CN115120701A

CN115120701A - Depression improving composition and preparation method and application thereof

Info

Publication number: CN115120701A
Application number: CN202210870973.5A
Authority: CN
Inventors: 张丹; 明磊国; 董玲娟; 王清霞
Original assignee: Shaanxi Zhonghong Kerui Institute Of Regenerative Medicine Co ltd
Current assignee: Shaanxi Zhonghong Kerui Institute Of Regenerative Medicine Co ltd
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2022-09-30

Abstract

The invention discloses a composition for improving depression, a preparation method and application thereof, and belongs to the technical field of biological medicines. The composition is liposome obtained after the phospholipid bilayer is embedded with the palmitoyl tripeptide-8 and EUK-134; wherein the mass part ratio of the palmitoyl tripeptide-8 to the palmitoyl tripeptide EUK-134 is (1-3) to (1-3). Adding palmitoyl tripeptide-8 into a phospholipid mixture in a stirring state, and uniformly mixing to obtain a mixture A; EUK-134 is added into water to be mixed evenly to obtain solution B; and slowly injecting the solution B into the mixture A under the stirring state, continuously stirring after the injection is finished, and then carrying out high-pressure homogenization treatment to prepare liposome suspension, namely the composition for improving depression. The liposome can be added with medical adjuvants to make into nasal drop, spray, gel, cream, powder or powder, and can be absorbed by nose to effectively improve chronic depression.

Description

Depression improving composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a composition for improving depression as well as a preparation method and application thereof.

Background

Depression is also called depressive disorder, and is a common mental disease related to emotion in clinic, and the typical clinical manifestations of depression are emotional depression, pessimistic boredom, cognitive and sleep disorders, and patients often fall into melancholy, uncontrollable self-meditation and negative thinking in the past, present and future. Patients with depression often have poor physiological function, high risk of relapse, and even have extremely high suicide rate.

At present, the treatment of depression in western medicine is mainly based on oral antidepressant drugs or psychotherapy in clinic. There are many drugs for treating depression in clinic, such as tricyclic and tetracyclic antidepressants, monoamine oxidase inhibitors, selective 5-hydroxytryptamine (5-HT) reuptake inhibitors, etc. However, the first-line clinical treatment only shows obvious improvement effect on 1/3 patients, and the drug has a latent period of several weeks to several months in the onset effect, short duration of curative effect, easy relapse and certain adverse reaction. Therefore, a product which is convenient to use, safe and effective in relieving depression is needed.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a composition for improving depression, a preparation method and application thereof, which are convenient to use, safe and capable of effectively improving depression.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the composition for improving depression is a liposome obtained after palmitoyl tripeptide-8 and EUK-134 are embedded in phospholipid bilayers;

wherein the mass part ratio of the palmitoyl tripeptide-8 to the palmitoyl tripeptide EUK-134 is (1-3) to (1-3).

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

Preferably, the liposome is added with medical auxiliary materials to be prepared into nasal drops, spray, gel, cream, powder or powder spray.

The invention also discloses a preparation method of the composition for improving depression, which comprises the steps of adding palmitoyl tripeptide-8 into a phospholipid mixture in a stirring state, and uniformly mixing to obtain a mixture A; EUK-134 is added into water and evenly mixed to obtain solution B; and injecting the solution B into the mixture A under the stirring state, continuing stirring, and then carrying out high-pressure homogenization treatment to obtain liposome suspension.

Preferably, the phospholipid mixture is Pro-Lipo ^TM Neo。

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

Preferably, after the solution B is injected, stirring is continued for 20 min.

Preferably, on the basis of preparing the liposome suspension, 10% of D-mannitol is added, and freeze-drying is carried out after uniform mixing, so as to obtain the liposome freeze-dried powder.

The invention also discloses application of the composition for improving depression in preparation of a medicament for improving depression.

Preferably, the route of administration of the medicament is nasal administration.

Compared with the prior art, the invention has the following beneficial effects:

the palmitoyl tripeptide-8 and EUK-134 can play a role in fundamentally improving depression for a long time by removing free radicals, resisting inflammation, nourishing cranial nerves and inhibiting cranial nerve apoptosis; the composition is encapsulated into liposome and administered through nasal cavity, so that irritation and toxicity of the composition to the nasal cavity can be effectively reduced. The liposome can increase the absorption rate of the medicine, and the phospholipid can nourish nerves, activate nerve cells of a human body, improve brain functions, and improve the information transmission speed and accuracy among nerve cells. Toxicological experiments prove that compared with a Control group, the administration group has no significant difference and has no obvious toxic effect on L929 cells, which indicates that the composition has good biocompatibility. Animal experiments prove that compared with a Model group, the administration group has significant difference and the depression symptom of the mice is relieved. The composition is absorbed through nose when in use, and the palmitoyl tripeptide-8 and EUK-134 are absorbed through nasal mucosa, can be directly connected to the skull through olfactory nerve pathway and trigeminal nerve pathway, targets the brain, avoids the first pass effect and blood brain barrier of the liver, and thus can quickly take effect and improve the utilization rate of effective components. Therefore, the composition is safe and effective, is convenient to use, can quickly take effect, and can be applied to the improvement of depression.

Drawings

FIG. 1 is a graph of toxicity of liposomes of the invention at different concentrations on L929 cells;

FIG. 2 is a graph of the effect of the liposome lyophilized powder of the present invention on mouse depressive-like behavior; wherein, A is a sweet water preference experiment, B is a tail suspension experiment, and C is a forced swimming experiment; relative to Model groups, the values indicate that P is less than 0.01, three groups of LDG, MDG and HDG are compared with each other, the value # indicates that P is less than 0.05, and the value # indicates that P is less than 0.01;

FIG. 3 is a graph showing the effect of the lyophilized liposome powder of the present invention on the level of mouse hippocampal inflammatory factor; wherein A is the effect on IL-1 beta; b is the effect on TNF-alpha; c is the effect on IL-10; relative to the Model, # indicates P < 0.01, and three groups of LDG, MDG, and HDG are compared with each other, # indicates P < 0.05, and # indicates P < 0.01.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, if not otherwise specified, are commercially available; palmitoyl tripeptide-8 is purchased from Shanxi Hao Sen Biotech limited, and the purity is more than or equal to 98 percent; EUK-134 is purchased from Purui biological medicine technology GmbH, purity is more than 98%; the phospholipid mixture was Pro-Lipo available from Sage Chemical (Group) Inc ^TM Neo。

Example 1

A composition for improving depression is liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 (manganese chloride ethyl bisiminomethyl guaiacol) in phospholipid bilayer. Wherein the mass part ratio of the palmitoyl tripeptide-8 to the palmitoyl tripeptide EUK-134 is 1: 2.

1. Preparation of composition for improving depression

The preparation method comprises the following steps: first, 0.5 part of palmitoyl tripeptide-8 was added to 10 parts of the phospholipid mixture under stirring to mix them uniformly, thereby obtaining a mixture a. 1 part of EUK-134 was added to 188.5 parts of water and mixed well to obtain solution B. And slowly injecting the solution B into the mixture A under the stirring state, wherein the stirring speed of the mixture A is 800-1000 rpm/min, the injection speed is 1-4% V/min, stirring is continued for 20min after injection is finished, and then high-pressure homogenization treatment is carried out to obtain the liposome suspension (namely the composition for improving depression). The final mass concentration of phospholipid in the liposome suspension is 5%, the final concentration of palmitoyl tripeptide-8 is 0.25%, and the final concentration of EUK-134 is 0.5%.

2. Evaluating the performance of the prepared composition for improving depression

1) Cell assay

Accurately sucking the liposome suspension prepared above, and diluting with DMEM medium by 10 times, 20 times and 50 times to obtain HDG (high dose), MDG (medium dose) and LDG (low dose) suspensions for use.

The mouse fibroblast cells (L929) frozen in liquid nitrogen tank were revived in DMEM medium supplemented with 1% double antibody and 10% fetal bovine serum at 37 deg.C, 95% humidity and 5% CO ₂ For a period of time. After the cells grew well adherent, the L929 cells were treated according to 1.2 x 10 ⁴ Density per well was seeded in 96-well plates and Blank (medium only, no cell and liposome suspension in wells), Control (cell and medium only, no liposome suspension in wells) and LDG, MDG and HDG experimental groups were set up with 5 replicates each.

After 24h incubation, the medium was aspirated from the wells and 200 μ L of the corresponding concentration gradient liposome suspension was added again. Blank and Control groups do nothing.

After 24h of further cell culture, 20. mu.L of MTT solution (5mg/mL, i.e., 0.5% MTT) was added to each well and after 4h of incubation, the medium was carefully aspirated from the wells. 200uL of DMSO was added to each well, the 96-well plate was gently shaken, and the OD value was measured at a wavelength of 490nm using a microplate reader. And calculating cell viability using the formula:

and (4) analyzing results:

as shown in fig. 1, compared with the Control group, there is no significant difference between the three experimental groups LDG, MDG, HDG and Control, which indicates that the low, medium and high liposome concentrations have no significant toxic effect on L929 cells, and the liposome has good biocompatibility.

2) Animal experiments

a. Laboratory animal

Healthy KM mice, 30 males, with a body mass (18. + -.2) g, were provided by the laboratory animal center of the university of air force military medical sciences. Before the experiment, the water is freely drunk under the conditions of constant temperature (24 +/-2) DEG C and relative humidity of 40-70 percent. After being adapted to feeding for one week, the animals were randomly divided into 5 groups, i.e., LDG group, MDG group, HDG group, Model group and Control group, and each group had 6 animals.

b. Material

ELISA kits for IL-1, TNF-alpha, IL-10 and the like are provided by Wuhan Huamei bioengineering Co., Ltd.

c. Establishing mouse chronic depression model

Control group 6 was maintained in one cage group without any external stimulus and was free to drink water for ingestion. The rest groups of mice are raised in an isolated culture mode, different stimulations are given every day, the stimulation is received, the contrast is larger as much as possible, and the stress sources comprise 7 types of horizontal shaking for 5min, fasting and water deprivation for 24h, tail clamping for 5min, cold water swimming for 5min at 4 ℃, oven heat drying for 5min at 45 ℃, day and night reversing for 12h and wet padding for 24 h. The groups of mice receiving the stimulation received one stimulation on a random schedule per day, one group of mice failed to receive the same stimulation continuously, and the same stimulation was used up to 3 times.

d. Test method

And adding 10% of D-mannitol into the prepared liposome suspension, uniformly mixing, and freeze-drying to obtain the liposome freeze-dried powder. The prepared liposome freeze-dried powder is redissolved by 100mL PBS to obtain redissolved stock solution which is used for intranasal instillation of HDG mice. The reconstituted stock solutions were diluted 5-fold and 20-fold respectively for intranasal instillation in mice in the MDG group and LDG group, respectively. Control and Model mice were instilled with PBS nasally. The 5 groups of mice were nasally administered 1 time a day, 20 μ L each time, for 21 days. Stimulation was performed simultaneously with the administration, and the mice in each group were stimulated except for the Control group, which did not require stimulation.

Sugar water preference test (SPT):

before testing, 1% sucrose water was adaptively drunk by each group of mice for 48h, so that fear of new things and preference of drinking water position are avoided as much as possible, and the position of a drinking water bottle needs to be changed once a day. During testing, the mouse is forbidden to be watered for 24 hours, two bottles of water (A bottle of water is tap water, and B bottle of water is 1% sucrose water) with the same volume are added, and the sugar water preference degree is calculated according to the sugar water intake and tap water intake of the mouse in a period of 5 hours. (sugar water preference [ ([ B/(a + B) ] × 100%).

Tail overhang experiments (TailSuspensionTest, TST):

the tail 1/3 of the mouse was taped to a distance of 50cm from the ground and suspended, and the sum of the immobility times of the mouse over 5min in this environment was measured by a trained blinded design observer.

Forced swimming test (forcedstimingtest, FST):

the experimental method is a behavior despair experimental method and is used for evaluating an animal model with the antidepressant effect of the medicine. The experiment was completed within two days. The first day: mice were placed in a cylindrical bucket 50cm high and 20cm in diameter for 10min, wherein the bucket was filled with water (temperature 25. + -. 1 ℃ C.). The next day: the mice were placed in a cylindrical bucket for 5 min. Immobility time within 5min was recorded for mice by an experimenter unfamiliar with the design of the trial.

Detection of inflammatory factors:

after sacrifice, the hippocampal tissue was homogenized (10% w/v) with ice-cold Phosphate buffer solution (PBS, pH 7.4), centrifuged (12,000rpm, 4 ℃, 20min), the supernatant was collected, and the contents of IL-1 β, TNF- α and IL-10 in hippocampal specimens were determined using an ELISA kit, using the exact procedures described in the kit.

e. Analysis of results

As shown in FIG. 2, in the sugar water preference experiment of FIG. 2A, sugar water intake was significantly reduced in the Model group compared to the Control group, indicating successful modeling of the chronic depression Model. Compared with the Model group, the LDG, MDG and HDG all have significant differences, which shows that the three groups can improve the condition that the sugar water intake of the Model group is reduced, namely the depression behavior of the mice. Meanwhile, the LDG group, the MDG group and the HDG group are compared with each other, and the intake of the sugar water in the MDG group is obviously higher than that in the HDG group, so that the effect of improving depression in the MDG group is better than that in the HDG group. In the tail suspension experiment of fig. 2B, compared with the Control group, the Model group showed a significant increase in the immobility time of the mice in the tail suspension experiment, indicating the success of modeling the chronic depression Model. Compared with the Model group, the mice have significantly reduced immobility time of LDG, MDG and HDG, which shows that the three groups can improve the depression behavior of the mice. Meanwhile, the LDG, MDG and HDG groups have no significant difference in comparison. In the forced swimming experiment of fig. 2C, compared with the Control group, the Model group significantly increases the immobility time of the mouse in the forced swimming experiment, which indicates that the Model of the chronic depression Model is successfully modeled. Compared with the Model group, the immobility time in swimming is obviously reduced in the LDG, MDG and HDG, which shows that the three groups can improve the depression condition of the mice. Meanwhile, three groups of LDG, MDG and HDG are compared with each other, and compared with the LDG and the HDG, the MDG has the advantages that the immobility time in a forced swimming experiment of the mouse is reduced, and the obvious difference is achieved. The MDG can obviously reduce the immobility time of the mice in the forced swimming experiment compared with the LDG and the HDG, and the MDG dosage group has better effect of reducing the immobility time of the mice in the forced swimming experiment.

As shown in FIG. 3, in FIG. 3A, the effect on IL-1 β was significantly different between the Model group and the Control group, and the inflammatory factor IL-1 β was significantly increased. Compared with the Model group, the contents of IL-1 beta of the LDG, the MDG and the HDG are obviously reduced and have significant difference, which shows that the three groups can reduce the content of IL-1 beta of the hippocampal tissue of the Model group. Meanwhile, the three groups of LDG, MDG and HDG are compared with each other, and no significant difference exists. In FIG. 3B, the effect on TNF-. alpha.showed that the level of inflammatory factor TNF-. alpha.was significantly increased in the Model group compared to the Control group. Compared with the Model group, the LDG, MDG and HDG are all obviously reduced, which shows that the three groups can reduce the content of inflammatory factor TNF-alpha of mouse hippocampal tissues; meanwhile, the three groups of LDG, MDG and HDG are compared with each other, and no significant difference exists. In FIG. 3C, the effect on IL-10 shows that there is a significant difference between the Model group and the Control group, and the anti-inflammatory factor IL-10 in the Model group is significantly reduced. Compared with the Model group, the LDG, MDG and HDG have significant differences, which shows that the three groups can increase the content of mouse hippocampal tissue anti-inflammatory factor IL-10. Meanwhile, the three groups of LDG, MDG and HDG are compared with each other, and compared with the LDG and the HDG, the MDG can obviously increase the content of the anti-inflammatory factor IL-10 in the hippocampal tissue of the mouse, and the effect of improving the content of the anti-inflammatory factor IL-10 in the MDG dosage group is also proved to be stronger compared with the LDG and the MDG.

The results show that the liposome embedding the palmitoyl tripeptide-8 and EUK-134 can be absorbed through the nose, so that the sugar water intake of the chronic depressed mice can be obviously increased, the standing time in tail suspension experiments and forced swimming experiments can be reduced, and the depression behavior of the chronic depressed mice can be improved. Of the three groups of doses, the MDG dose group was more effective in improving depression than LDG and HDG. Further analyzing the reasons of the liposome for improving depression, the experimental groups with different dosages are found to have obviously reduced inflammatory factors IL-1 beta and TNF-alpha of mouse hippocampal tissues, obviously increased content of anti-inflammatory factors IL-10 and higher content of IL-10 in MDG group than LDG and HDG group. The results show that the liposome of palmitoyl tripeptide-8 and EUK-134 can reduce the content of inflammatory factors IL-1 beta and TNF-alpha and increase the content of anti-inflammatory factor IL-10 to play an antidepressant effect.

Example 2

A composition for improving depression is liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 (manganese chloride ethyl bisiminomethyl guaiacol) in phospholipid bilayer. Wherein the mass part ratio of the palmitoyl tripeptide-8 to the palmitoyl tripeptide EUK-134 is 1: 3.

The preparation method comprises the following steps:

first, 0.5 part of palmitoyl tripeptide-8 was added to 10 parts of the phospholipid mixture under stirring to mix them uniformly, thereby obtaining a mixture a. 1.5 parts of EUK-134 was added to 188 parts of water and mixed well to obtain solution B. And slowly injecting the solution B into the mixture A under the stirring state, wherein the stirring speed of the mixture A is 800-1000 rpm/min, the injection speed is 1-4% V/min, stirring is continued for 20min after injection is finished, and then high-pressure homogenization treatment is carried out to obtain the liposome suspension (namely the composition for improving depression). The final mass concentration of phospholipid in the liposome suspension is 5%, the final concentration of palmitoyl tripeptide-8 is 0.25%, and the final concentration of EUK-134 is 0.75%.

Example 3

A composition for improving depression is liposome obtained by embedding palmitoyl tripeptide-8 and EUK-134 (ethyl bis-imino methyl guaiacol manganese chloride) in phospholipid bilayer. Wherein the mass part ratio of the palmitoyl tripeptide-8 to the palmitoyl tripeptide EUK-134 is 3: 1.

The preparation method comprises the following steps:

first, 1.5 parts of palmitoyl tripeptide-8 was added to 40 parts of the phospholipid mixture under stirring to be uniformly mixed, thereby obtaining a mixture a. 0.5 part of EUK-134 was added to 958 parts of water and mixed well to obtain solution B. And slowly injecting the solution B into the mixture A under the stirring state, wherein the stirring speed of the mixture A is 800-1000 rpm/min, the injection speed is 1-4% V/min, stirring is continued for 20min after injection is finished, and then high-pressure homogenization treatment is carried out to obtain the liposome suspension (namely the composition for improving depression). The final mass concentration of phospholipid 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%.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A composition for improving depression, which is a liposome obtained after palmitoyl tripeptide-8 and EUK-134 is embedded in phospholipid bilayer;

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

3. The composition for improving depression according to claim 1, wherein the liposome is mixed with a medicinal adjuvant to prepare nasal drops, spray, gel, cream, powder or powder.

4. A method for preparing a composition for improving depression according to any one of claims 1 to 3, wherein palmitoyl tripeptide-8 is added to a phospholipid mixture under stirring and mixed uniformly to obtain a mixture A; EUK-134 is added into water and evenly mixed to obtain solution B; and injecting the solution B into the mixture A under the stirring state, continuing stirring, and then carrying out high-pressure homogenization treatment to obtain liposome suspension.

5. The method for preparing a composition for improving depression according to claim 4, wherein the phospholipid mixture is Pro-Lipo ^TM Neo。

6. The method for preparing a composition for improving depression according to claim 4, wherein the step of injecting the solution B into the mixture A under stirring is as follows: slowly injecting the solution B into the mixture A with the stirring speed of 800-1000 rpm/min at the speed of 1-4% V/min.

7. The method for preparing a composition for improving depression according to claim 4, wherein the stirring is continued for 20min after the injection of solution B is completed.

8. The method for preparing a composition for improving depression according to claim 4, wherein the liposome lyophilized powder is obtained by adding 10% of D-mannitol to the liposome suspension, mixing uniformly and then lyophilizing.

9. Use of the composition for improving depression according to any one of claims 1 to 3 in the preparation of a medicament for improving depression.

10. The use according to claim 9, wherein the drug is administered nasally.