Amomum villosum essential oil liposome with anti-inflammatory and anti-depression effects as well as preparation method and application thereof

Abstract

The invention discloses an amomum villosum essential oil liposome with anti-inflammatory and anti-depression effects as well as a preparation method and application of the amomum villosum essential oil liposome. The amomum villosum essential oil liposome provided by the invention is prepared by injecting the lipid phase obtained by mixing the wall material, the emulsifier and the amomum villosum essential oil into the water phase through an ethanol injection method, the particle size distribution is uniform, the encapsulation efficiency is relatively high, and the stability and bioavailability of the amomum villosum essential oil are improved; meanwhile, the fructus amomi essential oil has better biological safety and no side effect, can be used for in-vivo drug-loading treatment, has a very good anti-inflammatory effect, can remarkably relieve the depressive symptom of a model mouse, further improves the in-vivo treatment effect of the fructus amomi essential oil, has better anti-inflammatory and anti-depression effects compared with original essential oil, and can be used for preparing a medicine. And more products and methods are provided for developing more anti-inflammatory or anti-depression drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to an Amomum villosum essential oil liposome with anti-inflammatory and anti-depressant effects, and a preparation method and application thereof. Background Art

[0002] Depression, also known as depressive disorder, is a mental disorder with high incidence, low treatment acceptance rate and high recurrence rate. Its main feature is significant and persistent low mood. Some patients may have self-harm, suicidal behavior, and may even be accompanied by psychotic symptoms such as delusions and hallucinations. Depression seriously endangers human physical and mental health. Clinically, depressive disorders can be divided into mild, moderate and severe according to the number, type and severity of symptoms. They are respectively targeted at different groups such as the elderly, children, and maternal women, and are called elderly depression, childhood depression, postpartum depression, etc. The exact cause and pathogenesis are still unclear, but a large amount of data has suggested that genetic factors, neurochemical factors and psychosocial factors have a significant impact on the occurrence of depression. At present, the treatment of depression is mainly based on drug therapy, but the antidepressants currently on the market are not ideal. Commonly used antidepressant Western medicines have certain side effects, adverse reactions, slow onset and low efficiency. Therefore, the development of new antidepressants with higher efficacy, obvious characteristics and fewer adverse reactions is the main goal of new drug research and development.

[0003] With the research on traditional Chinese medicine, many natural Chinese medicinal plants with antidepressant effects have been discovered in recent years, such as Hypericum perforatum, Morinda officinalis, ginseng, etc., as well as Chinese herbal compound drugs, such as Xiaoyaosan and Shugan Jieyu Capsules, which have little side effects and a low relapse rate while improving depression-like symptoms, and are more effective than Western medicine. However, at present, there are still few natural Chinese medicinal drugs that can be used to treat depression, and their efficacy needs to be improved. More antidepressant drugs need to be developed urgently.

[0004] Amomum L., a member of the genus Cardamom in the Zingiberaceae family, refers to the dried fruits or seeds of the endemic species of Amomum hainanensis, Amomum villosum Lour., and Amomum villosum Lour.. Among them, Amomum villosum Lour. is known as the "medical treasure" of traditional Chinese medicine due to its wide planting area, high yield and remarkable medicinal quality. It is the largest species of Amomum. As one of the "four major southern medicines" in ancient China, Amomum villosum has established its priority in nourishing the spleen, stomach, lungs, kidneys and other organs due to its functionality in stimulating appetite, invigorating the spleen, relieving pain, and calming the fetus. Amomum villosum essential oil has become a hot topic in current medical research because it contains many bioactive substances, such as limonene, bornyl acetate and linalool. Studies have shown that Amomum villosum essential oil has significant anti-inflammatory and antibacterial effects, and can treat gastrointestinal diseases such as gastric and duodenal ulcers, enteritis, pediatric abdominal pain, chronic diarrhea, stomach pain, diarrhea, abdominal distension, and stomach weakness. Its external use has a certain effect on herpes around the lips, skin eczema, and tinea corporis.

[0005] However, due to the poor stability and low bioavailability of Amomum villosum essential oil, it is not conducive to drugability research and clinical application, and the drug efficacy is unstable in vivo and the effect is not ideal. In addition, existing studies all use Amomum villosum essential oil as a patch for external use, and improve its effect by encapsulating it with a specific carrier, such as the prior art encapsulating Amomum villosum volatile oil in nano-liposomes to improve the permeability of Amomum villosum volatile oil through the stratum corneum and increase the drug penetration; and the external use of Amomum villosum volatile oil nanoliposomes can increase the food intake and weight gain of anorexic animals. At present, it is rarely used in the study of depression, and there are still few Amomum villosum essential oil drugs, and there is still a lack of drugs containing Amomum villosum essential oil that can be used for in vivo treatment. In order to improve the poor stability and poor bioavailability of Amomum villosum essential oil, it is necessary to develop Amomum villosum essential oil drugs with better efficacy, expand the scope of antidepressant Chinese medicine, improve the in vivo therapeutic effect of Amomum villosum essential oil, and provide more effective and safe treatment plans for the Chinese medicine Amomum villosum to treat various diseases. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the problems that the existing Amomum villosum essential oil has poor stability and bioavailability and is not conducive to in vivo treatment, and to provide an Amomum villosum essential oil liposome with anti-inflammatory and antidepressant effects, as well as a preparation method and application thereof.

[0007] The invention aims to provide a method for preparing Amomum villosum essential oil liposome.

[0008] Another object of the present invention is to provide a kind of Amomum villosum essential oil liposome.

[0009] Another object of the present invention is to provide application of Amomum villosum essential oil liposome.

[0010] Another object of the present invention is to provide a medicine.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a method for preparing Amomum villosum essential oil liposomes, comprising the following steps:

[0013] S1. Take the wall material, emulsifier, and Amomum villosum essential oil and add them to an organic solvent, stir and heat to dissolve the lipid phase;

[0014] The wall material is selected from lanosterol or cholesterol, and the emulsifier is selected from one or more of diglyceride, cephalin, egg yolk lecithin, and phosphatidylcholine; the mass ratio of the wall material: emulsifier: Amomum villosum essential oil is: (1-4): (4-17): (1-5);

[0015] S2. inject the lipid phase into the aqueous phase, stir, remove the organic solvent, and obtain Amomum villosum essential oil liposomes after ultrasonication.

[0016] The Amomum villosum liposome prepared by the present invention is mainly composed of a specific wall material and an emulsifier, which is responsible for forming a stable double-layer lipid structure to protect and carry inclusions; the Amomum villosum essential oil is an active ingredient encapsulated by the liposome, which is rich in various bioactive substances, and the liposome encapsulation prepared by the present invention can strengthen the stability and bioavailability of the Amomum villosum essential oil. The particle diameter of the prepared Amomum villosum essential oil liposome is 50-150nm, and the particle size distribution is uniform, and the stability is good, and it has good biosafety, can be used for in vivo treatment, has good anti-inflammatory effect, can significantly reduce NO concentration, inhibit the expression of ROS, and can significantly reduce the depressive symptoms of model mice, and has a more excellent anti-inflammatory and antidepressant effect than the original essential oil, and further improves the in vivo therapeutic effect of the Amomum villosum essential oil.

[0017] Preferably, in S1, the wall material is selected from lanosterol or cholesterol, and the emulsifier is selected from diglyceride or lecithin.

[0018] Preferably, in S1, the mass ratio of wall material: emulsifier: Amomum villosum essential oil is: 1: (4-17): (1-5).

[0019] Preferably, in S1, the mass ratio of wall material: emulsifier: Amomum villosum essential oil is: 1: (4-6): (1.25-1.5).

[0020] More preferably, in S1, the mass ratio of wall material: emulsifier: Amomum villosum essential oil is 1:4:1.25.

[0021] More preferably, the wall material in S1 is lanosterol and the emulsifier is diglycerol. Lanosterol has more methyl groups and double bonds in its molecule, and this structure makes it more gentle in regulating the fluidity of the lipid system.

[0022] Preferably, the heating temperature in S1 is 35-40°C.

[0023] More preferably, the heating temperature is 35°C

[0024] Preferably, the ultrasonic conditions in S2 are: power 330-400 W, time 30-60 min, temperature -5-0°C.

[0025] More preferably, the ultrasonic power is 330 W and the time is 40 min.

[0026] Preferably, the organic solvent in S1 is ethanol, and the solid-liquid ratio of ethanol to the wall material, emulsifier and Amomum villosum essential oil is 40-80:1 mg / mL.

[0027] More preferably, the solid-liquid ratio is 60-80:1 mg / mL.

[0028] The invention provides a Amomum villosum essential oil liposome, which is prepared by the method.

[0029] The invention provides application of Amomum villosum essential oil liposome in preparing anti-inflammatory or antidepressant medicine.

[0030] The invention provides a medicine containing Amomum villosum essential oil liposome.

[0031] The present invention has the following beneficial effects:

[0032] The Amomum villosum essential oil liposome provided by the invention is prepared by injecting a fat phase obtained by mixing a specific wall material, an emulsifier and the Amomum villosum essential oil into a water phase by an ethanol injection method and then ultrasonically preparing the liposome. The liposome has uniform particle size distribution and high encapsulation rate, thereby improving the stability and bioavailability of the Amomum villosum essential oil. The liposome has good biosafety and no side effects, can be used for in vivo drug delivery therapy, has good anti-inflammatory effect, can significantly alleviate the depression symptoms of model mice, further improves the in vivo therapeutic effect of the Amomum villosum essential oil, has better anti-inflammatory and antidepressant effects than the original essential oil, and provides more products and methods for developing more anti-inflammatory or antidepressant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The particle size distribution diagram of Amomum villosum essential oil liposome

[0034] Figure 2 This is the standard curve of bornyl acetate.

[0035] Figure 3 This is the result of MTT experiment.

[0036] Figure 4 Figure 2 shows the body weight changes in the depression mouse model.

[0037] Figure 5 TST is the immobility time of mice.

[0038] Figure 6 The immobility time of mice in FST.

[0039] Figure 7 The results of COX-2 immunohistochemistry are shown in Figure 2.

[0040] Figure 8 The figure shows the results of Bax immunohistochemistry. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] The ripe Amomum villosum Lour. fruits were from Yangchun, Yangjiang, Guangdong Province.

[0044] Embodiment 1 Extraction of Amomum villosum essential oil

[0045] (1) Material preparation: Screen and wash the Amomum villosum to remove impurities and residual soil. Dry the Amomum villosum evenly at room temperature. Turn it over regularly during the drying process to ensure that all the Amomum villosum is evenly heated. After drying to an appropriate moisture content, weigh 500 g of the processed Amomum villosum and crush it finely using a high-speed grinder.

[0046] (2) Extraction operation: The crushed Amomum villosum was transferred to a 10 L round-bottom flask, and 5 L of deionized water was slowly added to ensure that the Amomum villosum was completely immersed.

[0047] (3) Distillation extraction: Place the round-bottom flask device containing the material and deionized water in the electric heating mantle, and properly connect the essential oil extractor and the condenser to ensure that the system is sealed and the channel is unobstructed. The power of the electric heating mantle is set to 1 kW, the heating temperature is controlled, and the essential oil components in Amomum villosum are gradually extracted by steam distillation. The entire extraction process is maintained for 4 hours to ensure the maximum extraction of the essential oil components.

[0048] (4) Essential oil collection and drying: During the extraction process, the essential oil and water mixture condensed by the condenser will be collected. In the collected mixture, the essential oil and water will naturally separate due to their different densities. At this time, the essential oil layer can be separated using a separatory funnel. The extracted Amomum villosum essential oil is then dried with anhydrous sodium sulfate (Na 2 SO 4) to remove moisture, and then filter with a 0.45 μm filter membrane to obtain slightly yellowish and transparent Amomum villosum essential oil.

[0049] (5) Storage: After extraction, the Amomum villosum essential oil should be transferred to a clean and sealed brown bottle and stored in a refrigerator at 4°C.

[0050] Embodiment 2 Synthesis of Amomum villosum essential oil liposome

[0051] The ethanol injection method was used to prepare Amomum villosum liposomes with a drug-lipid ratio of 1:4. First, 50 mg of wall material and 200 mg of emulsifier were accurately weighed, and then 62.5 mg of Amomum villosum essential oil was added to 5 mL of ethanol. The mixture was heated to 35-40°C using a magnetic stirrer and stirred until completely clear to ensure that all ingredients were evenly mixed and completely dissolved to form a uniform oily mixture and a lipid phase.

[0052] The dissolved lipid phase is slowly injected into the pre-prepared aqueous PBS buffer through a syringe. The injection process needs to be controlled at a moderate rate to avoid the formation of large lipid vesicles or excessive foaming. After the injection is completed, the mixture needs to be stirred at room temperature for a period of time to facilitate the evaporation of the organic solvent.

[0053] Finally, the sample is placed in an ultrasonic instrument for ultrasonication. The treatment process is usually carried out in an ice bath to prevent the sample from overheating. The ultrasonic conditions are: power 330-400W, time 30-60min, temperature -5-0℃. Ultrasonic waves can help break up larger lipid vesicles and form liposomes of uniform size. The time and power of ultrasonic treatment need to be adjusted according to the required size and uniformity of the liposomes. After the ultrasonic treatment is completed, wait for the foam to dissipate, and finally obtain clear and uniform Yangchun Amomum villosum essential oil liposomes.

[0054] According to the above method, Amomum villosum essential oil liposomes with different liposome formulas in Table 1 were simultaneously prepared.

[0055] Table 1 Configuration of different wall materials and emulsifiers

[0056]

[0057] Example 3 Characterization of liposomes

[0058] 1. Liposome particle size analysis

[0059] The particle size of liposomes is measured using a particle size analyzer. First, the pre-made liposome sample (combination 1-4 in Example 2) is diluted to an appropriate concentration to ensure that it is suitable for measurement. The diluted sample is loaded into a special cell suitable for particle size measurement. Dynamic light scattering technology is used to monitor the change in the intensity of scattered light after the sample is irradiated with a light beam, thereby measuring the speed of the Brownian motion of the particles. The data obtained are analyzed and processed by the built-in software of the instrument, and the particle size and distribution of the liposomes are finally calculated.

[0060] The results showed that the molecular particle size of the liposomes of different combinations prepared in Example 2 was between 50-150 nm and could be absorbed by cells; the Amomum villosum essential oil liposomes prepared in combination 4 had an average particle size (Mean Diameter) of 112.0 nm, a polydispersity index (polydispersity) of 0.14, a standard deviation (Geometric Standard Deviation, GSD) of 1.436, and a particle size distribution diagram as shown in FIG. Figure 1 As shown, it shows that the size distribution of the liposome particles is relatively uniform, and the liposome essential oil is more easily absorbed by cells after being encapsulated.

[0061] 2. Liposome encapsulation efficiency determination

[0062] First, prepare different concentrations of gentian acetate standard, under fixed detection wavelength, measure its peak area by high performance liquid chromatography (HPLC), construct the standard curve between gentian acetate concentration and peak area.Separate liposomes (combination 1-4 in embodiment 2) by high speed centrifugation, and discard the free borneol acetate in the supernatant.Remaining liposome sediment is redissolved in ethanol and again by ultrasonic wave crushing, releases the borneol acetate encapsulated in liposome.The sample is diluted 50 times, and the borneol acetate concentration after dissolution is measured using HPLC under the same chromatographic conditions, and the content of the borneol acetate encapsulated by the actual liposome is obtained by calculation.

[0063] The liposome encapsulation efficiency (EE) is calculated by the following formula:

[0064]

[0065] The standard curve of gentianyl acetate monomer is as follows Figure 2 As shown, according to the content of gentian acetate contained in the samples obtained by standard curve and GC-MS analysis, the sample concentration measured by HPLC, and the encapsulation efficiency of Amomum villosum essential oil liposomes of combinations 1-4 were calculated according to the above formula. As shown in Table 2, the encapsulation efficiency of Amomum villosum essential oil in different liposomes was 53-64.3%.

[0066] Table 2 Encapsulation efficiency of different liposome raw materials

[0067]

[0068] Example 4 In vitro effect of Amomum villosum essential oil liposome

[0069] 1. Biosafety of Amomum villosum essential oil liposomes

[0070] (1) Cell culture: The BV2 cells used in this experiment were provided by the Natural Products Laboratory of Guangdong University of Technology. BV2 microglial cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% double antibody) at 37°C and 5% CO. 2 When the cells grew to 90% confluence, they were digested with trypsin and then resuspended in complete medium.

[0071] (2) MTT method to test cell proliferation inhibition rate: MTT method is used to determine cell activity and proliferation inhibition at a specific concentration. The specific steps are: BV2 cells with good growth condition are blown down, the cell density is adjusted to 30,000 per well, inoculated into a 96-well plate, and placed in an incubator for 24 hours. The Amomum villosum essential oil liposomes to be tested (combination of Example 2) are dissolved in DMSO to prepare a dilution with a sample concentration of 200 mg / mL. At the same time, MTT powder is dissolved in PBS, and a 0.5 mg / mL MTT solution is prepared and stored in the dark. A total of 6 drug administration groups are set up, and Amomum villosum essential oil liposome samples are added to the 96-well plate by a half-dilution method and placed in an incubator. After 24 hours, the culture medium is sucked out, 100 μL of MTT solution is added to the 96-well plate, and placed in an incubator. After 4 hours, the MTT solution is sucked away, and 100 μL of DMSO solvent is added to the 96-well plate. The detection wavelength was set at 570 nm, and the absorbance was detected by an enzyme-labeled instrument to determine the survival rate of cells in samples with a concentration of 6.25 μg / mL-200 μg / mL.

[0072] The results of the test are as follows Figure 3 As shown in the figure, when the sample concentration is 6.25-100μg / mL, the activity of BV2 cells is close to that of the blank group, indicating that the toxicity is low at this concentration and has good biosafety. When the sample concentration is 200μg / mL, BV2 cells are significantly inhibited and produce certain cytotoxicity, so the concentration below 100μg / mL is selected for subsequent experiments.

[0073] 2. Anti-inflammatory effect of Amomum villosum essential oil liposomes

[0074] (1) Determination of nitric oxide (NO) content: During inflammation, the NO content will increase relatively, and is often used as an important indicator of cellular inflammation expression. Griess reagent is composed of naphthylethylenediamine sulfate and acidic phosphate or sulfate solution. This reagent can react with nitrite (NO) in cells. 2-) reacts to generate a purple compound, whose absorbance can be measured with the aid of an enzyme-labeled instrument. The specific steps are:

[0075] BV2 cells in good growth condition were blown down, the cell density was adjusted to 300,000 per well, inoculated into a 96-well plate, and placed in an incubator. After 24 hours, the culture medium was discarded, and 50 μg / mL and 100 μg / mL of sample diluent were added to each well (combination 1-4 in Example 2 was diluted with PBS). After 2 hours, 1 mg / mL LPS solution was added to the 96-well plate and placed in an incubator. After 24 hours, 50 μL of supernatant was aspirated, inoculated into another 96-well plate, and 50 μL of Griess Reagent I and II were added. The absorbance was detected with an enzyme-labeled instrument, and the detection wavelength was 562 nm. 50 μg / mL and 100 μg / mL were selected as high and low concentration groups for nitric oxide testing.

[0076] 50μg / mL and 100μg / mL were selected as high and low concentration groups for nitric oxide testing. The results are shown in Table 3. Compared with the blank group, the NO concentration in the model group increased significantly, indicating that the cellular inflammatory effect in this group was very significant. Different Amomum villosum essential oil liposomes can significantly reduce the NO concentration, and the high concentration group is more significant.

[0077] Table 3 Experimental results of NO in different liposomes

[0078]

[0079] (2) Detection of ROS levels: Reactive oxygen species (ROS) are closely related to inflammation. Detection of ROS levels is crucial for understanding cell physiological functions and cell damage caused by environmental factors. The DCFH-DA fluorescent probe was used to detect the changes in ROS levels in BV2 microglia after treatment with different Amomum villosum essential oil liposomes. This experiment needs to be carried out in the dark. The specific steps are as follows:

[0080] BV2 cells with good growth were blown down, the cell density was adjusted to 300,000 per well, inoculated into a 6-well plate, and placed in an incubator. After 24 hours, the culture medium was discarded, and 50 μg / mL and 100 μg / mL sample diluents were added to each well (combination 1-4 in Example 2 was diluted with PBS). After 2 hours, 1 mg / mL LPS solution was added to the 6-well plate and placed in an incubator. After 24 hours, the culture medium in the 6-well plate was sucked out, rinsed with basal culture medium, and then 1 mL of DCFH-DA fluorescent probe was added and placed in an incubator. After 30 minutes, rinsed three times with basal culture medium, and then the cells were blown down. Fluorescence intensity was detected by flow cytometer. The excitation light wavelength was 488 nm, and the fluorescence wave emitted was 530 nm.

[0081] The measurement results are shown in Table 4. The ROS content in the model group increased significantly, while the fluorescence intensity of the different liposome treatment groups decreased significantly, and the inhibitory effect of the 100 μg / mL concentration group was more significant, indicating that the Amomum villosum essential oil liposomes prepared by the present invention can significantly inhibit the expression of ROS.

[0082] Table 4 ROS experimental results of different liposomes

[0083]

[0084] Example 5 Effect of Amomum villosum essential oil liposomes on fat accumulation

[0085] (1) Cell culture: The 3T3-L1 cells used in this experiment were provided by the Natural Products Laboratory of Guangdong University of Technology. 3T3-L1 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% double antibody) at 37°C and 5% CO. 2 When the cells grew to 90% confluence, they were digested with trypsin and then resuspended in complete medium.

[0086] (2) Pipette the 3T3-L1 cells that are growing well and place them at 2×10 4 / well was inoculated in a 12-well plate. After 24 hours, 0.25M 3-isobutyl-1-methylxanthine (IBMX) solution, 10mg / mL insulin and 1mM dexamethasone solution (DXM) were taken, and 10% CS-DMEM culture medium was used at a ratio of 1:500, mixed evenly, and prepared into an induction solution. The experiment was divided into a blank group, a model group, and an experimental group. The induction solution was added to the model group, and the liposomes (100μg / mL) of combination 1-4 in Example 2 were added to the experimental group and cultured for 2 days.

[0087] (3) Oil Red O staining: After 2 days of differentiation, remove the cell culture medium, wash the cells twice with PBS, add Oil Red O fixative to just cover the cells, and fix at room temperature for 30 minutes. Prepare Oil Red O staining solution according to the instructions, A solution: B solution = 3:2 (vol:vol), mix well and let stand for 10 minutes, then filter through filter paper to obtain clear Oil Red staining solution. Discard the fixative solution, wash twice with distilled water, and then add 60% isopropanol to soak for 5 minutes. Add freshly prepared Oil Red O staining solution, soak for 20 minutes, discard the staining solution, and wash 2-5 times with distilled water until there is no excess staining solution. Add Mayer's hematoxylin staining solution, counterstain the nucleus for 1 minute, discard the staining solution and wash with water until there is no excess staining solution. Finally, add Oil Red buffer solution, incubate for 1 minute and discard, add distilled water to cover the cells and observe and photograph under a live cell imager.

[0088] (4) Enzyme-linked immunosorbent assay for triglyceride: Triglyceride is a fat molecule formed by long-chain fatty acids and glycerol, and is one of the most abundant lipids in the human body. By testing its content, we can determine the effect and function of using Amomum villosum essential oil liposomes on fat accumulation in the body.

[0089] Take the triglyceride standard provided by the kit and dilute it step by step with the sample diluent according to the instructions to prepare a series of gradient concentrations (such as 0, 10, 20, 50, 100, 200 μg / mL). Add the diluted standard to the corresponding wells of the ELISA plate (at least 3 replicate wells are set up for each concentration). Mark the position of each well to facilitate the drawing of the standard curve during analysis.

[0090] Preparation of antibody-antigen-enzyme-labeled antibody complex and color development reaction: Purified lipase antibody is coated on microplate to make solid-phase antibody, lipase is added to the microwells coated with monoclonal antibody in sequence, and then combined with horseradish peroxidase (HRP)-labeled lipase antibody to form lipase antibody-lipase antigen-enzyme-labeled antibody complex, and after thorough washing, substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and the reaction is terminated under the action of acid and converted into the final yellow.

[0091] Use an ELISA reader to measure the OD value of each well in sequence at a wavelength of 450nm, and adjust the blank well to zero. The measurement should be performed within 15 minutes after adding the stop solution. Use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, and calculate the concentration of triglycerides.

[0092] The linear regression equation is: y = 0.0368x + 0.0130, the correlation coefficient R 2 =0.9990. The test results are shown in Table 5. The normal value of serum triglyceride is generally in the range of 0.4-1.7mmol / L. The results show that the concentration of triglyceride after the action of the Amomum villosum essential oil liposomes in combination 1 is within the normal range, and the triglyceride concentration will not increase, causing fat accumulation; the Amomum villosum essential oil liposomes in other combinations will slightly increase the triglyceride concentration, but it is lower than the model group, and will not cause excessive increase in triglyceride concentration and fat accumulation. Therefore, the use of the Amomum villosum essential oil liposomes prepared by the present invention for in vivo metabolism will not increase the burden on the body.

[0093] Table 5 Experimental results of ELISA

[0094]

[0095] Example 6 Antidepressant Activity of Amomum villosum Essential Oil Liposomes

[0096] 1. Animal experiment grouping and construction of LPS-induced depression model

[0097] C57BL / 6J mice were adaptively fed for one week, then ear-tagged and weighed, and randomly divided into groups. All experimental groups had 5 mice in each group, and the blank group had 3 mice in each group. All mice in the experimental group were weighed and recorded every day within a fixed time period. The total duration of the experiment was two weeks. In the first week, only nasal drops were administered. In the second week, LPS was injected intraperitoneally at the same time as the administration for modeling. Each administration time was the same time in the afternoon. LPS was dissolved in 0.9% saline. The experimental drugs used the Amomum villosum essential oil prepared in Example 1 and the Amomum villosum liposomes of combination 1 in Example 2.

[0098] Experimental procedure: The blank control group only needs to be injected with 0.9% saline intraperitoneally, and the other groups are injected with LPS solution at a dose of 100 μL / 10g for modeling 1.5 hours after intranasal administration according to the dose in Table 6. After two weeks of administration, the mouse behavioral experiments - tail suspension test and forced swimming test - are carried out every other day.

[0099] Table 6 Experimental groups and dosage

[0100]

[0101] 2. Weight changes of experimental mice

[0102] From one week before the experiment to the first week of intranasal administration (-7 to 7 days), the weight of mice increased steadily. Figure 4 As shown in the figure, it shows that Amomum villosum essential oil and Amomum villosum essential oil liposomes did not have a negative impact on the overall physiological condition of mice during this period. In the subsequent LPS injection modeling stage, except for the control group, the weight gain of mice in each experimental group slowed down, among which the weight gain slowdown in the LPS model group was the most significant, while the weight gain slowdown in the AV-H and AV Lip-H groups was relatively mild. This shows that high doses of Amomum villosum essential oil and Amomum villosum essential oil liposomes can slow down the LPS-induced weight gain slowdown to a certain extent without any side effects.

[0103] 3. Tail suspension test (TST)

[0104] Transfer C57BL / 6J mice that had been given the drug for one week from the animal room to the laboratory. Take the mice out of the cage and stick them with medical tape 1 cm away from the tip of the tail so that the animal's tail can be suspended from the hanging rod of the device. The operation process must not cause excess pressure to the mice to prevent them from entering a state of stress. Put the mice in a head-down position, with the tip of the mouse's tail about 30 cm above the ground. Aim the camera lens horizontally at the body of the mouse so that the field of view of the video is complete. The experiment was recorded for a total of 6 minutes, and the animal's immobility time was recorded for the last 4 minutes. After the experiment, the mice were released and put back in the cage.

[0105] The tail suspension test evaluation results are as follows Figure 5 As shown in the figure, lipopolysaccharide (LPS) significantly promoted the immobility (i.e., despair) behavior of mice in the TST, which is consistent with the known effect of LPS in inducing depressive symptoms. In contrast, the Amomum villosum essential oil group and the Amomum villosum essential oil liposome group were able to effectively reduce the immobility time in a dose-dependent manner, and the antidepressant effect of the Amomum villosum essential oil group encapsulated by liposomes was more significant.

[0106] 4. Forced Swim Test (FST)

[0107] The mice were placed in a 1000 mL transparent glass beaker for swimming. The water temperature in the beaker was 25°C and the water height was 35 cm, so that the mice could not stand in the beaker. The experimental groups were separated by opaque baffles and did not affect each other. The entire experimental process was kept quiet to avoid external interference. Each mouse was recorded for 6 minutes, and the time the mouse did not move for 4 minutes after the experiment was recorded, that is, the time the mouse did not struggle to escape and floated on the water surface. After the experiment, the mice were fished out, wiped dry with a towel, and then placed under a fluorescent lamp to dry.

[0108] The results of the FST experiment are as follows Figure 6 As shown in the figure, the swimming immobility time of depression model mice was significantly higher than that of the blank group, and the depression mouse model was successfully established. After drug treatment, the survival desire of mice increased, especially after high-dose Yangchun Amomum villosum essential oil group (AV-H) and high-dose Yangchun Amomum villosum liposome group (AV Lip-H), the immobility time of mice was significantly reduced, indicating that the essential oil and liposome of Yangchun Amomum villosum have significant therapeutic effects on depression model mice, and the liposome group has less immobility time than the essential oil group at the same dose, and the effect is better.

[0109] 5. Immunohistochemistry (IHC)

[0110] (1) Treatment of mice in each group and specimen preparation

[0111] After the behavioral test, the mouse dissection experiment was performed the next day. All experimental supplies were disinfected with alcohol before the experiment. The mice in each group were dissected and the whole brain was carefully removed. The mouse brain was placed in a 5mL EP tube containing 4% paraformaldehyde, fixed in a 4℃ refrigerator for 12h, and replaced with 30% sucrose until the brain tissue sank to the bottom of the EP tube. Then it was transferred to a -80℃ refrigerator for long-term storage.

[0112] (2) Preparation of paraffin sections

[0113] a. Tissue fixation: The freshly removed brain tissue is immersed in 4% paraformaldehyde solution for 24 to 48 hours.

[0114] b. Tissue dehydration: After washing the paraformaldehyde from the sample, immediately start the step-by-step dehydration process to avoid damage to tissue morphology due to rapid water loss. Use gradient alcohol concentrations for continuous dehydration, the specific steps are as follows: 70% alcohol for 6 hours → 85% alcohol for 12 hours → 90% alcohol for 1 hour → 95% alcohol for 1 hour → anhydrous ethanol I for 1 hour → anhydrous ethanol II for 1 hour.

[0115] c. Tissue clearing: Replace the alcohol with xylene solution, and soak the tissue in anhydrous ethanol:xylene (1:1) for 25 minutes → soak the tissue in xylene I for 15 minutes; soak the tissue in xylene II for 10 minutes.

[0116] d. Tissue paraffin immersion: Using the principle that xylene and paraffin are miscible, xylene is removed from the tissue. The operation is carried out at a constant temperature of 60°C, and the tissue is placed in wax liquid I, wax liquid II, and wax liquid III for 1 hour each.

[0117] e. Tissue embedding: Preheat the embedding box in a 60°C incubator and add hot melt paraffin to fix the tissue position for subsequent sectioning. After embedding, the sample is quickly placed in ice water to solidify the paraffin block.

[0118] f. Tissue sectioning: Place the embedded paraffin block on a microtome for cutting. The thickness of the slice is 4 μm.

[0119] g. Slice processing: Spread the slices on the water surface, pick them up with a glass slide and bake them in an oven overnight.

[0120] (3) Experimental operation

[0121] First, dewax the slices and soak them in xylene I and II for 30 minutes to achieve transparency. Then soak the slices in anhydrous ethanol I and II for 10 minutes each, and then in 95%, 90%, 85%, and 70% alcohol for 5 minutes each. Finally, wash with distilled water for 2 minutes.

[0122] a. Perform antigen repair: Place the slices in a beaker filled with citric acid antigen repair solution, heat at 60°C for 8 minutes, let stand for 8 minutes, then adjust the temperature to 50°C and heat for 7 minutes. After heating, remove the slices and cool them down. Finally, place the slices in a culture dish and fix them on a shaker. Rinse the slices with PBS buffer for a total of 3 washes, 5 minutes each time.

[0123] b. Block endogenous peroxidase: soak the slices with 3% hydrogen peroxide and incubate in the dark for 20 minutes. After incubation, place the slices in a culture dish and fix them on a shaker. Rinse the slices with PBS buffer for 3 times, 5 minutes each time.

[0124] c. Blocking serum: Mix 5% animal serum with TBST buffer (the animal source must match the secondary antibody), use a special histochemical pen to draw a circle around the slice on the slide to prevent the added reagent from overflowing. Then add enough serum in this circle and incubate at room temperature for one hour.

[0125] d. Incubate with primary antibody: After the blocking step, gently shake off the serum solution from the slide and add the primary antibody solution to the same area. The dilution factor of the primary antibody solution is 1:100.

[0126] e. Incubation with secondary antibody: After the primary antibody is incubated and bound, place the slide in TBST buffer and wash it 3 times with a shaker at a constant shaking speed, 5 minutes each time. After washing, add the secondary antibody for incubation.

[0127] f. Data analysis: Excel was used to calculate and organize the data, ZEN was used to take screenshots and ipwin32 was used to calculate the optical density, and Graphpad Prism 9 was used for statistical analysis and drawing. "*" indicates p < 0.05, "**" indicates p < 0.01, and p < 0.05 indicates that the difference is statistically significant.

[0128] We analyzed the expression of COX-2 and Bax proteins in the CA3 region of the hippocampus and the cerebral cortex using immunohistochemistry. Figure 7 and Figure 8 As shown in the figure, in the blank control group, the number of positive cells for COX-2 and Bax was relatively small, while in the LPS-induced mouse model group, the number of positive cells for the two proteins increased significantly. Compared with the LPS model group, the positive expression of COX-2 and Bax in the mice treated with Amomum villosum essential oil group and Amomum villosum essential oil liposome group was significantly reduced, showing a dose-dependent improvement effect, and at the same dose, the expression of COX-2 and Bax proteins in the liposome group was more significantly reduced, and the therapeutic effect was significantly improved.

[0129] In summary, the Amomum villosum essential oil liposomes provided by the present invention have uniform particle size distribution, good stability, high embedding rate, and can improve the stability and bioavailability of Amomum villosum essential oil. The Amomum villosum essential oil liposomes prepared by the present invention are used as in vivo therapeutic drugs, which can significantly reduce LPS-induced cell inflammation and mouse depression symptoms, and have more excellent anti-inflammatory and antidepressant effects than Amomum villosum essential oil, not only improve the stability and bioavailability of Amomum villosum essential oil, but also further improve the effect of Amomum villosum essential oil, for preparing more methods for treating inflammation or depression, and for developing new antidepressant therapies based on natural medicines. Provide an important scientific basis.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing Amomum villosum essential oil liposomes, characterized in that: The steps include: S1. Take the wall material, emulsifier, and Amomum villosum essential oil and add them to an organic solvent, stir and heat to dissolve the lipid phase; The wall material is selected from lanosterol or cholesterol, and the emulsifier is selected from one or more of diglyceride, cephalin, egg yolk lecithin, and phosphatidylcholine; the mass ratio of the wall material: emulsifier: Amomum villosum essential oil is: (1-4): (4-17): (1-5); S2. inject the lipid phase into the aqueous phase, stir, remove the organic solvent, and obtain Amomum villosum essential oil liposomes after ultrasonication.

2. The method according to claim 1, characterized in that: In S1, the wall material is selected from lanosterol or cholesterol, and the emulsifier is selected from diglyceride or lecithin.

3. The method according to claim 2, characterized in that: The mass ratio of wall material: emulsifier: Amomum villosum essential oil in S1 is: 1: (4-17): (1-5).

4. The method according to claim 3, characterized in that: The wall material in S1 is lanosterol and the emulsifier is diglyceride.

5. The method according to claim 1, characterized in that: The heating temperature in S1 is 35-40°C.

6. The method according to claim 3, characterized in that: The organic solvent in S1 is ethanol, and the solid-liquid ratio of ethanol to the wall material, emulsifier and Amomum villosum essential oil is 40-80:1 mg / mL.

7. The method according to claim 1, characterized in that: The ultrasonic conditions in S2 are: power 330-400 W, time 30-60 min, temperature -5-0°C.

8. A kind of Amomum villosum essential oil liposome, characterized in that: The invention is prepared by the method according to any one of claims 1 to 7.

9. Use of the Amomum villosum essential oil liposomes according to claim 8 in the preparation of anti-inflammatory or antidepressant drugs.

10. A drug, characterized in that Containing the Amomum villosum essential oil liposomes as claimed in claim 8.

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