CN114306409B - Extraction method of maca exosomes, maca exosomes extracted by extraction method and application of maca exosomes - Google Patents

Abstract

The invention discloses an extraction method of maca exosomes, maca exosomes extracted by the maca exosomes and application of the maca exosomes, wherein the extraction method comprises the following steps: s1, taking fresh maca fruits, removing root hairs, cleaning with tap water, and then washing with deionized water for three times; finally washing the mixture with ultrapure water for three times; s2, cutting fresh maca fruits into blocks, continuously squeezing for three times, filtering, continuously centrifuging juice, performing differential centrifugation, taking supernate, continuously centrifuging the supernate at an ultrahigh speed, taking precipitate, and obtaining precipitate suspension; s3, adding a sucrose solution into a centrifugal tube according to gradient, adding a precipitate suspension into the uppermost layer, and then centrifuging at a super high speed; s4, respectively collecting all the layers after centrifugation, adding ice PBS (phosphate buffer solution) into the washed centrifuge tubes, uniformly mixing, and then centrifuging at a super high speed to remove sucrose; s5, taking and subpackaging the precipitate, resuspending the precipitate with PBS, filtering and subpackaging the precipitate, and storing the filtrate at-80 ℃. The extraction method utilizes sucrose gradient purification, the purity of maca exosomes is improved from 50.1% to 100%, and the yield is improved by nearly 6 times; the maca exosome can be used for preparing medicaments for preventing depression and antidepressants.

Description

Extraction method of maca exosomes, maca exosomes extracted by extraction method and application of maca exosomes

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for extracting maca exosomes, the maca exosomes extracted by the maca exosomes and application of the maca exosomes.

Background

Depression is a common psychological disorder, and is a mental disorder with depressed mood, loss of interest, pessimism, negativity, lack of subjective motility, self-responsibility or self-guilt as main clinical manifestations, and the serious patients may have biased behaviors such as suicide. In China, about 3000 thousands of depression patients are diagnosed at present, the incidence rate reaches 6%, WTO tissue prediction shows that depression becomes the second major clinical chronic disease after hypertension in recent years. Although traditional antidepressant drugs have certain curative effect, most of them have the disadvantages of slow response speed, weak efficacy strength and short action duration. At present, only half of patients have better response to the antidepressant, and more than half of patients with the antidepressant cannot be completely relieved. Therefore, it is of great significance to actively search for effective antidepressants and to explore the mechanism of action.

Chronic, unresolved stress is a major risk factor for the development of depression in the clinic. On this basis, willner et al, 1987, proposed a "Chronic unpredicted Mild stress model" (CUMS), where variability and unpredictability of stress stimuli are critical to the success of model fabrication. After CUMS, behavioral symptoms associated with clinical depression, such as anhedonia, altered grooming behavior, acquired helplessness, and other symptomatic manifestations of major depressive disorder, such as decreased motor ability, social interaction ability, a deficit in aggression ability, etc., are present in rodents. Typically, CUMS-induced behavioral abnormalities can persist for several months, and these induced depressive-like symptoms can be ameliorated by chronic rather than acute treatments, such as the traditional 5 hydroxytryptamine reuptake inhibitor SSRIs. The model can simulate the environmental causes of depression, so that the behavior characteristics change of animals, the increase of plasma corticoids and the like are similar to endogenous depression symptoms, and the classical anti-depression treatment is effective, so that the model has higher value as a depression model and is one of depression models widely used at present.

Maca (Lepidium meyenii, maca) is a medicinal and edible plant of the family Brassicaceae in south America, is native to the 4000-4500 m altitude of the mountain range Peru Andes, and is also named as 'Peru ginseng'. Maca has rich components, mainly comprising: the nutrient supplement comprises a primary metabolite and a secondary metabolite, wherein the primary metabolite mainly contains nutrient components such as polysaccharide, protein and amino acid; the secondary metabolites comprise maca amide, glucosinolate, benzyl isothiocyanate, carboline alkaloid, pyridine alkaloid, imidazole alkaloid, macaene and the like, and are main chemical substances with the pharmacodynamic action of maca. Maca has multiple active functions including blood fat reduction, fatigue resistance, tumor resistance and the like, however, researches on maca exosomes in the aspect of depression resistance are rarely reported. Exosomes are small membrane vesicles (30-150 nm) secreted by cells containing complex micrornas (mirnas) and proteins. Exosome-like nanoparticles containing proteins and mirnas are found in many plants, and these plant exosomes have the potential for large-scale production due to lack of toxicity, and their intrinsic properties have the potential to carry other compounds such as drugs or miRNA molecules. The plant exosomes are about 40-150-nm in diameter, have a phospholipid bilayer structure, are saucer-like or cup-like, and consist of a large number of lipids, including mi RNA and proteins. Research shows that the plant exosome has the functions of resisting inflammation, resisting virus, resisting fibrosis, resisting tumor and the like, and can also participate in defense reaction of pathogen invasion. However, there have been no reports of the use of plant exosomes, particularly maca exosomes, for the prevention or treatment of depression.

Disclosure of Invention

In order to solve the defects of slow effect taking, weak drug effect and short action time of the existing antidepressant drugs, the invention provides a method for extracting maca exosomes, the maca exosomes extracted by the same and application of the maca exosomes.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for extracting maca exosomes comprises the following steps:

s1, taking a certain amount of fresh maca fruits, removing root hairs, washing the maca fruits clean with tap water, and then washing the maca fruits with deionized water for three times, wherein each time lasts for 1-3min; washing with ultrapure water for three times, each for 1-3min;

s2, cutting the cleaned fresh maca fruits into blocks, mixing and crushing the blocks by a juicer, continuously squeezing the blocks for three times, filtering the crushed maca fruits, taking juice, and continuously centrifuging the juice for 1000g and 10min;3000g,20min;10000g,40min; centrifuging at differential speed, collecting supernatant, removing precipitate, centrifuging the supernatant at ultra high speed, centrifuging at 150000g at ultra high speed for 120min, collecting precipitate, and suspending in ice PBS solution to obtain precipitate suspension;

s3, adding a sucrose solution into a centrifugal tube according to gradient, adding the precipitate suspension obtained in the step S2 into the uppermost layer, and then carrying out ultra-high speed centrifugation at 150000g for 120min;

s4, respectively collecting all the layers into a cleaned centrifugal tube after centrifugation is finished, adding an ice PBS solution, uniformly mixing, and then carrying out ultra-high speed centrifugation at 150000g for 90min to remove sucrose;

s5, taking and subpackaging the precipitate, resuspending the precipitate with 1-3ml of PBS, filtering with a 0.22um filter membrane, subpackaging after filtering, and storing at-80 ℃.

Further, the sucrose concentration gradient in step S3 is: 8%, 30%, 45% and 60%.

Furthermore, 3ml of PBS is adopted in the centrifuge tube in the step S3, so that the concentration sucrose gradient purification of the precipitation suspension can be ensured.

The invention also provides the maca exosome obtained by the extraction method.

Furthermore, the purity of the maca exosomes is improved from 50.1% to 100%, and the yield is improved by nearly 6 times.

The invention also provides application of the maca exosomes obtained by the extraction method in preparation of medicaments for preventing depression and antidepressants.

Has the advantages that:

(1) The extraction method disclosed by the invention has the advantages that the sucrose is used for gradient purification, the purity and the yield of the maca exosomes can be greatly improved, and the purity is improved from 50.1% to 100%. The yield is improved by nearly 6 times;

(2) The maca exosome obtained by the invention is applied to preparation of a medicine for preventing depression and treating depression, a mouse depression model is established through a chronic mild unpredictable stress model, the extracted maca exosome is injected into a depressed mouse body in a tail vein injection mode, and the maca exosome is judged to have an anti-depression effect by observing the difference of the behavioural characteristics of the mouse and a fluoxetine group positive control drug and a normal saline negative control group. After the normal group is added with the medicine, the effect of obviously improving the ethology is achieved, and the effect of preventing depression is shown.

Drawings

Figure 1 is a graph demonstrating the results of sucrose gradient centrifugation to improve the purity and yield of exosomes:

fig. 2-5 demonstrate the successful establishment of a UCMS depression model:

FIG. 2 is a graph comparing results of evaluating a UCMS model by sugar water preference;

FIG. 3 is a graph comparing the results of evaluating the UCMS model by forced swimming;

FIG. 4 is a graph comparing results from evaluating a UCMS model by a tail-overhang experiment;

FIG. 5 is a graph comparing the results of evaluating a UCMS model by a novel feeding inhibition experiment;

figures 6-11 demonstrate the antidepressant and antidepressant effects of maca exosomes:

FIG. 6 is a comparison graph of the results of maca exosomes in mouse forced swimming experiments;

FIG. 7 is a comparison graph of the results of the mouse tail suspension experiment with maca exosomes;

FIG. 8 is a graph comparing the results of mouse novelty feeding inhibition experiments with maca exosomes;

FIG. 9 is a graph showing the results of the research on the effect of maca exosomes on the level of 5-hydroxytryptamine (5-HT) in UCMS model mice;

FIG. 10 is a graph showing the results of studies on the influence of maca exosomes on the level of Norepinephrine (NE) in UCMS model mice;

FIG. 11 is a graph showing the results of studies on the effect of maca exosomes on Dopamine (DA) levels in UCMS model mice;

fig. 12 is a parameter setting diagram.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Components not specifically shown in the examples of the present invention are commercially available products, and the equipment or apparatus used is conventional.

Example 1: maca exosome and extraction method thereof

A method for extracting maca exosomes comprises the following steps:

s1, taking 1kg of fresh maca fruits, removing root hairs, washing the maca fruits clean with tap water, and then washing the maca fruits with deionized water for three times, wherein each time lasts for 1-3min; washing with ultrapure water for three times, each for 1-3min;

s2, cutting the cleaned fresh maca fruits into blocks, mixing and crushing the blocks by a juicer, continuously squeezing the blocks for three times, filtering the crushed maca fruits, taking juice, and continuously centrifuging the juice for 1000g and 10min;3000g,20min;10000g,40min; centrifuging at differential speed, collecting supernatant, removing precipitate, centrifuging the supernatant at ultra high speed, centrifuging at 150000g at ultra high speed for 120min, collecting precipitate, and suspending in ice PBS solution to obtain precipitate suspension;

s3, in a centrifugal tube, according to the sucrose concentration gradient: adding sucrose solution 8%, 30%, 45% and 60%, adding the precipitate suspension obtained in step S2 to the uppermost layer, and centrifuging at 150000g at ultra high speed for 120min; meanwhile, 3ml of PBS is adopted in a centrifugal tube, so that the concentration sucrose gradient purification of the precipitation suspension can be ensured;

s4, respectively collecting all the layers into a cleaned centrifugal tube after centrifugation is finished, adding an ice PBS solution, uniformly mixing, and then carrying out ultra-high speed centrifugation at 150000g for 90min to remove sucrose;

s5, taking and subpackaging the precipitate, resuspending the precipitate with 1-3ml of PBS, filtering with a 0.22um filter membrane, subpackaging after filtering, and preserving at-80 ℃; finally obtaining the maca exosome.

And (3) detecting the purity and yield of the obtained maca exosomes:

1.1 The working principle is as follows: the Particle size distribution and density of the exosome particles are measured by a nanoparticle tracking analyzer (Particle metric GmbH, germany), the purity of the exosome particles is preliminarily judged according to the distribution of peaks, and the less the number of peaks, the higher the purity; the yield of the exosome granules is judged by the number of exosomes per ml, and the larger the number is, the larger the yield is.

1.2 detection method:

(1) After sucrose gradient purification centrifugation, the exosome purity and yield are improved: respectively preparing sucrose gradient solutions (8%, 30%, 45% and 60%) by adopting a Tris-HCl solution with the concentration of 20mmol/L, pH being 7.2, adding the exosomes extracted primarily, and performing gradient centrifugation;

(2) The Particle size distribution and purity were analyzed by a nanoparticle tracking analyzer (Particle metric GmbH, germany): 5 microliter of exosome is absorbed and added into an instrument, a laser light source is used for irradiating the nanoparticle suspension, the Brownian motion and electrophoresis phenomena of single nanoparticles can be observed by using a full black background, and tracking, particle size measurement, zeta potential measurement, concentration measurement and the like of the single nanoparticles can be realized.

As shown in FIG. 1, the study results show that 3 peaks appear in the particle size before purification, the particle size is 135.5, 158.3, 347.3nm, the corresponding purity is 50.1, 46.6, 3.2%, and the number of vesicles per ml is 1.1E +6, 1.0E +6, 1.1E +6, respectively. According to the particle size (40-150 nm), the maca exosome size in the determination is 135.5nm, the corresponding purity is 50.1%, and the number of exosomes per ml is 1.1E +6 respectively. After the sucrose gradient centrifugal purification, 1 peak appears in the particle size, the particle size is 137.7nm, the corresponding purity is 100%, and the number of exosomes per ml is 6.3E +6. Therefore, the purity is improved to 100% from 50.1%, the amount of exosome is improved to 6.3E +6 from 1.1E +6, and the yield is improved by nearly 6 times.

Example 2: application of maca exosomes in preparation of medicines for preventing depression and antidepressants

In the embodiment of the invention, a C57BL/6J mouse chronic stress depression model (CUMS) is adopted, antidepressant drugs (fluoxetine, FLUU) are used as positive control drugs, PBS is used as a negative control group, and maca exosomes are injected intravenously, namely the experimental groups are as follows: normal group (control), control + maca (100 ug/kg), UCMS + PBS, UCMS + maca (50 ug/kg), UCMS + maca (100 ug/kg), UCMS + maca (200 ug/kg), and FLU (10 mg/kg), and their depression-preventing and depression-preventing effects were observed and compared in different experiments.

2.1 materials of investigation

2.1.1 animals: the clean grade C57BL/6J mice are fed with 18-25g of normal-temperature normal-humidity sterile feed all around, and all experiments follow the ethical specifications of experimental animals of Xuzhou medical university;

2.1.2 reagents: maca exosomes (obtained using the method in example 1), fluoxetine (10 mg/kg), PBS;

2.1.3 Instrument: tail-hanging apparatus, forced swimming apparatus, open field apparatus, 50cm × 50cm × 20cm white plastic box, 50ml sugar water bottle, 14cm glass beaker and shaker.

2.2 Research method

2.2.1 establishment of model of Chronic stress Depression (CUMS)

Mice in the UCMS group are randomly stressed by two or three types of stresses with different properties every day for 8 weeks, so that the animals cannot expect the stimulation, and the adaptation is avoided. Random stress was done in the following manner: feeding all model groups of mice in a single cage; clean water was sprayed to each standard cage to wet the bedding and the animals were placed in the wet cages for 24h. The amount of sprayed water can be adjusted according to the size of the squirrel cage so that the padding is completely wet but no water accumulation is caused; each cage was removed for 24h of bedding, after which the animals were transferred to the empty cages 12h required for another stressor; the cage is inclined for 12h at an angle of 45 degrees, the mouse cage can lean against a stable object, and the animal can be ensured to move in the cage without influencing the position of the mouse cage; the normal 12h day/12 h night cycle was changed to a continuous cycle every 30min for 8h. Subsequently, the animals were returned to the normal day-night cycle; transferring each animal from its own cage to the cages of adjacent animals, and then separating them; all bedding was removed and water was added at a depth of 0.25 feet (mouse) or 0.5 feet (rat) for 4 hours. Care was taken to maintain the water temperature-30 ℃ to minimize the potential for body temperature drop, while animals were simply dried with a soft towel before being placed in the new cage; exposing the animal to an environment containing predator scent/sound for 24 hours by randomly spreading fur samples or urine (10-20 ml) originating from natural predators into the cage, or broadcasting natural predator growling or predatory noise adjacent to the cage for 24 hours; swimming with ice water (4 deg.C) for 5min twice a day; fasting and water prohibition are carried out for 24 hours; a flash lamp 12h; at the end of each day of stress, all animals were placed in clean cages and returned to their rearing status.

2.2.2 Evaluation experiment of mouse depression model

(1) Preference of sugar water

Animals were habituated to drinking 1% sucrose solution 48h before testing, acclimated to rearing and provided two bottles (1% sucrose solution VS drinking water) for 24h, recording the consumption volumes of 1% sucrose and drinking water, respectively, and calculating according to the formula: [ sugar water consumption/(drinking water consumption + sugar water consumption) ]. Times.100%;

(2) Forced swimming experiment

The mice were swimming trained 24 hours in advance, and placed in a 2L beaker containing 1200ml at 22-24 ℃ for 10 minutes. On the day of the experiment, the mice were placed in large beakers under the same conditions, observed for 6 minutes in total, and the "immobility" time 5 minutes after the observation was recorded;

(3) Tail suspension experiment

The tail of the tail-suspended mouse is adhered to a stable rod (20 cm from the ground, and 2cm from the tail tip part of the tail is adhered by an adhesive tape); observing the behavior of the experimental animal within 6 minutes, and recording the sum of the 'immobility' time of the mouse within 5 minutes;

(4) Novel feeding inhibition experiment

The mice were fasted 24 hours earlier and the next day Jiang Xiaoshu was placed in a large open topped box prepared with some food such as melon seeds or nuts and jelly in the middle. Mice fasted for 24 hours were then placed along the box edges and a timer was started, recording the time at which the mice began to eat within ten minutes (recorded as 10 minutes over 10 minutes before they had not eaten). The food intake is calculated only when the mice begin to bite the melon seeds, and the food intake is not calculated when the mice are grabbed and licked or smelled; after recording, the mice were placed in individual cages, weighed feed was added and the feed consumption was recorded over five minutes.

2.2.3 behavioural assays

(1) Open field experiment

Open field testing is a commonly used behavioural test for determining the general motor capacity, anxiety level and search desire of animals. The bottom area is 96cm multiplied by 96cm, and the height of the four-wall wood board is 50cm. After 1h acclimation of the animals in the test room, the mice were taken out of the squirrel cage and placed in the center of the bottom of the open field, allowed to freely explore the mine field for 5 minutes, and the distance explored was recorded. After each rat experiment was completed, the whole open field was cleaned with 75% alcohol, and after the alcohol smell completely dissipated, the next rat was tested. Recording and analyzing the distance of the rat open field experiment movement by using an AnyMaze video tracking system and a video tracking system;

(2) Water maze

The Morris Water Maze (MWM) is an experiment for forcing an experimental animal to swim, observing and recording the time and the track of the animal hidden in a water platform after entering the water, and deducing the learning, memory, spatial cognition and other aspects of the animal after analysis. The memory capacity of rats was determined by Morris water maze test in 14d experimental groups after ischemia/reperfusion in this study. Acquired training was performed on days 10-13 post-ischemia, starting at 9 am, 00, 4 times daily, with intervals of about 1h, for 4 consecutive days. In water, the rats were placed in the water with either quadrant facing the pool wall, which looked for an underwater platform. On the 14 th day, the platform is removed, all rats are placed into the water from the water inlet point of the third quadrant facing the pool wall, and the monitoring system automatically tracks and records the movement condition within 90 s;

(3) Elevated cross maze

The elevated plus maze is about 70cm above the ground and consists of two opaque closed arms 14cm high in opposite directions and two open arms in opposite directions (30 cm. Times.5 cm). At the beginning of the experiment, mice were placed in the center of the maze facing one open arm, and the duration of the experiment was recorded for 3 minutes. After each test, 50% ethanol solution was used to eliminate the off-flavor left by the subjects before. The time in the closed and open arms of the mice within 3 minutes was recorded using a stopwatch.

2.2.4 Tissue sample testing

The grouping method is the same as above, and the chemical component analysis is performed by taking blood from mouse eyeball, and the chromatographic separation is performed on the target compound through a liquid chromatographic column by using an ultra performance liquid chromatograph (UPLC-MS/MS). The content of 5-HT, NE and DA which are closely related to depression in each group is respectively determined and compared.

2.2.4.1 Experimental procedure

(1) Extraction of metabolites: precipitating 10 μ L serum sample with 40 μ L methanol, vortexing at 2000 rpm for 3min, centrifuging at 4 deg.C and 13000 rpm for 5min, collecting supernatant 40 μ L, and placing on sample injection vial;

(2) Preparation of a standard solution: diluting the stock solution of the standard substance by a certain multiple of times with methanol to prepare a mother solution of the standard substance, and configuring a working curve of the standard substance by using the mother solution of the standard substance, wherein the concentration is 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 300 in ng/mL.

2.2.4.2 UPLC-MS/MS parameter settings

(1) UPLC parameters, see FIG. 12 (a)

Liquid phase gradient is shown in FIG. 12 (b)

(2) MS parameters, see fig. 12 (c).

2.3 results of the study

2.3.1 mouse Depression model evaluation results

(1) Evaluation of UCMS model by syrup preference

And (4) verifying the success of CUMS modeling of the mouse through a sugar water preference experiment. As shown in fig. 2, the Control group (i.e., the unstressed group) shows a high sugar water preference rate, while the UCMS group (i.e., the model group) shows a low sugar water preference rate, but the individual difference is large, and different individuals show different sugar water and are distributed unevenly.

(2) Evaluation of UCMS model by forced swimming

And (4) verifying the success of CUMS modeling of the mouse through a forced swimming experiment. As shown in FIG. 3, the forced swimming time of the Control group and UCMS group was compared, and it was shown that the mice in the model group had long immobility time, while those in the Control group had short immobility time.

(3) Evaluation of UCMS model by tail suspension experiment

Through a tail suspension experiment, the success of CUMS modeling of the mouse is verified. As shown in fig. 4, the tail suspension experiment of the mice in the Control group and the UCMS group showed that the immobility time of the model group was longer and the immobility time of the Control group was shorter as a whole.

(4) Evaluation of UCMS model by novel feeding inhibition experiment

The success of CUMS modeling of the mice is verified through a novel feeding inhibition experiment. As shown in fig. 5, the mice of the Control group and the UCMS group compared with the novel feeding inhibition experiment, which is shown in that the incubation period for eating in the model group is longer than that for eating in the Control group within ten minutes.

2.3.2 Post-dose ethological testing of groups

(1) Performance of maca exosomes in forced swimming experiments of mice

The experiment is divided into 5 groups, the grouping method is the same as the above, the forced swimming experiment of the mice is carried out, as shown in fig. 6, compared with the Control group, the Control + maca group has obviously less immobility time, and the maca exosomes have the effect of preventing depression; compared with UCMS, the immobility time of UCMS + maca (50, 100 and 200 mu g/kg) is obviously shortened, which indicates that maca exosomes have the effect of treating depression; compared with the positive control drug FLU group, UCMS + maca (50, 100 and 200 mu g/kg) has obviously shorter time, which indicates that the effect of maca exosomes on treating depression is better than that of the FLU. Therefore, the maca exosomes have the advantages of reducing the immobility time of mice in a forced swimming experiment of the mice, and preventing and resisting depression.

(2) Performance of maca exosomes in mouse tail suspension experiment

The experimental groups are the same as the above, and the tail suspension experiment of the mice is carried out, as shown in fig. 7, compared with the Control group, the tail suspension time of the Control + maca group is obviously reduced, which indicates that maca exosomes have the effect of preventing depression; compared with UCMS, the time of UCMS + maca (50, 100 and 200 mu g/kg) is obviously shortened, and the dose dependence is realized, which indicates that maca exosomes have the effect of treating depression; compared with the positive control drug FLU group, UCMS + maca (200 mug/kg) has less mouse immobility time, which indicates that the effect of maca exosome on treating depression is better than that of the FLU. Therefore, the maca exosomes have the advantages of reducing the immobility time of mice in a mouse tail suspension experiment, and preventing and resisting depression.

(3) Performance of maca exosomes in mice novelty inhibition feeding experiments

The experimental groups are the same as the above, and a mouse novelty inhibition feeding experiment is carried out, as shown in fig. 8, compared with the Control group, the Control + maca group has obviously reduced feeding latency, which indicates that maca exosomes have a depression preventing effect; compared with UCMS, UCMS + maca (50, 100 and 200 mu g/kg) has a latent eating period and dose dependence, and shows that maca exosomes have a depression treatment effect; compared with the positive control drug FLU group, UCMS + maca (100, 200 mu g/kg) has obviously reduced feeding latency, which indicates that the effect of maca exosome on depression treatment is better than that of the FLU. Therefore, the maca exosomes have obvious advantages of preventing depression and resisting depression in a mouse novel feeding inhibition experiment.

The method establishes a mouse depression model through chronic mild unpredictable stress, and verifies whether the molding is successful or not through ethological detection such as sugar water preference, forced swimming, tail suspension test, novel inhibition feeding test and the like; maca exosomes are injected into veins of a normal group or a model group, and the effects of preventing depression and resisting depression are observed through ethological detection such as forced swimming, tail suspension test, novelty inhibition feeding test and the like. The modeling results of this experiment are consistent with the results of the earlier expected study.

2.3.3 Targeted Metabonomics detection of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA)

Known researches show that the expression of 5-HT, NE and DA in depression patients is low, the content of the 5-HT, NE and DA is lower than that of normal people, and the lower the content of the 5-HT, NE and DA, the more serious the clinical symptoms are, the expression and significance reaction of the 5-HT, NE and DA in the depression patients are found, so that the values of the 5-HT, NE and DA can be further researched, the research evidence is better provided for clinical treatment, and the experimental results are as follows.

(1) The maca exosomes can improve the level of 5-hydroxytryptamine (5-HT) in UCMS model mice

As shown in FIG. 9, the control + maca (100 ug/kg) group slightly increased the level of 5-HT in mice under normal conditions as compared to the control group; compared with UCMS model group, tail vein injection of 50ug/kg,100ug/kg and 200ug/kg maca exosomes obviously improves 5-HT level and has statistical significance.

(2) Maca exosomes can increase Norepinephrine (NE) levels in UCMS model mice

As shown in FIG. 10, under normal conditions, the NE levels in mice did not differ much from the control + maca (100 ug/kg) compared to the control group; compared with UCMS model group, 100ug/kg and 200ug/kg maca exosomes injected into tail vein can obviously improve NE level of model mice, and have statistical significance.

(3) Maca exosomes can improve Dopamine (DA) level of UCMS model mice

As shown in FIG. 11, under normal conditions, the DA levels in mice were not significantly different when control + maca (100 ug/kg) was compared to the control group; compared with UCMS model groups, the tail vein injection of maca exosomes improves the DA level of the model mice to different degrees, has more obvious effect of 200ug/kg, and has statistical significance.

The results show that the 5-HT, NE and DA are obviously reduced after the exosome is added into the model, and the model has a better anti-depression effect.

Claims (4)

1. The extraction method of the maca exosomes is characterized by comprising the following steps:

s1, taking 1kg of fresh maca fruits, removing root hairs, washing the maca fruits clean with tap water, and then washing the maca fruits for three times with deionized water for 1-3min each time; washing with ultrapure water for three times, each for 1-3min;

s2, cutting the cleaned fresh maca fruits into blocks, mixing and crushing the blocks by a juicer, continuously squeezing the blocks for three times, filtering the crushed maca fruits, taking juice, and continuously centrifuging the juice for 1000g and 10min;3000g,20min;10000g,40min; centrifuging at differential speed, collecting supernatant, removing precipitate, centrifuging the supernatant at ultra high speed, centrifuging at 150000g at ultra high speed for 120min, collecting precipitate, and suspending in ice PBS solution to obtain precipitate suspension;

s3, adding a sucrose solution into a centrifugal tube according to a gradient, wherein the sucrose concentration gradient is as follows: 8%, 30%, 45% and 60%; adding the precipitate suspension obtained in the step S2 into the uppermost layer, and then performing ultra-high speed centrifugation at 150000g for 120min; 3ml of PBS is adopted in a centrifugal tube, so that the concentration sucrose gradient purification of the precipitation suspension can be ensured;

s4, respectively collecting all the layers into a cleaned centrifugal tube after centrifugation is finished, adding an ice PBS solution, uniformly mixing, and then carrying out ultra-high speed centrifugation at 150000g for 90min to remove sucrose;

s5, taking and subpackaging the precipitate, resuspending the precipitate with 1-3ml of PBS, filtering with a 0.22um filter membrane, subpackaging after filtering, and storing at-80 ℃.

2. A maca exosome, which is prepared by the extraction method of the maca exosome according to claim 1.

3. The maca exosome according to claim 2, wherein the purity of the maca exosome is increased from 50.1% to 100%, and the yield is increased by approximately 6 times.

4. Use of maca exosomes according to claim 2 or 3 in the preparation of a medicament for the prevention of depression, antidepressants.

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