Nano self-assembly particle with anti-depression effect and preparation method thereof

Abstract

The invention relates to a nano self-assembly particle with an anti-depression effect and a preparation method thereof. According to the nano self-assembled particle prepared by the invention, paeoniflorin and coniferyl ferulate are compatible, so that the nano self-assembled particle can resist neuroinflammation and depression, has a remarkable anti-depression effect and has an obvious synergistic interaction effect. The nano self-assembled particles are free of other carrier components, high in drug loading capacity, low in toxicity, good in safety, small and uniform in particle size, high in stability and long in in-vivo circulation time; the traditional Chinese medicine composition has the advantages that the used raw materials are few in flavor, beneficial to quality control, all natural product extracts, excellent in safety and suitable for long-term use, has great potential in the aspect of preventing and improving depression, and has important clinical significance and application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nano self-assembled particle with an antidepressant effect and a preparation method thereof. BACKGROUND

[0002] With the development of human beings and the progress of society, the working and living style is constantly changing, the social competition is increasingly intensified, and the mental and psychological diseases frequently disturb the health of people.

[0003] Depression patients mainly have symptoms such as long-term low mood, slow thinking, reduced will activity, sleep disorders and the like. However, the mainstream antidepressant drugs currently used in clinical practice, such as serotonin selective reuptake inhibitors (SSRIs), including Fluoxetine, Paroxetine, Sertraline and the like, have some limitations, such as delayed onset of action, long treatment period, high recurrence rate after treatment and the like. Compared with the delayed onset of action of 3 to 6 weeks, ketamine as an N-methyl-D-aspartate receptor (NMDARs) antagonist can rapidly act after a single administration and can last for several days. SUMMARY

[0004] The present application aims to solve the problems existing in the prior art, and provides a nano self-assembled particle with an antidepressant effect and a preparation method thereof, which uses paeoniflorin active intermediates and coniferyl ferulate active intermediates as main active ingredients for compounding, can not only resist neural inflammation but also resist depression, has a significant antidepressant effect, and has a significant synergistic effect, so as to achieve safe and rapid-acting antidepressant treatment.

[0005] In order to solve the above technical problems, the present application is realized by the following technical scheme.

[0006] The present application provides a nano self-assembled particle in the first aspect, which comprises paeoniflorin active intermediates and coniferyl ferulate active intermediates.

[0007] The paeoniflorin active intermediates are prepared by the following method: taking paeoniflorin (Pae oniflorin (PF), chemical formula C 23 H 28 O 11 , CAS number 23180-57-6) is added to an organic solvent 1 for reflux extraction, the filtrate is filtered and concentrated; then an extractant is added for extraction, and further concentrated to a extract; finally dried and crushed to obtain;

[0008] The coniferyl ferulate active intermediates are prepared by the following method: taking coniferyl ferulate (Coniferyl ferulate (CF), chemical formula C20 H 20 O6, CAS No.63644-62-2) is soaked in organic solvent 2, and then water is added for water bath extraction; then the filtrate is obtained by filtration, centrifuged and concentrated, and then organic solvent 2 is added again for precipitation, and the filtrate is collected after standing and washing with organic solvent 2, and finally dried to obtain.

[0009] Preferably, the organic solvent 1 is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, chloroform; more preferably, the organic solvent 1 is selected from methanol.

[0010] Preferably, the mass ratio of the organic solvent 1 to paeoniflorin is 10-15:1.

[0011] Preferably, the reflux extraction is performed 1-5 times, each time for 1-1.5h.

[0012] Preferably, the extractant is prepared by adding ethyl acetate into HCl to a pH value of 2-3.

[0013] Preferably, the organic solvent 2 is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran, chloroform; more preferably, the organic solvent 2 is selected from ethanol.

[0014] Preferably, the mass ratio of the organic solvent 2 to cimicifugic acid cimicifugyl ester is 3-6:1.

[0015] Preferably, the soaking time is 3-6h.

[0016] Preferably, the mass ratio of water to cimicifugic acid cimicifugyl ester is 10-20:1.

[0017] Preferably, the water bath extraction temperature is 80-100℃, the water bath extraction is performed 1-3 times, and each time for 1-2h.

[0018] Preferably, the mass ratio of the paeoniflorin active intermediate to the cimicifugic acid cimicifugyl ester active intermediate is 1-1.5:1.

[0019] The second aspect of the present application provides a preparation method of the above-mentioned nano self-assembled particles, comprising the following steps:

[0020] (1) The paeoniflorin active intermediate and the cimicifugic acid cimicifugyl ester active intermediate are respectively dissolved in a solvent to prepare a paeoniflorin active intermediate solution and a cimicifugic acid cimicifugyl ester active intermediate solution;

[0021] (2) adding water into the container, and adding the paeoniflorin active intermediate solution and the ferulic acid coniferyl ester active intermediate solution prepared in step (1) into the water respectively under the condition of magnetic stirring at room temperature;

[0022] (3) continuing to stir for 5-30 min under the condition of room temperature and light shielding, and then standing to obtain the product.

[0023] Preferably, the solvent in step (1) is one or more selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran and chloroform; more preferably, the solvent is tetrahydrofuran.

[0024] Preferably, the concentration of the paeoniflorin active intermediate solution in step (1) is 3-5 mM; and the concentration of the ferulic acid coniferyl ester active intermediate solution is 3-6 mM.

[0025] Preferably, the volume ratio of the paeoniflorin active intermediate solution to the ferulic acid coniferyl ester active intermediate solution in step (2) is 1:0.5-2; more preferably, the volume ratio of the paeoniflorin active intermediate solution to the ferulic acid coniferyl ester active intermediate solution is 1:1.

[0026] Preferably, the volume ratio of the water to the paeoniflorin active intermediate solution in step (2) is 10-50:1.

[0027] Preferably, the rotating speed of the magnetic stirring in step (2) is 500-1000 rpm.

[0028] The third aspect of the present application provides an application of the above-mentioned nano self-assembled particles in the preparation of a drug for treating depression.

[0029] The fourth aspect of the present application provides a pharmaceutical preparation comprising the above-mentioned nano self-assembled particles.

[0030] Preferably, the dosage form of the pharmaceutical preparation is one or more selected from the group consisting of a powder, an ointment, a gel and a cataplasm.

[0031] Preferably, the pharmaceutical preparation optionally further comprises a pharmaceutically acceptable carrier.

[0032] Preferably, the pharmaceutically acceptable carrier is one or more selected from the group consisting of a filler, a binder, a disintegrant, a lubricant, a preservative, an antioxidant, a chelating agent, a coloring agent, a flavoring agent, a solvent, a base, a gelling agent, an adhesive, a humectant, an emollient, a plasticizer, a crosslinking agent, a pH regulator and an aromatic agent.

[0033] Paeoniflorin as the main active ingredient of Chinese medicine Paeonia lactiflora Pall. and other plants, usually has anti-inflammatory, analgesic, liver protection, immune regulation and neuroprotective effects and functions, and its pharmacological activity has application value in traditional Chinese medicine and modern medicine research. Its commonly used dose is usually more than 5g / kg, and there are reports that excessive consumption of Paeonia lactiflora Pall. total glycoside capsules may cause a series of adverse reactions in patients. Among them, soft stool is one of the most common symptoms of discomfort. In addition, if the dose exceeds the normal load of the gastrointestinal tract, it will also increase the patient's gastrointestinal digestion and absorption burden, causing symptoms such as nausea and vomiting. The nano self-assembled particles prepared by the present application can have a significant antidepressant effect, which can significantly increase the sugar water intake of depressed mice, reduce the immobility time of the tail suspension of depressed mice, and reduce the immobility time of forced swimming of depressed mice, and the effect is obviously better than that of single paeoniflorin, ferulic acid pinus picea ester or positive drug fluoxetine. In addition, the present application obtains paeoniflorin active intermediates and ferulic acid pinus picea ester active intermediates by pretreating paeoniflorin and ferulic acid pinus picea ester respectively, and uses them as active ingredients to prepare nano self-assembled particles, which can further improve the antidepressant activity. Compared with the nano particles prepared by using conventional untreated paeoniflorin and ferulic acid, the nano self-assembled particles of the present application have a significantly stronger antidepressant effect and a significantly faster onset time, and compared with single active ingredient, the combination of paeoniflorin active intermediates and ferulic acid pinus picea ester active intermediates can produce a significant synergistic effect. A significantly better therapeutic activity can be achieved with a significantly lower dose, and the safety is better, avoiding various adverse reactions and cumulative toxicity caused by long-term large-dose taking. In general, the nano self-assembled particles prepared by the present application are compatible with paeoniflorin and ferulic acid pinus picea ester, which can not only resist neural inflammation but also resist depression, and the antidepressant effect is significant and has a significant synergistic effect. The nano self-assembled particles of the present application have no other carrier components, high drug loading, low toxicity, good safety, small and uniform particle size, high stability, long in vivo circulation time, and the like. The raw drug used has a small amount, which is conducive to quality control, and is a natural product extract with excellent safety, suitable for long-term use, and has great potential in preventing and improving depression, and has important clinical significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The TEM morphology characterization result schematic diagram of the nano self-assembled particles prepared by example 1.

[0035] Figure 2 The stability analysis result schematic diagram of the nano self-assembled particles prepared by example 1.

[0036] Figure 3 The self-assembly driving force detection result schematic diagram of the nano self-assembled particles prepared by example 1.

[0037] Figure 4 Figure for behavioral evaluation results of antidepressant effect of nano self-assembled particles. DETAILED DESCRIPTION

[0038] In order to make the objects, technical solutions and effects of the present application clearer and more explicit, the present application is further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0039] Without special instructions, the experimental animals used in the context of the present application are adult male C56BL6 / J mice, aged 2 months, weighing 22±2g, all from SCXK (Yue) 2022-0174. All experimental animals are bred in SPF level animal rooms, the temperature of the animal room is kept at 23±2℃, and the humidity is controlled at 50±15%. One week before the experiment, the animals are normally fed and adapted, and all animals are free to eat and drink. The experiments are carried out with the approval of the animal experiment ethics committee, and all behavioral experiments are carried out from 8.00 to 18.00 every day. The various experimental methods used in the present application, including animal experiments, are conventional methods and techniques in the art.

[0040] Example 1

[0041] First, the preparation of nano self-assembled particles is carried out, and the specific steps are as follows:

[0042] (1) Take 10g of paeoniflorin and add 150mL of methanol for reflux extraction for 1.5h, filter the filtrate, repeat three times; combine the filtrate and concentrate to no alcohol taste, then add an extractant for extraction, and further concentrate to extract; finally dry and crush to obtain; wherein the extractant is prepared by adding ethyl acetate to HCl to pH 2.5. Take 10g of ferulic acid pinocresyl ester and soak in 50mL of 90% concentration ethanol for 5h, then drain and add 150mL of distilled water to extract at 90℃ water bath for 1.5h, filter the filtrate, repeat twice; combine the filtrate, centrifuge and concentrate, then add 90% concentration ethanol for precipitation, stand overnight (12h) and filter, collect the filter residue and wash with 90% concentration ethanol, and finally dry. Dissolve the paeoniflorin active intermediate and the ferulic acid pinocresyl ester active intermediate in tetrahydrofuran respectively to prepare a paeoniflorin active intermediate solution and a ferulic acid pinocresyl ester active intermediate solution, wherein the concentration of the paeoniflorin active intermediate solution is 2.40g / L, and the concentration of the ferulic acid pinocresyl ester active intermediate solution is 1.78g / L.

[0043] (2) In a round-bottom flask, 2 mL of deionized water was added, and 100 μL of the paeoniflorin active intermediate solution and 100 μL of the ferulic acid coniferyl ester active intermediate solution prepared in step (1) were added dropwise into the water under magnetic stirring at room temperature using a pipette. The rotation speed was 750 rpm.

[0044] (3) The stirring was continued at room temperature in the dark for 10 min, and then the product was obtained by standing.

[0045] Example 2

[0046] The nanoscale self-assembled particle solution prepared in Example 1 was ultrasonically dispersed in a water bath for 5 min, and 10 μL of the solution was added dropwise onto a 300-mesh carbon film electron microscope copper grid 20 times. The sample was placed under the selected area of a transmission electron microscope (TEM) to observe the sample morphology, and the detection results are shown in FIG. 2. Figure 1 The results show that the nanoscale self-assembled particles prepared in the application have a spherical structure, clear boundaries, uniform dispersion, and an average size of 229.4 nm, without obvious size difference.

[0047] The nanoscale self-assembled particle solution prepared in Example 1 was ultrasonically dispersed in a water bath for 5 min, and the sample was placed in the dark at 4°C. The hydrated particle size of the nanomedicine was measured using a laser particle size analyzer (Malvern) for seven consecutive days, and the detection results are shown in FIG. 3. Figure 2 The results show that after seven days of particle size measurement, the particle size of the nanomedicine remained basically unchanged after being stored in the dark at 4°C for 7 days, indicating good particle stability.

[0048] Further, the nanoscale self-assembled particle solution prepared in Example 1 was subjected to self-assembly driving force detection, and the specific steps were as follows:

[0049] (1) Covalent bond detection: 500 μL of the nanoscale self-assembled particle solution prepared in Example 1 was added with 2.5 mL of tetrahydrofuran, and the detection was performed using a UV-Vis spectrophotometer (UV-Vis) in the wavelength range of 200-500 nm.

[0050] (2) Hydrogen bond detection: 500 μL of the nanoscale self-assembled particle solution prepared in Example 1 was added with 2.5 mL of a 0.6% w / v urea (Urea) solution, and the detection was performed using a UV-Vis spectrophotometer (UV-Vis) in the wavelength range of 200-500 nm.

[0051] (3) Hydrophobic force detection: 500 μL of the nanoscale self-assembled particle solution prepared in Example 1 was added with 2.5 mL of a 0.6% w / v sodium dodecyl sulfate (SDS) solution, and the detection was performed using a UV-Vis spectrophotometer (UV-Vis) in the wavelength range of 200-500 nm.

[0052] Test results as follows Figure 3 As shown in the figure. The results show that the UV-Vis absorption spectrum of ferulic acid coniferyl ester (CF) has characteristic absorption peaks at 272 nm and 279 nm, and the UV-Vis absorption spectrum of paeoniflorin (PF) has a characteristic absorption peak at 313 nm. The maximum absorption peaks of the synthesized self-assembled nanoparticles are at 277 nm and 303 nm, proving the successful loading of the two components CF and PF in the nanomedicine.

[0053] The UV-Vis spectrum of the self-assembled nanoparticles changed significantly after the addition of tetrahydrofuran. The positions of the maximum absorption peaks changed to 270 nm and 313 nm, almost coinciding with the maximum absorption peak wavelengths of CF and PF after 250 nm. Furthermore, compared to the pure self-assembled nanoparticle solution, the solution containing tetrahydrofuran became clearer and more transparent. This is because tetrahydrofuran disrupts the non-covalent bonds between the nanoparticle molecules. This demonstrates that the self-assembly process of these nanoparticles is driven by non-covalent bonds.

[0054] The absorption spectrum of the self-assembled nanoparticles changed significantly after the addition of urea, with a marked decrease in the absorption peak and the appearance of a new absorption peak at 352 nm. Simultaneously, the nanoparticle solution with urea added was more transparent than the pure self-assembled nanoparticle solution, indicating that hydrogen bonding is one of the non-covalent bonds driving nanoparticle self-assembly. After the addition of SDS, the absorption peaks of the self-assembled nanoparticles blue-shifted to 275 nm and 300 nm, and the absorbance decreased significantly. Furthermore, the solution of the self-assembled nanoparticles with SDS became more transparent, indicating that hydrophobic interactions are also one of the non-covalent bonds driving nanoparticle self-assembly.

[0055] Example 3

[0056] First, the chronic emergency depression model is constructed, and the chronic unpredictable mild stress (CUMS) modeling method is currently widely used in depression research. The pathophysiological changes caused by this modeling method are similar to human depression, and it can be used for the screening of antidepressants and experimental research on the pathophysiological mechanisms of depression. The antidepressants can reverse the depressive behavior changes in stressed mice. The types of stress received by the mice are as follows: water deprivation for 24 hours, food deprivation for 24 hours, 12 hours of day-night reversal, 6 hours of restraint, 18 hours of 45-degree inclined mouse cage, 24 hours of wet cage, and 24 hours of unfamiliar objects. The stress cycle is 4 weeks; during this period, the blank group mice do not receive any stress, and they can eat and drink freely. After the modeling is completed, the mice are randomly divided into 7 groups, each containing 12 mice, which are blank group, model group, control group 1, control group 2, control group 3, experimental group, and positive control group.

[0057] Among them, the blank group is given 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. The model group is given 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. The test drug of control group 1 is paeoniflorin active intermediate, which is diluted to 0.8 mg / kg with 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. The test drug of control group 2 is ferulic acid pinoceryl ester active intermediate, which is dissolved in 0.05% anhydrous ethanol and diluted to 0.8 mg / kg with 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. The test drug of control group 3 is a nanoparticle of paeoniflorin + ferulic acid pinoceryl ester (the preparation of the nanoparticle refers to the method in Example 1, except that the active intermediates of paeoniflorin and ferulic acid are not treated). It is diluted to 1.6 mg / kg with 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. Experimental group: the nanoparticle solution prepared in Example 1 is diluted to 1.6 mg / kg with 0.9% physiological saline, and the intraperitoneal injection is given according to the mouse body weight of 0.1 mL / 10 g. Positive control group: fluoxetine is diluted to a concentration of 15 mg / kg with 0.9% physiological saline, and the intragastric administration is given according to the mouse body weight of 0.1 mL / 10 g.

[0058] Subsequently, the above-constructed chronic stress depression model mice were subjected to sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST), respectively, to evaluate the antidepressant effect of the nano self-assembled particles.

[0059] Among them, the sucrose preference test is an evaluation index of anhedonia, and the specific test method is as follows: 72 h before the test, two identical bottles containing 1% sucrose solution were placed in each cage to eliminate the influence of animal habits on the experiment. Before the test, all mice were deprived of water and food for 16 h. On the test day, each mouse was individually housed and obtained two identical bottles containing 100 mL of 1% sucrose solution or 100 mL of water. The test duration was 6 h. All bottles used in the test were weighed before and after the test. After the test, the sucrose preference was calculated: sucrose preference (%) = sucrose intake / (sucrose intake + water intake) x 100%, and was standardized by the body weight of each animal.

[0060] Tail suspension behavior detection is widely used in the process of basic research of antidepressants, which is a classic method for rapid evaluation of the efficacy of antidepressants, stimulants and sedatives. The principle is to use the mouse to try to escape after the tail is suspended, but it cannot escape, so it gives up struggling and enters a unique depressive state of immobility. The immobility time of the animal during the experiment reflects the depressive state, and antidepressants and stimulants can significantly shorten the state. The tail of the experimental animal is fixed, and the head is suspended downward. The animal struggles in this environment, trying to get rid of the predicament, and after efforts still cannot escape, intermittent immobility appears, showing a "behavioral despair" state. The animal struggles desperately in this environment to try to escape and cannot escape, thus providing an inescapable oppressive environment. After a period of experiment, a series of parameters of the animal's despair immobility state in this environment are recorded. The typical "immobility state" exhibited by the animal reflects a state called "behavioral despair state". This behavioral despair model is similar to depression and is sensitive to most antidepressants, and its efficacy is significantly correlated with clinical efficacy, so it is widely used for the initial selection of antidepressants. The specific test method is as follows: the mouse is placed in a sealed soundproof box isolated from hearing and vision, and the tail tip is about 1 cm away from the mouse. The hook is adhered to the suspension with tape at a distance of 50 cm from the ground, and the animal's activity is recorded. The ANY-maze software (Stoeling Co. Ltd.; USA) is used to record and analyze the total immobility time in the last 4 min of the 6 min test time.

[0061] The forced swim test is one of the techniques used to evaluate the standard of depression in animal models of depression research, which is a desperate experimental method. Animals are placed in water at a suitable temperature, and in this environment, one of the animals' common behaviors is to struggle desperately. Another behavior is to escape but often fail to escape or be unable to escape, and an inevitable oppressive environment is thus generated. After a period of time, the animals exhibit a typical "immobile state". A series of parameters are observed and recorded during the movement and floating of the experimental animals, which can be used to evaluate the efficacy of antidepressants. The specific test method is as follows: the mice are taken out of the cage and placed in a transparent glass jar (50 cm high and 30 cm in diameter), which is filled with water about 30 cm high (23±2℃), and the mice are allowed to swim freely for 6 min. When the mouse floats on the water surface without struggling, it is considered to be immobile. At the end of the test, the mouse is removed from the water, dried and dried, and then placed back in the cage. The total immobility time during the last 4 min of the 6 min test is recorded and analyzed by ANY-maze software.

[0062] The results are expressed as mean ± standard error (Mean ± SEM) and analyzed using SPSS 21.0 software (SPSS, Armonk, USA). For comparison of more than two groups, one-way ANOVA was used, followed by LSD post-hoc analysis. All data are expressed as mean ± SD, and p<0.05 is considered statistically significant. Drug interaction index (CDI): calculation method: CDI = AB / (A x B), where AB is the ratio of the test index of the combination group to the control group, and A and B are the ratios of the test index of the single drug group to the control group. If CDI <1, it indicates that the two drugs have a synergistic effect; when CDI <0.7, the synergistic effect is very significant; CDI =1 is additive effect; and CDI >1 is antagonistic effect.

[0063] The experimental results are shown in Tables 1-3 and Figure 4The experimental results show that, in the sugar water experiment (see Table 1 for results), compared with the blank group, after 4 weeks of stress, the sugar water intake of the model group mice is significantly reduced (p<0.05); compared with the model group, after 4 weeks of modeling, the single administration of control group 1 or control group 2 cannot significantly increase the sugar water intake after 30 minutes (p>0.05); the single administration of control group 3 or the experimental group can increase the sugar water intake after 30 minutes (p<0.05), but is significantly less than the experimental group (p<0.05). And after the single administration of the positive control group drug fluoxetine for 30 minutes, the sugar water intake cannot be significantly increased (p<0.05). It is shown that the nano self-assembled particles prepared by the application have obvious antidepressant effect and the antidepressant effect is better than that of the compound without nano treatment. Further synergistic effect analysis finds that the average interaction index CDI of the experimental group drug is CDI = experimental group / (control group 1 + control group 2) = 0.72402, the average interaction index CDI of the control group 3 drug is CDI = control group 3 / (control group 1 + control group 2) = 0.5466, and the CDI of the control group 3 is 0.5466 <1, indicating that the two drugs have synergistic effect, the CDI of the experimental group is 0.72402 <1, the result is very close to 0.7, and the CDI of the experimental group is greater than that of the control group 3, indicating that the nano self-assembled particle drug combination of the application has better synergistic effect.

[0064] Table 1 Sugar water intake of mice after single administration of drugs in each group for 30 minutes (%)

[0065]

[0066] In the above results, *p<0.0001 compared with the model group, & p<0.0001 compared with control group 2, @ p<0.0001 compared with control group 3, $ p<0.0001 compared with the positive control group, # p<0.0001.

[0067] In the tail suspension test (see Table 2 for results), compared with the blank group, after 4 weeks of modeling, the tail suspension immobility time of the model mice was significantly increased (p<0.05), indicating that the model was successful; compared with the model group, after 4 weeks of modeling, single administration of control group 1 or control group 2 or control group 3 could not significantly reduce the immobility time of the mice after 2 hours (p>0.05). However, compared with the model group, single administration of the experimental group 10 hours later could significantly reduce the immobility time of the mice in the tail suspension test (p<0.05). At the same time, single administration of the positive control group drug fluoxetine 10 hours later also could not improve the immobility time of the mice in the tail suspension test (p>0.05). In addition, the experimental group could significantly reduce the immobility time of the mice in the tail suspension test compared with control group 1 or control group 2 or control group 3 (p<0.05), suggesting that the experimental group had obvious rapid antidepressant effect.

[0068] Table 2 Immobility time of mice in tail suspension test after single administration of drugs in each group for 2 hours

[0069]

[0070] In the above results, compared with the model group, * p<0.001, compared with control group 1, & p<0.001, compared with control group 2, @ p<0.001, compared with control group 3, $ p<0.001, compared with the positive control group, # p<0.001.

[0071] In the forced swimming test (see Table 3 for results), compared with the blank group, after 4 weeks of modeling, the forced swimming immobility time of the model mice was significantly increased (p<0.05), indicating that the model was successful; compared with the model group, after 4 weeks of modeling, single administration of control group 1 or control group 2 or control group 3 could not significantly reduce the immobility time of the mice after 28 hours (p>0.05). However, compared with the model group, single administration of the experimental group 28 hours later could significantly reduce the immobility time of the mice in the forced swimming test (p<0.05). At the same time, single administration of the positive control group drug fluoxetine 28 hours later also could not improve the immobility time of the mice in the forced swimming test (p>0.05). In addition, the experimental group could significantly reduce the immobility time of the mice in the forced swimming test compared with control group 1 or control group 2 or control group 3 (p<0.05), suggesting that the experimental group had obvious antidepressant effect and the curative effect was stable and superior to other administration groups.

[0072] Table 3 Immobility time of mice in tail suspension test after single administration of drugs in each group for 28 hours

[0073]

[0074] In the above results, compared with the model group, * p<0.001, compared with control group 1, & p<0.001, compared with control group 2, @ p<0.001, compared with control group 3, $ p<0.001, compared with positive control group, # p<0.001.

[0075] In summary, the nano self-assembled particles prepared by the present application have significant antidepressant effect, which can significantly increase the sugar intake of depressed mice, reduce the immobility time of the tail suspension of depressed mice, and reduce the immobility time of forced swimming of depressed mice, and the effect is significantly better than the improvement effect of single paeoniflorin, ferulic acid pinocerebrosides or positive drug fluoxetine. In addition, the present application obtains paeoniflorin active intermediates and ferulic acid pinocerebrosides active intermediates by pretreating paeoniflorin and ferulic acid pinocerebrosides respectively, and uses them as active ingredients to prepare nano self-assembled particles, which can further improve the antidepressant activity. Compared with the nano particles prepared by using conventional untreated paeoniflorin and ferulic acid, the nano self-assembled particles of the present application have significantly stronger antidepressant effect and significantly faster onset time, and compared with single active ingredient, the combination of paeoniflorin active intermediates and ferulic acid pinocerebrosides active intermediates can produce obvious synergistic effect. At the same time, the nano self-assembled particles prepared by the present application have good stability, the raw materials used are natural product extracts, have excellent safety, are suitable for long-term use, have great potential in preventing and improving depression, and have important clinical significance and application value.

[0076] The above specific embodiment part specifically introduces the analysis method involved in the present application. It should be noted that the above introduction is only to help those skilled in the art better understand the method and idea of the present application, and is not a limitation on the related content. Those skilled in the art can also make appropriate adjustments or modifications to the present application without departing from the principles of the present application, and the above adjustments and modifications should also belong to the protection scope of the present application.

Claims

1. A nano-self-assembling particle, characterized in that, The paeonol active intermediate and the ferulic acid coniferyl ester active intermediate; The paeonol active intermediate is prepared by the following method: refluxing paeonol in an organic solvent 1, filtering the filtrate and concentrating; then extracting with an extractant and further concentrating to a extract; and finally drying and crushing to obtain the paeonol active intermediate; The ferulic acid coniferyl ester active intermediate is prepared by the following method: soaking ferulic acid coniferyl ester in an organic solvent 2, draining and then extracting with water in a water bath; then filtering the filtrate, centrifuging and concentrating, adding the organic solvent 2 again for precipitation, filtering after standing, washing the filter residue with the organic solvent 2, and finally drying to obtain the ferulic acid coniferyl ester active intermediate.

2. The nanoassemblies of claim 1, wherein, The extractant is prepared by the following method: adding ethyl acetate into HCl until the pH value is 2-3.

3. The nano-self-assembled particles according to claim 1, characterized in that, The organic solvent 1 is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran and chloroform.

4. The nanoassemblies of claim 1, wherein, The organic solvent 2 is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran and chloroform.

5. The nano-self-assembled particles according to claim 1, characterized in that, The mass ratio of the paeonol active intermediate to the ferulic acid coniferyl ester active intermediate is 1-1.5:

1.

6. Process for the preparation of nano- self-assembled particles according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) dissolving the paeonol active intermediate and the ferulic acid coniferyl ester active intermediate in a solvent respectively to obtain a paeonol active intermediate solution and a ferulic acid coniferyl ester active intermediate solution; (2) adding water into a container, and adding the paeonol active intermediate solution and the ferulic acid coniferyl ester active intermediate solution prepared in step (1) into the water drop by drop under the condition of room temperature and magnetic stirring; (3) continuing to stir under the condition of room temperature and light shielding for 5-30 min, and then standing to obtain the product.

7. The preparation method according to claim 6, characterized in that, The solvent in step (1) is selected from one or more of methanol, ethanol, isopropanol, tetrahydrofuran and chloroform.

8. Use of the nano self-assembled particles according to any one of claims 1-5 and / or the nano self-assembled particles prepared by the method according to any one of claims 6-7 in the preparation of a medicament for treating depression.

9. A pharmaceutical preparation, characterized by, The medicament comprises the nano self-assembled particles according to any one of claims 1-5 and / or the nano self-assembled particles prepared by the method according to any one of claims 6-7.

10. The pharmaceutical preparation according to claim 9, characterized in that, The pharmaceutical preparation further comprises a pharmaceutically acceptable carrier.

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