A composition for improving depression, its preparation method and application

Abstract

The present invention discloses a composition for improving depression, its preparation method and application, belonging to the technical field of biomedicine. The composition is a liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 in a phospholipid bilayer; the mass ratio of palmitoyl tripeptide-8 to EUK-134 is (1-3):(1-3). Palmitoyl tripeptide-8 is added to the stirred phospholipid mixture and mixed evenly to obtain mixture A; EUK-134 is added to water and mixed evenly to obtain solution B; then solution B is slowly injected into mixture A under stirring, and after the injection is completed, stirring is continued, and then high-pressure homogenization treatment is carried out to obtain a liposome suspension, that is, the composition for improving depression. The liposome can be added with medical excipients to be made into nasal drops, sprays, gels, creams, powders or powder aerosols. After nasal absorption, it can effectively improve chronic depression.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a composition for improving depression, a preparation method thereof, and an application thereof. Background Art

[0002] Depression, also known as depressive disorder, is a common emotion-related mental illness in clinical practice. Its representative clinical manifestations include low mood, pessimism, cognitive and sleep disorders. Patients often fall into melancholy, uncontrollable self-reflection, and negative thinking about the past, present, and future. Depressed patients often have poor physiological functions, a high recurrence risk, and even a very high suicide rate.

[0003] At present, the clinical treatment of depression by Western medicine mainly focuses on oral antidepressant drugs or psychotherapy. There are many antidepressant drugs in clinical practice, such as tricyclic and tetracyclic antidepressants, monoamine oxidase inhibitors, selective serotonin (5-HT) reuptake inhibitors, etc. However, only 1 / 3 of the patients show obvious improvement in the first-line clinical treatment, and there is a latency period of several weeks to several months for the drug to take effect. The curative effect lasts for a short time, is easy to relapse, and has certain adverse reactions. Therefore, a product that is convenient to use, safe, and can effectively improve depression is needed. Summary of the Invention

[0004] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a composition for improving depression, a preparation method thereof, and an application thereof, which are convenient to use, safe, and can effectively improve depression.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A composition for improving depression provided by the present invention, wherein the composition is a liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 in a phospholipid bilayer;

[0007] Among them, the mass ratio of palmitoyl tripeptide-8 to EUK-134 is (1-3):(1-3).

[0008] Preferably, the mass ratio of palmitoyl tripeptide-8 to EUK-134 is 1:2.

[0009] Preferably, medical adjuvants are added to the liposome to prepare nasal drops, sprays, gels, creams, powders, or powder aerosols.

[0010] The present invention also discloses a preparation method of the above-mentioned composition for improving depression. Palmitoyl tripeptide-8 is added to the phospholipid mixture under stirring to be uniformly mixed to obtain mixture A; EUK-134 is added to water and uniformly mixed to obtain solution B; then solution B is injected into mixture A under stirring, and stirring is continued, followed by high-pressure homogenization treatment to obtain a liposome suspension.

[0011] Preferably, the phospholipid mixture is Pro-Lipo TM Neo.

[0012] Preferably, the specific steps for slowly injecting solution B into mixture A under stirring are as follows: Solution B is slowly injected into mixture A with a stirring speed of 800-1000 rpm at a rate of 1-4% V / min.

[0013] Preferably, after the injection of solution B is completed, stirring is continued for 20 min.

[0014] Preferably, based on the obtained liposome suspension, 10% of D-mannitol is added, and after uniform mixing, freeze-drying is carried out to obtain liposome lyophilized powder.

[0015] The present invention also discloses the application of the above-mentioned composition for improving depression in the preparation of a drug for improving depression.

[0016] Preferably, the administration route of the drug is nasal administration.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] A composition for improving depression provided by the present invention, palmitoyl tripeptide-8 and EUK-134 can fundamentally play a long-term role in improving depression by scavenging free radicals, anti-inflammation, nourishing brain nerves, and inhibiting apoptosis of brain nerves; encapsulating the composition into liposomes and administrating through nasal cavity can effectively reduce the irritation and toxicity of the drug to the nasal cavity. The liposome itself can increase the absorption rate of the drug, and the phospholipids in it can nourish nerves, activate nerve cells in the human body, improve brain function, and increase the speed and accuracy of information transmission between nerve cells. Through the toxicological experiment verification of the prepared composition, there is no significant difference compared with the Control group, and there is no obvious toxic effect on L929 cells, indicating that the composition has good biocompatibility. Through the animal experiment verification of the prepared composition, the sucrose preference test, tail suspension test, forced swimming test, and inflammatory factor detection test all prove that there is a significant difference between the administration group and the Model group, and the depressive symptoms of mice are alleviated. When the composition is used, it is absorbed through the nasal cavity. Palmitoyl tripeptide-8 and EUK-134 are absorbed through the nasal mucosa, and can directly enter the skull through the olfactory nerve pathway and trigeminal nerve pathway, targeting the brain, avoiding the first-pass effect of the liver and the blood-brain barrier, so as to quickly take effect and improve the utilization rate of the active ingredients. Therefore, the composition is not only safe and effective, convenient to use, but also can quickly take effect, and can be applied to the improvement of depression. Description of the Drawings

[0019] Figure 1 It is a toxicity diagram of liposomes with different concentrations of the present invention on L929 cells;

[0020] Figure 2 It is an influence diagram of the freeze-dried liposomes of the present invention on the depressive-like behavior of mice; among them, A is the sucrose preference test, B is the tail suspension test, and C is the forced swimming test; compared with the Model group, ** indicates P < 0.01, and when comparing the three groups of LDG, MDG, and HDG, # indicates P < 0.05, ## indicates P < 0.01;

[0021] Figure 3 It is an influence diagram of the freeze-dried liposomes of the present invention on the levels of inflammatory factors in the hippocampus of mice; among them, A is the influence on IL-1β; B is the influence on TNF-α; C is the influence on IL-10; compared with the Model, ** indicates P < 0.01, and when comparing the three groups of LDG, MDG, and HDG, # indicates P < 0.05, ## indicates P < 0.01. Detailed Embodiments

[0022] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] It should be noted that the terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not necessarily limit to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0025] In the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained in the market unless otherwise specified; Palmitoyl Tripeptide-8 is purchased from Shaanxi Haosen Biotechnology Co., Ltd., with a purity of ≥98%; EUK-134 is purchased from Pure Biotechnology Co., Ltd., with a purity of >98%; the phospholipid mixture is Pro-Lipo TM Neo.

[0026] Example 1

[0027] A composition for improving depression, which is a liposome obtained by embedding Palmitoyl Tripeptide-8 and EUK-134 (Ethylbisiminomethylguaiacol Manganese Chloride) in a phospholipid bilayer. The mass ratio of Palmitoyl Tripeptide-8 to EUK-134 is 1:2.

[0028] 1. Preparation of the composition for improving depression

[0029] The specific preparation method is as follows: First, 0.5 part of palmitoyl tripeptide-8 is added to 10 parts of phospholipid mixture under stirring to make it evenly mixed, obtaining mixture A. 1 part of EUK-134 is added to 188.5 parts of water to make it evenly mixed to obtain solution B. Then, solution B is slowly injected into mixture A under stirring. The stirring speed of mixture A is 800 - 1000 rpm / min, and the injection speed is 1 - 4% V / min. After the injection is completed, stirring continues for 20 min, and then high-pressure homogenization treatment is carried out to obtain a liposome suspension (i.e., the composition for improving depression). In the liposome suspension, the final mass concentration of phospholipid is 5%, the final concentration of palmitoyl tripeptide-8 is 0.25%, and the final concentration of EUK-134 is 0.5%.

[0030] 2. Performance evaluation of the prepared composition for improving depression

[0031] 1) Cell experiment

[0032] Accurately pipette the above-prepared liposome suspension, and dilute it 10 times, 20 times, and 50 times respectively with DMEM medium to obtain HDG (high dose), MDG (medium dose), and LDG (low dose) suspensions for use.

[0033] Resuscitate mouse fibroblasts (L929) frozen in a liquid nitrogen tank, and culture them in DMEM medium supplemented with 1% double antibody and 10% fetal bovine serum under the conditions of 37 °C, 95% humidity, and 5% CO2 for a period of time. After the cells adhere and grow well, inoculate L929 cells into a 96-well plate at a density of 1.2×10 4 / well, and set up a Blank group (only medium is added to the well, without cells and liposome suspension), a Control group (only cells and medium are added to the well, without liposome suspension), and LDG, MDG, and HDG experimental groups. Each group has 5 parallel samples.

[0034] After culturing for 24 h, aspirate the medium in the well and re-add 200 μL of liposome suspension with the corresponding concentration gradient. The Blank group and the Control group are not treated.

[0035] After the cells are cultured for another 24 h, add 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) to each well. After incubating for 4 h, carefully aspirate the medium in the well. Add 200 μL of DMSO to each well, gently shake the 96-well plate, and measure the OD value at a wavelength of 490 nm using an enzyme-labeling instrument. And calculate the cell survival rate using the formula:

[0036]

[0037] Result analysis:

[0038] AsFigure 1 As shown in the figure, compared with the Control group, there was no significant difference between the three experimental groups LDG, MDG, HDG and the Control group, indicating that low, medium and high liposome concentrations had no obvious toxic effect on L929 cells and the liposomes had good biocompatibility.

[0039] 2) Animal experiments

[0040] a. Experimental animals

[0041] Thirty healthy male KM mice, weighing (18 ± 2) g, were provided by the Experimental Animal Center of the Air Force Medical University. They were housed at a constant temperature of (24 ± 2)°C and a relative humidity of 40% to 70% with free access to food and water. After one week of acclimatization, they were randomly divided into five groups: LDG, MDG, HDG, Model, and Control, with six animals in each group.

[0042] b. Materials

[0043] ELISA kits for IL-1, TNF-α, IL-10, etc. were provided by Wuhan Huamei Bioengineering Co., Ltd.

[0044] c. Establishment of a chronic depression model in mice

[0045] The control group of six mice was group-housed in a cage, without any external stimulation, and had free access to food and water. The remaining groups of mice were housed individually and given different daily stimuli, with the goal of ensuring that the stimuli varied as much as possible. The stressors included 5 minutes of horizontal shaking, 24 hours of food and water deprivation, 5 minutes of tail clamping, 5 minutes of swimming in cold water at 4°C, 5 minutes of heating in a 45°C oven, 12 hours of day-night reversal, and 24 hours of damp bedding. Each group of mice received a randomly assigned stimulus each day. No group of mice could receive the same stimulus consecutively, and the same stimulus was applied a maximum of three times.

[0046] d. Test methods

[0047] 10% D-mannitol was added to the liposome suspension prepared above, mixed evenly and freeze-dried to obtain liposome freeze-dried powder. The prepared liposome freeze-dried powder was reconstituted with 100 mL of PBS to obtain a reconstituted stock solution, which was used for nasal instillation in the HDG group mice. The reconstituted stock solution was diluted 5 times and 20 times respectively and used for nasal instillation in the MDG group and LDG group mice, respectively. PBS was instilled into the control group and the model group mice through the nose. The 5 groups of mice were given nasal administration once a day, 20 μL each time, for 21 consecutive days. At the same time as the administration, stimulation was performed. Except for the control group, which did not require stimulation, the mice in other groups were stimulated.

[0048] Sucrose preference test (SPT):

[0049] Before the test, each group of mice was allowed to adapt to drinking 1% sucrose solution for 48 h to minimize fear of new things and preference for the drinking location. The position of the water bottle was changed daily. During the test, the mice were first deprived of water for 24 h and then given two bottles of water of equal volume, A (tap water) and B (1% sucrose solution). The sucrose preference was calculated based on the sucrose intake and tap water intake of the mice during a 5-h period. (Sucrose preference % = [B / (A + B)] × 100%).

[0050] Tail Suspension Test (TST):

[0051] The distal 1 / 3 of the mouse's tail was fixed with tape 50 cm above the ground. The total immobile time of the mouse within 5 min in this environment was measured by a trained blinded observer.

[0052] Forced Swimming Test (FST):

[0053] This experimental method is a behavioral despair experimental method used to evaluate the antidepressant effect of drugs in animal models. The experiment was completed in two days. On the first day, the mice were placed in a cylindrical bucket with a height of 50 cm and a diameter of 20 cm for 10 min. The bucket was filled with water (temperature 25 ± 1°C). On the second day, the mice were placed in the cylindrical bucket for 5 min. The immobile time of the mice within 5 min was recorded by an experimenter who was not familiar with this test design.

[0054] Detection of inflammatory factors:

[0055] After sacrificing the mice, the hippocampal tissue was homogenized (10% w / v) with ice-cold phosphate buffer solution (PBS, pH 7.4), centrifuged (12,000 rpm, 4°C, 20 min), and the supernatant was collected. The contents of IL-1β, TNF-α, and IL-10 in the hippocampal specimens were detected using an ELISA kit, and the specific operation steps were carried out strictly according to the instructions of the kit.

[0056] e. Result analysis

[0057] As Figure 2 shown Figure 2In the sucrose preference test of A, compared with the Control group, the sucrose intake in the Model group was significantly reduced, indicating that the chronic depression model was successfully established. Compared with the Model group, there were significant differences in LDG, MDG, and HDG, indicating that these three groups could all improve the reduction of sucrose intake in the model group, that is, they could improve the depressive behavior of mice. At the same time, when comparing LDG, MDG, and HDG with each other, the sucrose intake in the MDG group was significantly higher than that in the HDG group, indicating that the effect of MDG in improving depression was better than that of HDG. Figure 2 In the tail suspension test of B, compared with the Control group, the immobility time of mice in the tail suspension test in the Model group was significantly increased, indicating that the chronic depression model was successfully established. Compared with the Model group, the immobility time of mice in LDG, MDG, and HDG was significantly reduced, indicating that these three groups could all improve the depressive behavior of mice. At the same time, there was no significant difference when comparing LDG, MDG, and HDG with each other. Figure 2 In the forced swimming test of C, compared with the Control group, the immobility time of mice in the forced swimming test in the Model group was significantly increased, indicating that the chronic depression model was successfully established. Compared with the Model group, the immobility time during swimming in LDG, MDG, and HDG was significantly reduced, indicating that these three groups could all improve the depressive condition of mice. At the same time, when comparing LDG, MDG, and HDG with each other, compared with LDG and HDG, the immobility time of mice in the forced swimming test in MDG was reduced, with a significant difference. This indicates that in the forced swimming test, compared with LDG and HDG, MDG could significantly reduce the immobility time of mice, and it also shows that the MDG dose group had a better effect in reducing the immobility time of mice in the forced swimming test.

[0058] As Figure 3 shown, Figure 3 In A, in the effect on IL-1β, there was a significant difference between the Model group and the Control group, and the inflammatory factor IL-1β was significantly increased. Compared with the Model group, the content of IL-1β in LDG, MDG, and HDG was significantly reduced, and there were significant differences, indicating that these three groups could all reduce the content of IL-1β in the hippocampal tissue of the model group. At the same time, when comparing LDG, MDG, and HDG with each other, there was no significant difference. Figure 3 In B, the results of the effect on TNF-α showed that compared with the Control group, the content of the inflammatory factor TNF-α in the Model group was significantly increased. Compared with the Model group, it was significantly reduced in LDG, MDG, and HDG, indicating that these three groups could all reduce the content of the inflammatory factor TNF-α in the hippocampal tissue of mice; at the same time, when comparing LDG, MDG, and HDG with each other, there was no significant difference. Figure 3In C, the impact on IL-10 showed that there were significant differences between the Model group and the Control group, and the anti-inflammatory factor IL-10 in the Model group was significantly decreased. Compared with the Model group, there were significant differences in the LDG, MDG, and HDG groups, indicating that all three groups could increase the content of the anti-inflammatory factor IL-10 in the hippocampal tissue of mice. At the same time, when comparing the three groups of LDG, MDG, and HDG with each other, compared with the LDG and HDG groups, the MDG group could significantly increase the content of the anti-inflammatory factor IL-10 in the hippocampal tissue of mice, which also indicated that the MDG dose group had a stronger effect on increasing the content of the anti-inflammatory factor IL-10 compared with the LDG and MDG groups.

[0059] The above results showed that the liposomes encapsulating palmitoyl tripeptide-8 and EUK-134, after nasal absorption, could significantly increase the sucrose intake of chronically depressed mice and reduce the immobility time in the tail suspension test and forced swimming test, that is, improve the depressive behavior of chronically depressed mice. Among the three doses, the MDG dose group had a better effect on improving depression than the LDG and HDG groups. Further analyzing the reasons for the improvement of depression by liposomes, it was found that compared with the Model group, the inflammatory factors IL-1β and TNF-α in the hippocampal tissue of mice in the experimental groups with different doses were significantly decreased, and the content of the anti-inflammatory factor IL-10 was significantly increased, and the content of IL-10 in the MDG group was higher than that in the LDG and HDG groups. It indicated that the liposomes of palmitoyl tripeptide-8 and EUK-134 exerted antidepressant effects by reducing the content of the inflammatory factors IL-1β and TNF-α and increasing the content of the anti-inflammatory factor IL-10.

[0060] Example 2

[0061] A composition for improving depression is a liposome obtained by encapsulating palmitoyl tripeptide-8 and EUK-134 (ethyl bisiminomethyl guaiacol manganous chloride) with a phospholipid bilayer. The mass fraction ratio of palmitoyl tripeptide-8 and EUK-134 is 1:3.

[0062] The specific preparation method is as follows:

[0063] First, 0.5 parts of palmitoyl tripeptide-8 was added to 10 parts of phospholipid mixture under stirring to make it evenly mixed to obtain mixture A. 1.5 parts of EUK-134 was added to 188 parts of water and evenly mixed to obtain solution B. Then, solution B was slowly injected into mixture A under stirring, the stirring speed of mixture A was 800 - 1000 rpm / min, the injection speed was 1 - 4% V / min, and after the injection was completed, stirring continued for 20 min, and then high-pressure homogenization treatment was carried out to obtain a liposome suspension (i.e., the composition for improving depression). The final mass concentration of phospholipids in the liposome suspension was 5%, the final concentration of palmitoyl tripeptide-8 was 0.25%, and the final concentration of EUK-134 was 0.75%.

[0064] Example 3

[0065] A composition for improving depression, which is a liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 (ethyl bisiminomethyl guaiacol manganese chloride) in a phospholipid bilayer. The mass fraction ratio of palmitoyl tripeptide-8 to EUK-134 is 3:1.

[0066] The specific preparation method is as follows:

[0067] First, 1.5 parts of palmitoyl tripeptide-8 are added to 40 parts of phospholipid mixture under stirring to make it uniformly mixed, obtaining mixture A. 0.5 part of EUK-134 is added to 958 parts of water to make it uniformly mixed to obtain solution B. Then, solution B is slowly injected into mixture A under stirring. The stirring speed of mixture A is 800-1000 rpm / min, and the injection speed is 1-4% V / min. After the injection is completed, stirring continues for 20 min, and then high-pressure homogenization treatment is carried out to obtain a liposome suspension (i.e., the composition for improving depression). The final mass concentration of phospholipids in the liposome suspension is 4.0%, the final concentration of palmitoyl tripeptide-8 is 0.15%, and the final concentration of EUK-134 is 0.05%.

[0068] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A composition for improving depression, characterized in that, The composition is Pro-Lipo TM Liposomes obtained by Neo encapsulating palmitoyl tripeptide-8 and EUK-134; Among them, the mass ratio of palmitoyl tripeptide-8 to EUK-134 is (1~3):(1~3).

2. The composition for improving depression according to claim 1, wherein The mass ratio of palmitoyl tripeptide-8 to EUK-134 is 1:

2.

3. The composition for improving depression according to claim 1, wherein Medical excipients are added to the liposomes to prepare nasal drops, sprays, gels, creams, powders or powder aerosols.

4. A method for preparing a composition for improving depression according to any one of claims 1 to 3, characterized in that, Add palmitoyl tripeptide-8 to Pro-Lipo under stirring TM Neo and mix well to obtain mixture A; add EUK-134 to water and mix well to obtain solution B; then inject solution B into mixture A under stirring, continue stirring, and then perform high-pressure homogenization treatment to prepare a liposome suspension.

5. The preparation method of a composition for improving depression according to claim 4, characterized in that, The specific steps for injecting the solution B into the mixture A under stirring are as follows: The solution B is slowly injected into the mixture A with a stirring speed of 800~1000 rpm / min at a rate of 1~4% V / min.

6. The preparation method of a composition for improving depression according to claim 4, characterized in that, After the injection of the solution B is completed, stirring is continued for 20 min.

7. The preparation method of a composition for improving depression according to claim 4, characterized in that, On the basis of the prepared liposome suspension, 10% of D-mannitol is added, and after mixing evenly, freeze-drying is carried out to obtain liposome lyophilized powder.

8. Use of a composition for improving depression according to any one of claims 1~3 in the preparation of a drug for improving depression.

9. The application according to claim 8, wherein The administration route of the drug is nasal administration.

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