Brassica rapa L. polysaccharide nano-liposome as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of Brassica rapa L. polysaccharide nano-liposome. The preparation method comprises the following steps: 1) dissolving lecithin, cholesterol and Tween 80 in an organic solvent to form an organic phase; 2) dissolving the Brassica rapa L. polysaccharide in a PBS solution to prepare a Brassica rapa L. polysaccharide aqueous solution; (3) adding a common turnip polysaccharide aqueous solution into the organic phase prepared in the step (1) while stirring, ultrasonically dispersing uniformly, and fully emulsifying to form a W / O type emulsion; 4) carrying out rotary evaporation on the formed W / O emulsion to form a colloidal state, and adding a PBS solution for rotary hydration to obtain a suspension; and 5) carrying out ultrasonic treatment on the suspension prepared in the step 4), and then filtering to obtain the common turnip polysaccharide nano-liposome. The Brassica rapa L. polysaccharide nano-liposome prepared by the invention has relatively high membrane permeability, excellent electronegativity, stability and drug slow release ability, and can be used in drugs for protecting plateau hypoxia cognitive impairment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical materials, and particularly relates to a radix brassicae napiformis polysaccharide nano-liposome, a preparation method thereof, and an application thereof in the preparation of a drug for protecting against hypoxic cognitive impairment on the plateau. Background Art

[0002] Plateau workers in a hypobaric and hypoxic working environment are prone to central nervous system damage due to insufficient oxygen intake by the body.

[0003] Previous neuroprotective drugs for plateau hypoxia, such as acetylcholinesterase inhibitors and hormonal drugs, can relieve cognitive impairment caused by plateau hypoxia by improving neurotransmitter metabolism and structural plasticity, but continuous administration has adverse reactions. In recent years, plant herbs have received high attention in the prevention and treatment of plateau hypoxic injury and plateau diseases. Rhodiola rosea oral liquid / capsules, Compound Danshen Dripping Pills, anti-plateau disease formula, etc. The traditional Chinese medicine components cover Salvia miltiorrhiza, Panax notoginseng, Borneol, Santalum album, Amomum villosum, American ginseng, etc., and have played an active role in improving hypoxia, promoting blood circulation to remove blood stasis, and relieving symptoms. However, according to the feedback from plateau people, Rhodiola rosea has a slow onset and limited efficacy for acute hypoxia, and its protective effect on neurocognitive impairment in plateau work is also unclear. Moreover, the growth period of Rhodiola rosea is four years. Due to its developed root system, each plant uprooted causes varying degrees of vegetation damage. Therefore, it is urgent to explore new safe and effective neurocognitive impairment protection components.

[0004] On the other hand, radix brassicae napiformis in Tibet, as a cruciferous brassica plant that can be used as both food and medicine, has the effects of being sweet in taste, warm in nature, clearing heat and detoxifying, and tonifying and increasing oxygen. It is generally believed in Tibet that radix brassicae napiformis can prevent hypoxia and resist fatigue. The research on the main component, radix brassicae napiformis polysaccharide, of its freeze-dried powder and water extract confirms that compared with the positive control Dinocton (mainly Rhodiola rosea), radix brassicae napiformis can relieve acute hypoxia (producing a protective effect after 1 day of administration), prolong the hypoxia survival time, and reduce the damage to the structure and function of the hippocampus of the brain caused by hypobaric hypoxia. However, as a plant polysaccharide preparation, radix brassicae napiformis has disadvantages such as poor stability, low bioavailability, easy degradation, and weak cell penetration ability, which affect the exertion of the polysaccharide drug effect. The reason is that most of the conventional dosage form polysaccharides are degraded and metabolized before reaching the action site, so large doses need to be continuously administered.

[0005] Therefore, how to obtain radix brassicae napiformis active components with higher anti-hypoxic activity and improve their pharmacological activity through chemical modification is the key issue for radix brassicae napiformis extracts to enhance anti-hypoxia and improve neurocognitive impairment. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the main object of the present invention is to provide a radix brassicae rapa polysaccharide nano-liposome (BRLP-PL), a preparation method thereof, and an application thereof in the preparation of a drug for protecting against high-altitude hypoxia-induced cognitive loss.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A preparation method of a radix brassicae rapa polysaccharide nano-liposome, comprising the following steps:

[0009] 1) Dissolve lecithin, cholesterol, and Tween 80 in an organic solvent to form an organic phase;

[0010] 2) Dissolve radix brassicae rapa polysaccharide (BRLP) in a PBS solution to prepare an aqueous radix brassicae rapa polysaccharide solution;

[0011] 3) Add all of the aqueous radix brassicae rapa polysaccharide solution to the organic phase prepared in step 1) under stirring, and ultrasonically disperse and emulsify it uniformly to form a W / O emulsion;

[0012] 4) Rotate evaporate the formed W / O emulsion until a colloid is formed, and then add a PBS solution for rotary hydration to obtain a suspension;

[0013] 5) Ultrasonically treat the suspension prepared in step 4), and then filter it to obtain a radix brassicae rapa polysaccharide nano-liposome.

[0014] In some specific embodiments, the mass ratio of lecithin to cholesterol in step 1) is (1-2):(1-4), and the mass concentration of lecithin is 2-6 mg / ml.

[0015] In some specific embodiments, the mass ratio of lecithin to radix brassicae rapa polysaccharide is (3-20):1.

[0016] In some specific embodiments, the organic solvent in step 1) is a chloroform and methanol solution with a volume ratio of (2-6):(1-3).

[0017] In some specific embodiments, the ultrasonic process in step 3) is: the ultrasonic power is 250-350 W, and the ultrasonic treatment time is 2-18 min.

[0018] In some specific embodiments, the temperature of the rotary evaporation in step 4) is 30-50 °C.

[0019] As the same inventive concept, the present invention also provides a radix brassicae rapa polysaccharide nano-liposome.

[0020] As the same inventive concept, the present invention also provides an application of a radix brassicae rapa polysaccharide nano-liposome in the preparation of a drug for protecting against high-altitude hypoxia-induced cognitive impairment.

[0021] Compared with the prior art, the present invention has at least the following advantages:

[0022] 1) In the preparation method of the present invention, by selecting raw materials such as radix raphani polysaccharide and lecithin, and adopting the reverse evaporation-microporous membrane extrusion method, radix raphani polysaccharide nano-liposomes are prepared;

[0023] 2) The radix raphani polysaccharide nano-liposomes in the present invention have a particle size of 100-200 nm, with an obvious layer membrane structure, being spherical vesicles, and having high membrane permeability; the absolute value of their Zeta potential is far higher than 20 mV, indicating that the liposome system has excellent electronegativity and stability; furthermore, by using a mouse in vivo imaging system, it is confirmed that the radix raphani polysaccharide nano-liposomes have excellent drug sustained-release ability, can effectively maintain the drug concentration in the body and extend its action time.

[0024] 3) In the application of the radix raphani polysaccharide nano-liposomes in the present invention for protecting against hypoxic cognitive impairment on the plateau, through testing the effects of radix raphani polysaccharide nano-liposomes on the proliferation of HT22 hippocampal neurons and the damage of HT22 hippocampal neurons under hypobaric hypoxia, the potential application value of BRLP-LP in protecting neurons from hypoxic damage is further confirmed.

[0025] 4) It has been verified that the treatment with BRLP-PL shows significant effects in improving the activity ability, motor ability, exploratory behavior and depressive-like behavior of mice in the hypobaric hypoxia model, providing an experimental basis for the application of BRLP-PL in the treatment of hypoxic cognitive impairment on the plateau. And through the Morris water maze experiment verification, the spatial learning and memory ability of the mice in the BRLP-PL treatment group is significantly improved, providing an important experimental basis for the application of BRLP-PL in improving spatial cognitive dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0027] Figure 1 This is the morphological result of the radix raphani polysaccharide nano-liposomes in the present invention, where A is the apparent morphology of BRLP-PL; B is the transmission electron micrograph of BRLP-PL and Blank-PL;

[0028] Figure 2 This is the particle size distribution diagram of BRLP-PL and Blank-PL in the present invention;

[0029] Figure 3 This is the whole body dynamic distribution characteristic diagram of mice after intragastric administration of DiR fluorescent probe-labeled BRLP-PL and Blank-PL in the present invention;

[0030] Figure 4 The effects of BRLP-PL on the proliferation of HT22 hippocampal neurons and its protective effect on the injury of HT22 hippocampal neurons under hypobaric hypoxia in the present invention. Among them, A is the cytotoxicity test result of BRLP-PL; B is the protective effect of BRLP and BRLP-PL at the same dose on the hypobaric hypoxia injury of HT22 neurons.

[0031] Figure 5 Graphs showing the improvement of the activity, motor ability, exploratory behavior, and depressive-like behavior of mice in the hypobaric hypoxia model by BRLP-PL. Among them

[0032] A is the movement trajectory graph of mice in the open field; B is the total distance; C is the average speed; D is the movement distance in the central grid; E is the number of grid crossings; F is the number of grid crossings in the central grid; G is the curling time; H is the stretching time.

[0033] Figure 6 Graphs showing that BRLP-PL improved the spatial learning and memory impairment of mice acutely exposed to hypobaric hypoxia. Among them, A is the movement trajectory graph of experimental mice in the water maze during the spatial exploration period; B is the percentage of residence time in the target quadrant during the spatial exploration period; C is the number of platform crossings during the spatial exploration period; D is the platform latency during the place navigation period. Detailed implementation manners

[0034] The present invention will be further described in detail below with reference to the drawings and embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0035] When a quantity, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper and lower limit of a value, it should be understood that any range is equivalently disclosed by combining any upper limit or preferred value of the range with any lower limit or preferred value of the range, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values listed herein include the endpoints of the range and all integers and fractions within the range.

[0036] Unless otherwise specified, all percentages, parts, ratios, etc. in this article are by weight.

[0037] The materials, methods, and embodiments in this article are all exemplary and should not be construed as restrictive unless otherwise specified.

[0038] In the following examples, primary hippocampal neurons were extracted by the following method: The cell culture plates were pre-coated with 0.1 mg / mL polylysine and incubated overnight at 4°C. Pregnant rats were anesthetized with 1% sodium pentobarbital, and the abdominal skin was disinfected with 75% alcohol. The fetal rats were transferred from the uterus to a pre-cooled HBSS culture dish. After the fetal rats were dissected, the hippocampi were isolated, collected, minced, digested with 0.125% trypsin for 15 min, the supernatant was aspirated, NB medium was added, and the cell suspension was gently pipetted several times to obtain a cell suspension. After filtering through a 70 μm filter tip, the cell suspension was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended and mixed evenly. The cell suspension was diluted according to the experimental needs and then inoculated onto the pre-coated plates or cell slides. The plates were gently shaken to evenly distribute the cells, and then placed in an incubator for adherent culture. After 4 h, the inoculation medium in the wells was aspirated and replaced with fresh NB maintenance medium at room temperature for routine culture. After 1 day of in vitro culture, most of the primary hippocampal neurons had adhered, and the cell density and distribution were relatively uniform. Some cells had extended 3 - 4 processes, but connections had not yet formed between the processes. On the 3rd day of culture, the neuronal processes were significantly extended, the cell bodies were significantly enlarged, and a sparse network of process connections began to form. On the 7th day of culture, the neuronal cell bodies were plump and began to aggregate, the halos were clearly visible, the processes were thickened and extended, and finally a complex neural network was formed, indicating that the hippocampal neurons had become mature.

[0039] In the following examples, the experimental animals used were healthy C57 / BL6J male mice 8 - 10 weeks old after birth. The experimental mice were housed in a specific pathogen-free animal room with a 12-hour light-dark cycle (7 am to 7 pm), a room temperature of (22 ± 1°C), and an air humidity of 50 - 60%. The food and drinking water for the mice were provided routinely. All animal experiments were conducted in accordance with the relevant regulations of the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Army Medical University.

[0040] Example 1

[0041] This example provides a method for preparing radix brassicae napiformis polysaccharide nano-liposomes, specifically as follows:

[0042] 1) 120 mg of soybean phospholipid, 60 mg of cholesterol, and 20 μL of Tween 80 were dissolved in 8 mL of chloroform and 4 mL of methanol to form an organic phase.

[0043] 2) 5 mg of radix brassicae napiformis polysaccharide was dissolved in 5 mL of PBS to prepare 5 mL of 1 mg / mL radix brassicae napiformis polysaccharide aqueous solution (aqueous phase).

[0044] 3) The radix brassicae napiformis polysaccharide aqueous solution was added to the organic phase prepared in step 1) under stirring and sonicated at a power of 290 W for 10 min to mix evenly and fully emulsify to form a W / O emulsion.

[0045] 4) The formed W / O emulsion was rotary evaporated at a constant temperature of 35 °C until it formed a colloid, and then 12 mL of PBS solution was added and rotary hydrated for 20 min to obtain a suspension;

[0046] 5) The suspension prepared in step 4) was ultrasonically treated for 2 min, and then successively filtered through 0.45 μm and 0.22 μm filter membranes to obtain radix raphani polysaccharide nano-liposomes (abbreviated as BRLP-PL).

[0047] Example 2

[0048] In this test example, the encapsulation efficiency and drug loading of the radix raphani polysaccharide nano-liposomes prepared in step 1) were tested, specifically as follows:

[0049] Materials and test methods:

[0050] The low-temperature high-speed centrifugation method was used to determine the encapsulation efficiency (EE) and drug loading (DL). The nanoparticles and free polysaccharides were separated by centrifugation at 12000 r / min for 30 min at 4 °C, and the content of free polysaccharides in the supernatant was determined by the sulfuric acid-phenol method. The polysaccharide encapsulation efficiency was calculated as shown in formula (1), and the drug loading was calculated as follows:

[0051] Encapsulation efficiency = [(total polysaccharide content - supernatant polysaccharide content) / total polysaccharide content] × 100%

[0052] Drug loading rate = [(total polysaccharide content - supernatant polysaccharide content) / sum of the masses of lecithin and cholesterol] × 100%

[0053] The test showed that the encapsulation efficiency of the radix raphani polysaccharide nano-liposomes in this application was 80.87%, and the drug loading rate was 300 μg of radix raphani polysaccharide per milliliter of radix raphani polysaccharide nano-liposomes (BRLP-PL).

[0054] Example 3

[0055] In this example, the morphology of the prepared radix raphani polysaccharide nano-liposomes was observed and analyzed, specifically as follows:

[0056] In this application, blank liposomes were also introduced as a control group. The preparation method was basically the same as that of the radix raphani polysaccharide nano-liposomes (Example 1), except that in the preparation process, the aqueous solution was replaced with an equal amount of PBS solution to obtain blank nano-liposomes (abbreviated as Blank-PL).

[0057] The radix pseudostellariae polysaccharide liposomes prepared in this application were diluted 10 times with PBS. The diluted radix pseudostellariae polysaccharide liposomes were sucked up with a dropper onto a copper mesh. After natural drying, a 2% phosphotungstic acid aqueous solution was dropped onto the carbon film for negative staining. After the negative staining solution dried, it was placed under a transmission electron microscope (TEM) to observe and photograph the ultrastructure of the liposomes.

[0058] The test results are as Figure 1 shown. The liposomes prepared by the optimal process had a transparent and clear appearance and showed a faint blue opalescence. When observed under a transmission electron microscope, the liposomes had a relatively obvious lamellar structure ( Figure 1 A), and both BRLP-PL and Blank-PL were spherical vesicles ( Figure 1 B).

[0059] Example 4

[0060] In this example, the particle size, polydispersity index, and Zeta potential of the prepared liposomes were characterized and tested. Specifically:

[0061] Materials and test methods;

[0062] The radix pseudostellariae polysaccharide liposomes were measured at a laser intensity of 90° and a temperature of 25°C: diluted with PBS solution, and the average particle size, polydispersity index (PDI), and particle size distribution of the polysaccharide liposomes were measured using a nanoparticle analyzer; the radix pseudostellariae polysaccharide liposomes were measured at an electrode pressure of 1.3 V and a temperature of 25°C, with water as the dispersion medium, and the Zeta potential distribution of the polysaccharide liposomes was measured.

[0063] After diluting the radix pseudostellariae polysaccharide liposomes prepared in Example 1 by 1 time, the particle size, polydispersity coefficient (PDI), and Zeta potential were measured using a laser particle size analyzer. Each group of samples was measured in parallel 3 times, and the results showed ( Figure 2) The average particle size, PDI, and Zeta potential of the blank liposomes (Blank-PL) were 188.66 ± 1.23 nm, 0.15 ± 0.03, and (-57.57 ± 0.23) mV, respectively, while those of the liposomes loaded with radix pseudostellariae polysaccharide (BRLP-PL) were 199.02 ± 1.31 nm, 0.19 ± 0.04, and (-57.63 ± 0.27) mV, respectively. Although the particle size of BRLP-PL increased compared with that of Blank-PL, it still remained within the range of 100 - 200 nm, indicating that it still had high membrane permeability. As a dimensionless parameter reflecting the particle size distribution width, the value of PDI ranges from 0 to 1, and the smaller the value, the more uniform the particle size distribution. Although the PDI of BRLP-PL increased slightly compared with that of Blank-PL, it was still within the normal range, indicating that its particle size distribution was relatively concentrated. The Zeta potential was used to characterize the interaction strength between liposome particles. The larger the absolute value, the higher the stability of the liposomes in the system. Generally, when the absolute value of the Zeta potential is greater than 20 mV, the liposome system is relatively stable. The experimental results showed that the absolute values of the Zeta potential of Blank-PL and BRLP-PL were both much higher than 20 mV, indicating that this liposome system had excellent electronegativity and stability, providing an important physicochemical basis for its application in drug delivery systems.

[0064] Example 5

[0065] This example verified the slow-release performance of radix pseudostellariae polysaccharide nano-liposomes for brain protection, specifically as follows:

[0066] Materials and testing methods: Nano-liposomes were prepared with DiR fluorescent dye, and the final concentration of DiR fluorescent dye was 20 μg / mL. When preparing DiR fluorescently labeled liposomes, 300 μg of DiR fluorescent dye and 15 ml of radix pseudostellariae polysaccharide nano-liposome solution were dissolved together in chloroform solution to prepare nano-liposomes labeled with DiR fluorescent dye (BRLP-PL-DiR), and the final concentration of DiR fluorescent dye was 20 μg / mL.

[0067] The mice were depilated, anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and gavaged with Free-DiR (blank group, DiR fluorescent dye) and BRLP-PL-DiR (0.1 mL / 10 g), with 3 mice in each group. The in vivo status of radix pseudostellariae polysaccharide nano-liposomes labeled with DiR dye in mice was observed using a mouse in vivo fluorescence imaging system at 1, 3, 6, 12, and 24 h.

[0068] Results and discussion: The results are as Figure 3As shown, it can be seen from the figure that as time goes by, the fluorescent enrichment area in the mouse body gradually decreases. Among them, the fluorescent signal in the Free-DiR group almost completely disappears at 24 h, while the BRLP-PL-DiR group still shows significant fluorescent enrichment( Figure 3 ). This result confirms that BRLP-PL has excellent drug sustained-release ability, can effectively maintain the drug concentration in the body and extend its action time, thus providing an important experimental basis for the application of radix cruciferae polysaccharide nano-liposomes in drug delivery systems.

[0069] Example 6

[0070] This example verified the promoting effect of radix cruciferae polysaccharide nano-liposomes on the proliferation of HT22 hippocampal neurons and the protective effect on the injury of HT22 hippocampal neurons under low-pressure hypoxia.

[0071] Materials and testing methods:

[0072] To detect the cytotoxicity of BRLP-PL, based on the results of preliminary pre-experiments (each milliliter of BRLP-PL contains 300 μg of radix cruciferae polysaccharide), different concentrations of BRLP-PL (equivalent to 0 - 300 μg of radix cruciferae polysaccharide) were added to HT22 cells with a confluence of 30%. At the same time, a normal group, a model group, and a blank control group (containing only drugs and culture medium) were set up, with 6 replicates in each group. After the drugs acted for 24 h, the cell viability was detected by the CCK-8 method; to further explore the preventive effects of the same dose of BRLP and BRLP-PL on low-pressure hypoxia nerve injury, after the HT22 cell confluence reached 30%, 1 μg / mL, 3 μg / mL, and 5 μg / mL of BRLP and BRLP-PL were added respectively, and a normal group, a model group, and a blank control group (containing only drugs and culture medium) were set up, with 6 replicates in each group. After the drugs acted for 24 h, a low-pressure hypoxia model was established for 24 h, and after the end, the cell viability was detected by the CCK-8 method;

[0073] All data are expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism software (version 9). The differences between multiple groups were analyzed by one-way analysis of variance (ANOVA). The significance was set at P < 0.05, and P < 0.01 was highly significant.

[0074] Results and discussion:

[0075] By detecting the maximum safe concentration of BRLP-PL, the results are shown( Figure 4A), when the concentration of BRLP-PL was in the range of 400 μg / mL to 18.75 μg / mL, the cell viability was significantly lower than that of the control group (P < 0.0001), indicating that higher concentrations of BRLP-PL had an obvious inhibitory effect on cell viability. However, when the concentration of BRLP-PL decreased to 9.38 μg / mL, although the neuronal viability was lower than that of the control group, the difference was not significant (P > 0.05), while BRLP-PL at other concentrations showed a trend of higher cell viability than the control group. Especially when the concentration of BRLP-PL was 4.69 μg / mL, the cell viability was significantly higher than that of the control group (P < 0.05), indicating that BRLP-PL at this concentration could significantly promote the proliferation and growth of HT22 hippocampal neurons.

[0076] Based on the above results, to simplify the experimental design and further explore the biological effects of BRLP-PL, three concentrations of BRLP-PL, namely 1 μg / mL, 3 μg / mL, and 5 μg / mL, were selected for subsequent experiments. The results are as Figure 4 shown in B. BRLP-PL had a significant preventive effect on HT22 hippocampal neurons damaged by hypobaric hypoxia, and at the same dose, BRLP-PL at 1 μg / mL, 3 μg / mL, and 5 μg / mL showed stronger anti-hypoxic ability than BRLP, further confirming the potential application value of BRLP-LP in protecting neurons from hypoxic damage.

[0077] Example 7

[0078] This example verified the protective effect of radix glucosinolata polysaccharide nanoliposomes on hypobaric hypoxia injury in mice, specifically as follows:

[0079] Materials and testing methods:

[0080] 1) Experimental animals

[0081] All the mice used in the experiment were healthy male C57 / BL6J mice at 8 - 10 weeks old after birth. Some were used for the preparation of a hypobaric hypoxia (HH) cognitive impairment model. The animals were purchased from the Experimental Animal Center of the Army Medical Center of Characteristic Medicine (Chongqing, China), with the certificate number (license number SCXK 2022 - 0011). The mice were housed in the institute's animal house with a 12-hour light and dark cycle, and had free access to food and water. All experimental protocols were approved by the Experimental Animal Welfare and Ethics Committee of the Army Medical University (number: AMUWEC20237402) and were carried out with humane care in accordance with institutional guidelines.

[0082] 2) Grouping of experimental animals, model preparation, and drug administration intervention

[0083] In this study, 72 mice were randomly divided into six groups: normal control group (Control, at an altitude of 390 m, given the same dose of PBS), model group (HH, at an altitude of 6000 m, given the same dose of PBS), model group + blank liposome group (HH + BL, at an altitude of 6000 m, 10 mg / kg / d), model group + salidroside group (HH + Sal, at an altitude of 6000 m, 10 mg / kg / d), model group + radix pseudostellariae polysaccharide group (HH + BRLP, at an altitude of 6000 m, 10 mg / kg / d), and model group + radix pseudostellariae polysaccharide liposome group (HH + BRLP-PL, at an altitude of 6000 m, 10 mg / kg / d). Fourteen days before the experiment, the mice in each group were pretreated with the corresponding drugs. Subsequently, the mice in the model group were placed in an animal experiment low-pressure oxygen chamber and ascended to an altitude of 6000 m at a speed of 300 m / min for a 7-day HH exposure. During the modeling period, drugs were administered at 9:00 am every day, and the low carbon dioxide concentration in the chamber was maintained through the ventilation function of the equipment. After the modeling was completed, behavioral tests were conducted.

[0084] 3) Behavioral tests

[0085] 3.1) Open field test

[0086] The open field test (OFT) is a neurobiological test method based on the study of anxiety. By observing and recording the behavioral performance and movement trajectories of experimental animals in the open field, it evaluates the movement and anxiety-like behaviors of rodents in the face of a novel environment. The experimental mice were transported to the periphery of the open field test area to familiarize themselves with the experimental environment for 2 h. The mice were placed with their backs to the experimenter at the center of a black square device with a bottom size of 100×100×50 cm (the bottom was divided into 16 equal 25×25 cm squares, and the central area consisted of 4 squares in the center), and the experimenter quickly left the test area. The TrackingMaster video recording and analysis software was used to record the real-time movement trajectories of the mice within 10 min. After each test was completed, the excrement of the experimental mice was wiped clean, and the open field was wiped with 75% alcohol solution to remove the residual odor to prevent interference with the results of the next experiment. The experimenter was unaware of the experimental grouping, and the activity data of the mice were recorded.

[0087] 3.2) Morris water maze

[0088] The Morris water maze (MWM) used a circular water tank with a diameter of 200 cm, filled with water at 22°C - 24°C to a depth of at least 30 cm. The pool was evenly divided into 4 quadrants (northeast NE, southeast SE, southwest SW, northwest NW) in the four directions of east, south, west, and north, and 4 fixed images (square, circle, triangle, and rhombus) were pasted at the corresponding positions on the pool wall. In the center of the SW quadrant, a circular platform with a diameter of 10 cm was placed 2 cm below the water surface, which the mice could not see. The water maze experiment consisted of two phases and lasted for 5 days in total. In the first 4-day training phase, each mouse completed four experiments at a fixed time every day, with an interval of 10 minutes between each experiment. In each daily trial, the experimenter placed each mouse facing the starting point on the pool wall into the pool in the set order. During the entire training process, the position of the platform remained unchanged. When the mouse found or did not find the hidden escape platform within the 60-second time limit, the experimenter guided it to the platform, where it stayed for 15 seconds, then was taken out of the water, dried with a towel, and put back into the cage until the next trial. On the 5th day, the escape platform in the pool was removed, and then a spatial exploration test was conducted to evaluate the spatial memory ability of the mice. In this test, the mice were released into the pool from the midpoint of the NE in turn and allowed to swim freely for 60 seconds. The time when the mouse first found the platform, the number of times it crossed the platform, the time it spent on the platform, and the distance it moved on the platform were recorded as parameters to evaluate the spatial memory ability. All behavioral tests were conducted in a dim light environment to ensure consistent experimental conditions.

[0089] 3.3) Statistical processing methods

[0090] All data were expressed as mean ± SEM and statistically analyzed using GraphPad Prism software (version 9). The differences between multiple groups were analyzed by one-way analysis of variance (ANOVA). The significance was set at P < 0.05, and P < 0.01 was highly significant.

[0091] Results and discussion:

[0092] 1) Results of the open field test

[0093] The effects of BRLP-PL on depressive-like behaviors in mice with hypobaric hypoxia-induced models were evaluated through the open field test (OFT) system. The behavioral trajectory graph ( Figure 5 A) showed that mice in the HH group and the Blank-PL+HH group exhibited obvious central avoidance behaviors, while the trajectory distributions of the Sal+HH group, BRLP+HH group, and BRLP-PL+HH group were between those of the control group and the model group, indicating that Sal, BRLP, and BRLP-PL treatments partially alleviated the behavioral abnormalities in the model group.

[0094] In the OFT test, compared with the Control group, the spontaneous activity ability of mice in the HH group and the Blank-PL+HH group was significantly reduced, manifested as a significant decrease in the total movement distance, average speed, and movement distance in the central square (P<0.0001, Figure 5 B-D). Among them, the total distance of the Sal+HH group and the BRLP+HH group was significantly higher than that of the HH group (P<0.001, Figure 5 B), and the total distance of the BRLP-PL+HH group was also significantly higher than that of the Blank-PL+HH group (P<0.0001, Figure 5 B), and BRLP-PL treatment was superior to Sal and BRLP treatments in improving behavior abnormalities induced by low-pressure hypoxia. In terms of average speed, the average speed of the Sal+HH group and the BRLP+HH group was significantly higher than that of the HH group (P<0.01, P<0.001, Figure 5 C), and the average speed of the BRLP-PL+HH group was also significantly higher than that of the Blank-PL+HH group (P<0.001, Figure 5 C). In addition, the movement distance of the BRLP-PL+HH group in the central grid was significantly higher than that of the Blank-PL+HH group (P<0.001, Figure 5 D), further confirming the significant improvement effect of BRLP-PL treatment on the exploration behavior of the model group (P<0.001, Figure 5 D). In terms of the number of grid crossings, the number of grid crossings in the HH group and the Blank-PL+HH group was significantly reduced (P<0.01, P<0.001, Figure 5 E), while the number of grid crossings in the Sal+HH group and the BRLP+HH group was significantly higher than that of the HH group (P<0.01, P<0.01, Figure 5 E), and the number of grid crossings in the BRLP-PL+HH group was also significantly higher than that of the Blank-PL+HH group (P<0.01, Figure 5 E). In terms of the number of central grid crossings, the BRLP+HH group was significantly higher than the HH group (P<0.01, Figure 5 F), and the BRLP-PL+HH group was also significantly higher than the Blank-PL+HH group (P<0.01, Figure 5 F). In terms of anxiety behavior, the huddling time of the Blank-PL+HH group was significantly higher than that of the control group (P<0.0001, Figure 5 G), while the huddling time of the BRLP-PL+HH group was significantly reduced (P<0.0001, Figure 5 G), indicating that BRLP-PL treatment effectively alleviated the anxiety behavior of the model group. At the same time, the stretching time of the HH group and the Blank-PL+HH group was significantly lower than that of the control group (P<0.01, P<0.0001, Figure 5H), while the stretching time in the BRLP-PL + HH group significantly increased (P < 0.0001, Figure 5 H), further confirming the improvement effect of BRLP-PL treatment on depressive-like behaviors. In summary, BRLP-PL treatment showed significant effects in improving the activity ability, motor ability, exploratory behavior, and depressive-like behaviors of mice in the hypobaric hypoxia model, providing an experimental basis for the application of BRLP-PL in the treatment of PTSD.

[0095] 2) Results of the Morris water maze experiment

[0096] In this study, the Morris water maze experiment system was used to evaluate the effects of BRLP-PL on the spatial learning and memory abilities of mice induced by HH. Through comprehensive analysis of indicators such as trajectory maps, residence time in the target quadrant, number of platform crossings, and escape latency, the results were as Figure 6 shown in A. The swimming trajectories of mice in the Control group were relatively concentrated, showing good spatial memory ability, while the trajectories of mice in the HH group and the Blank-PL + HH group were relatively dispersed, and their activities were far from the target quadrant, indicating that their spatial memory ability was significantly impaired. The Sal + HH group and the BRLP-PL group had limited improvement effects on spatial memory ability, while the trajectories of mice in the BRLP + HH group were more concentrated than those in the model group, and their main activities were in the target quadrant area, showing a stronger recovery of spatial memory ability.

[0097] In terms of the residence time in the target quadrant, the residence time of mice in the Control group was significantly longer than that in the HH group and the Blank-PL + HH group (P < 0.5, P < 0.001, Figure 6 B), the residence time of mice in the BRLP + HH group was significantly higher than that in the HH group (P < 0.5, Figure 6 B), and the BRLP-PL + HH group was also significantly higher than the Blank-PL + HH group (P < 0.01, Figure 6 B), indicating that BRLP-PL had a significant improvement effect on spatial memory ability. In terms of the number of platform crossings, the number of crossings of mice in the Control group was significantly higher than that in the HH group and the Blank-PL + HH group (P < 0.5, P < 0.5, Figure 6 C), the Sal + HH group was significantly higher than the HH group (P < 0.5, Figure 6 C), and although the number of crossings of mice in the BRLP-PL + HH group was not significantly different from that in the model group (P > 0.05, Figure 6 C), it was significantly higher than the Blank-PL + HH group (P < 0.01, Figure 6 C), further confirming the improvement effect of BRLP-PL on spatial memory ability.

[0098] In terms of the escape latency, the results of the place navigation experiment showed that the escape latency of mice in all groups gradually decreased over time. However, starting from the second day, the escape latency of mice in the HH group and the Blank-PL+HH group was significantly higher than that in the Control group (P<0.5, Figure 6 D), and this phenomenon persisted until the 4th day; in the Sal+HH group, BRLP group, and BRLP-PL group, the escape latency of mice was significantly shortened starting from the second day and continued until the 4th day, indicating that these treatments had a significant improvement effect on spatial learning ability. In summary, the results of the Morris water maze experiment showed that the spatial learning and memory abilities of mice in the HH group and the Blank-PL+HH group were significantly impaired, while the abilities of mice in the Sal+HH group and BRLP group were restored to some extent. The spatial learning and memory ability of mice in the BRLP-PL treatment group was significantly improved compared with that in the Blank-PL+HH group, providing an important experimental basis for the application of BRLP-PL in improving spatial cognitive dysfunction.

[0099] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered by the scope of the claims and the specification of the present invention.

Claims

1. A preparation method of radix raphani polysaccharide nano-liposomes, characterized in that, It includes the following steps: 1) Dissolve lecithin, cholesterol, and Tween 80 in an organic solvent to form an organic phase; 2) Dissolve the radix pseudostellariae polysaccharide in PBS solution to prepare an aqueous solution of radix pseudostellariae polysaccharide; 3) Add all the aqueous solution of radix pseudostellariae polysaccharide to the organic phase prepared in step 1) under stirring, and disperse it evenly and emulsify it sufficiently by ultrasonic treatment to form a W / O emulsion; 4) Rotate evaporate the formed W / O emulsion until it forms a colloid, and then add PBS solution for rotary hydration to obtain a suspension; 5) Ultrasonically treat the suspension prepared in step 4), and then filter it to obtain radix pseudostellariae polysaccharide nano-liposomes.

2. The preparation method of the radix brassicae napiformis polysaccharide nano-liposome according to claim 1, characterized in that, In step 1), the mass ratio of the lecithin to the cholesterol is (1-2):(1-4), and the mass concentration of the lecithin is 2-6 mg / ml.

3. The preparation method of the radix brassicae napiformis polysaccharide nano-liposome according to claim 1, wherein, The mass ratio of the lecithin to the radix pseudostellariae polysaccharide is (3-20):

1.

4. The preparation method of the radix brassicae napiformis polysaccharide nano-liposome according to claim 1, wherein, The organic solvent in step 1) is a chloroform and methanol solution with a volume ratio of (2-6):(1-3).

5. The preparation method of the radix brassicae napiformis polysaccharide nano-liposome according to claim 4, characterized in that, The ultrasonic process in step 3) is: the ultrasonic power is 250-350 W, and the ultrasonic treatment time is 2-18 min.

6. The preparation method of the radix brassicae napiformis polysaccharide nano-liposome according to claim 4, characterized in that The temperature of the rotary evaporation in step 4) is 30-50 °C.

7. A radix pseudostellariae polysaccharide nano-liposome prepared by the preparation method according to any one of claims 1-6.

8. Use of a radix pseudostellariae polysaccharide nano-liposome according to claim 7 in the preparation of a drug for protecting against hypoxic cognitive impairment on the plateau.

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