sprays it into calcium chloride solution, stands still, and filters to obtain the sodium alginate-coated SOD-LF conjugate.

[0039] By adopting the above technical solution, the present invention connects the activated SOD (containing maleimide group) with the reduced LF (containing free sulfhydryl group). LF binds to the low-density lipoprotein receptor (LRP1) on the surface of brain endothelial cells, triggers receptor-mediated endocytic transport, and carries SOD across the blood-brain barrier. After binding with SOD, LF reduces renal clearance and improves bioavailability.

[0040] The present invention uniformly disperses the SOD-LF conjugate in sodium alginate solution, then sprays it into calcium chloride solution, stands still, and filters to obtain the sodium alginate-coated SOD-LF conjugate.

[0041] By adopting the above technical solution, the present invention coats the SOD-LF conjugate with sodium alginate. Sodium alginate has good environmental tolerance and crosslinks with calcium ions to form a gel, which is released by bacterial enzyme hydrolysis in the colon.

[0042] The present invention disperses the sodium alginate-coated SOD-LF conjugate and fucoidan in chitosan solution, adds sodium tripolyphosphate for stirring reaction, and centrifuges to collect the composition based on the synergistic effect of the SOD-LF conjugate and neuromodulation.

[0043] In the present invention, the sodium alginate-coated SOD-LF conjugate and fucoidan are co-coated with chitosan; among them, the SOD-LF conjugate belongs to double-layer coating, and fucoidan belongs to single-layer coating. Chitosan enables the coated substance to pass through the gastric juice and release the two in the alkaline environment of the intestine. Among them, fucoidan is released first and plays a role. 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 can be used as "food" (prebiotic) for specific intestinal flora, especially butyrate-producing bacteria, and activate the metabolic pathways related to butyrate synthesis in the flora (such as the butyrate kinase pathway) to increase the production of butyrate. Then the SOD-LF conjugate is slowly released and acts synergistically with the production of butyrate; butyrate stimulates intestinal endocrine cells (such as enterochromaffin cells) to release 5-hydroxytryptamine (5-HT), and 5-HT activates the afferent fibers of the vagus nerve, transmits the signal to the nucleus of the solitary tract (NTS) in the brainstem, inhibits the hypothalamic-pituitary-adrenal (HPA) axis, reduces the release of stress hormones (cortisol), and relieves anxiety. Butyrate can enter the blood through passive diffusion or monocarboxylate transporter (MCT1), partially penetrate the blood-brain barrier, act directly on the central nervous system, reduce the release of pro-inflammatory factors, reduce neuroinflammation. Butyrate enhances the antioxidant capacity of mitochondria by upregulating SOD and reduces neuronal apoptosis. By enhancing the endogenous SOD expression through the gut microbiota-gut-brain axis, synergistically inhibiting oxidative stress, combining SOD-LF (exogenous antioxidant) and fucoidan (endogenous pro-antioxidant), blocking the vicious cycle of inflammation-oxidation.

[0044] This composition enhances the blood-brain barrier permeability through the SOD-LF conjugate, combines with fucoidan to regulate the gut microbiota-gut-brain axis, and synergistically inhibits neuroinflammation and oxidative stress, thereby improving anxiety and insomnia.

[0045] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0050] As used in this invention, "room temperature" and "normal temperature" are both calculated as 25 ± 2 °C unless otherwise specified.

[0051] Unless otherwise specified, the raw materials or instruments used in the following examples of this invention are all obtained commercially.

[0052] Example 1

[0053] This example 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 a thiol reducing agent (TCEP), incubate at 36 °C for 1 h, then let stand at 4 °C for 40 min, and purify through a desalting column to obtain reduced lactoferrin;

[0055] Wherein 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 and then add sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester (Sulfo-SMCC), stir at 25 °C for 2 h, then add glycine to terminate the reaction, and finally purify through a desalting column to obtain activated SOD;

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

[0058] S3. Mix reduced lactoferrin with activated SOD in PBS solution, stir and react, then add cysteine to terminate the reaction, and finally obtain the SOD-LF covalent conjugate through purification using a Superdex 200 column;

[0059] Among them, the molar ratio of reduced lactoferrin to activated SOD is 1:2.

[0060] S4. Uniformly disperse the SOD-LF covalent conjugate in sodium alginate solution, then spray it into calcium chloride solution, let it stand, and filter to obtain sodium alginate-coated SOD-LF covalent conjugate with a particle size of 15 μm;

[0061] Among them, the ratio of the SOD-LF covalent conjugate to the sodium alginate solution is 1 g:20 mL;

[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. Disperse the sodium alginate-coated SOD-LF covalent conjugate and fucoidan in chitosan solution, add sodium tripolyphosphate, stir and react, and collect by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation.

[0065] Among them, the ratio of the sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution is 2 mg:2 mg:15 mL;

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

[0067] Example 2

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

[0069] S1. Dissolve lactoferrin in PBS solution, then add a thiol reducing agent (TCEP), incubate at 35 °C for 0.5 h, then stand at 2 °C for 30 min, and obtain reduced lactoferrin through purification using a desalting column;

[0070] Among them, 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-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide (Sulfo-SMCC), stir at 20 °C for 1 h, then add glycine to terminate the reaction, and finally purify through a 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, stirred, and the reaction was terminated by adding cysteine. 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 evenly dispersed in a sodium alginate solution and then sprayed into a calcium chloride solution, allowed to stand, and filtered to obtain a sodium alginate-coated SOD-LF covalent conjugate with a particle size of 10 μm;

[0076] The ratio of SOD-LF covalent conjugate to sodium alginate solution is 1 g: 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. The sodium alginate-coated SOD-LF covalent conjugate and fucoidan are dispersed in a chitosan solution, sodium tripolyphosphate is added, stirred for reaction, and collected by centrifugation to obtain a composition based on the synergistic effect of the SOD-LF covalent conjugate and neuromodulation.

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

[0081] The chitosan solution has a concentration of 2 mg / mL and a dosage of sodium tripolyphosphate of 0.1 wt %.

[0082] Example 3

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

[0084] S1. Lactoferrin was dissolved in PBS solution and then a thiol reducing agent (TCEP) was added. The mixture was incubated at 37°C for 1.5 h and then allowed to stand at 5°C for 50 min. Reduced lactoferrin was purified using a desalting column.

[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-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimide (Sulfo-SMCC), stir at 25 °C for 3 h, then add glycine to terminate the reaction, and finally purify through a desalting column to obtain activated SOD;

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

[0088] S3. Mix reduced lactoferrin and activated SOD in PBS solution, stir and react, then add cysteine to terminate the reaction, and then purify through a Superdex 200 column to obtain SOD-LF covalent conjugate;

[0089] Among them, the molar ratio of reduced lactoferrin to activated SOD is 1:3.

[0090] S4. Uniformly disperse the SOD-LF covalent conjugate in sodium alginate solution, then spray it into calcium chloride solution, let it stand and filter to obtain sodium alginate-coated SOD-LF covalent conjugate with a particle size of 20 μm;

[0091] Among them, the ratio of SOD-LF covalent conjugate to sodium alginate solution is 1 g:30 mL;

[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. Disperse sodium alginate-coated SOD-LF covalent conjugate and fucoidan in chitosan solution, add sodium tripolyphosphate, stir and react, and centrifuge to collect the composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation.

[0095] Among them, the ratio of sodium alginate-coated SOD-LF covalent conjugate, fucoidan, and chitosan solution is 3 mg:3 mg:20 mL;

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

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

[0098] Comparative Example 1

[0099] In this comparative example, SOD and LF are not coupled (the dosages are the same as in Example 1), and the specific preparation method is as follows:

[0100] Disperse SOD and LF evenly in the sodium alginate solution, then spray it into the calcium chloride solution, let it stand, and filter to obtain SOD-LF covalent conjugate coated with sodium alginate with a particle size of 15 μm;

[0101] Among them, the ratio of the total amount of SOD and LF to the sodium alginate solution is 1 g: 20 mL;

[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. Disperse SOD and LF coated with sodium alginate and fucoidan in the chitosan solution, add sodium tripolyphosphate, stir and react, and collect by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation.

[0105] Among them, the ratio of SOD-LF covalent conjugate coated with sodium alginate, fucoidan, and chitosan solution is 2 mg: 2 mg: 15 mL;

[0106] The concentration of the chitosan solution is 3 mg / mL, and the dosage of sodium tripolyphosphate is 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] This comparative example uses single-layer coating. The specific method is as follows:

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

[0112] Among them, 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-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide (Sulfo-SMCC), stir at 25 °C for 2 h, then add glycine to terminate the reaction, and finally purify through a desalting column to obtain activated SOD;

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

[0115] S3. Mix reduced lactoferrin and activated SOD in PBS solution, stir and react, then add cysteine to terminate the reaction. After purification using a Superdex 200 column, a SOD-LF covalent conjugate is obtained;

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

[0117] S4. Disperse the SOD-LF covalent conjugate and fucoidan in chitosan solution, add sodium tripolyphosphate, stir and react, and collect by centrifugation to obtain a composition based on the synergistic effect of the SOD-LF covalent conjugate and neuromodulation.

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

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

[0120] Experimental Example 1

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

[0122] Drug preparation: Prepare the compositions obtained in Examples 1 - 3 and Comparative Examples 1 - 3 into a 10 mg / mL medicinal solution with distilled water.

[0123] Gavage dose: Each group was gavaged with 25 mL / kg of the medicinal solution of the example or comparative example, and the blank group was gavaged with an equal amount of distilled water.

[0124] Experimental method: Each mouse was gavaged with the corresponding medicinal solution 2 times a day. After 7 consecutive days, a light-dark box test (50 cm * 50 cm, half covered is the dark box, and half uncovered is the light box) was carried out. After the mouse was placed in the open box, the measurement started. Each time, the timing was 10 min and repeated 3 times. Record the total time the mouse stayed in the light box.

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

[0126] Table 1

[0127] Group Average residence time in the light box / 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] Experimental Example 2

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

[0130] Forced swimming test: Mice were placed in a water bucket where they could not touch the bottom, twice a day for 5 minutes each time, 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 formulated with distilled water into a 10 mg / mL liquid medicine.

[0132] Gavage dose: Each group was gavaged with the liquid medicine of the example or comparative example at 25 mL / kg, and the blank group was gavaged with an equal volume of distilled water. Each mouse was gavaged with the corresponding liquid medicine 2 times. After 3 days, the forced swimming test was carried out to record the swimming time, as shown in Table 2.

[0133] Table 2

[0134]

[0135]

[0136] Experimental Example 3

[0137] Mouse insomnia experiment

[0138] Experimental animals: SPF - level healthy male BALB / c mice, weighing 15 - 20 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; 70 mice were selected. After one - week adaptation, they were randomly divided into 7 groups.

[0139] Intraperitoneal injection of 400 mg / kg PCPA according to the mouse body weight, with a dosage of 0.1 mL / 10 g, for 3 consecutive days. When the mice showed disappearance of circadian rhythm, strong aggressiveness, messy hair, and weight loss, it indicated that the model was successfully established.

[0140] Drug preparation: The compositions obtained in Examples 1 - 3 and Comparative Examples 1 - 3 were formulated with distilled water into a 10 mg / mL liquid medicine.

[0141] Gavage dose: Each group was gavaged with the liquid medicine of the example or comparative example at 25 mL / kg, and the blank group was gavaged with an equal volume of distilled water. After continuous administration for 3 days, the sleep - onset latency was statistically analyzed, as shown in 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 test examples, the composition prepared by the present invention has an obvious effect of relieving anxiety and insomnia and improving sleep quality. Comparative Example 1 shows that the coupling of SOD and LF can significantly improve the effect of SOD; Comparative Examples 2-3 show that the combined effect of the SOD-LF conjugate and fucoidan is significantly improved. In Comparative Example 3, since the SOD-LF conjugate and fucoidan are released simultaneously, the SOD-LF conjugate is absorbed before fucoidan takes effect, making it difficult to truly achieve the simultaneous effect.

[0145] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation, characterized in that, It includes the following preparation steps: S1. Dissolve lactoferrin in PBS solution, then add TCEP, and incubate, stand, and purify in sequence to obtain reduced lactoferrin; S2. Dissolve SOD in PBS solution, then add Sulfo-SMCC for stirring reaction, then add glycine to terminate the reaction, and finally purify to obtain activated SOD; S3. Mix reduced lactoferrin and activated SOD in PBS solution, carry out stirring reaction, then add cysteine to terminate the reaction, and then purify to obtain SOD-LF covalent conjugate; S4. Uniformly disperse the SOD-LF covalent conjugate in sodium alginate solution, then spray it into calcium chloride solution, stand and filter to obtain sodium alginate-coated SOD-LF covalent conjugate; S5. Disperse the sodium alginate-coated SOD-LF covalent conjugate and fucoidan in chitosan solution, add sodium tripolyphosphate for stirring reaction, and collect by centrifugation to obtain a composition based on the synergistic effect of SOD-LF covalent conjugate and neuromodulation.

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

3. The composition according to claim 1, characterized in that, In S2, the molar ratio of SOD to Sulfo-SMCC is (10-20):1; The ratio of SOD to PBS solution is (4-8) mg / mL.

4. The composition according to claim 1, wherein In S2, the temperature of the stirring reaction is 20-25 °C and the time is 1-3 h.

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

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

8. The composition according to claim 1, characterized in that, 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 according to claim 1, wherein In S5, the dosage of sodium tripolyphosphate is 0.1-0.3 wt%.

10. Use of the composition according to any one of claims 1-9 in the preparation of a drug for relieving anxiety and insomnia and improving sleep quality.

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