Radix cynanchi bungei exosome as well as extraction method and application thereof

Abstract

The invention discloses a radix cynanchi bungei exosome as well as an extraction method and application thereof, and belongs to the technical field of biology. The extraction method of the radix cynanchi bungei exosome comprises the following steps: removing root hairs from fresh radix cynanchi bungei, cutting into blocks, juicing, taking filtrate, carrying out continuous centrifugation and differential centrifugation, taking supernate, continuously carrying out ultra-high-speed centrifugation on the supernate, taking precipitate, and resuspending the precipitate, so as to obtain the radix cynanchi bungei exosome extract. According to the application of the radix cynanchi bungei exosome in preparation of the medicine for preventing and treating the Alzheimer's disease, a 3 * TgAD transgenic homozygous mouse is applied, then the extracted radix cynanchi bungei exosome is injected into the AD mouse body in a tail vein injection mode, small animal behavioristics are adopted, and the application of the radix cynanchi bungei exosome in preparation of the medicine for preventing and treating the Alzheimer's disease is achieved. The cognitive behavioral change of the mouse is observed by methods such as open field opening, new object recognition, water maze and the like, and it is verified that the radix cynanchi bungei exosome improves the cognitive impairment of the AD mouse.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an exosome of Polygonum multiflorum, its extraction method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease in modern times. In 2018, the Alzheimer's Disease International (ADI) released a report indicating that there are currently more than 50 million AD patients worldwide, and with the aging population, the number is expected to increase to 152 million by 2050. In my country, as of 2024, there were already 15 million registered AD patients, accounting for 1% of the total population. AD has become a significant factor affecting the quality of life of the elderly. Currently, FDA-approved drugs for the treatment of AD mainly fall into two categories: acetylcholinesterase inhibitors (AChEIs), such as domperidone, rivastigmine, and galantamine; and NMDA receptor antagonists, such as cyproheptadine. However, these drugs only help improve the quality of life of AD patients and do not significantly improve or slow down the disease's root causes or progression. In addition, patients taking these drugs may experience adverse reactions such as vomiting, insomnia, and diarrhea, resulting in low patient compliance and unsatisfactory treatment outcomes. Therefore, developing novel anti-AD drugs with low toxicity and high efficacy is an urgent problem that needs to be solved.

[0003] Binhai white fleeceflower root is a traditional medicinal herb and economic crop of Jiangsu Province. Binhai County has cultivated white fleeceflower root for over 300 years and is a famous "hometown of fleeceflower root" in my country, accounting for over 95% of the national output. White fleeceflower root mainly comes from the tuberous roots of *Cynanchumauriculatum* Royleex Wight, *Cynanchum wilfordii*, and *Cynanchumbungei* Decne, all belonging to the Asclepiadaceae family. It has a sweet and bitter taste and is slightly warm in nature. Traditional Chinese medicine believes it has the effects of nourishing blood and liver, strengthening kidneys and replenishing essence, blackening hair, and prolonging life. It is described in Tang Dynasty texts such as *He Shou Wu Lu*, *Tu Jing Ben Cao*, *Zheng Lei Ben Cao*, and *Ben Cao Gang Mu*. Currently, it is mainly used as a tonic in Jiangsu, Shandong, and Jilin provinces. Binhai white fleeceflower root belongs to *Cynanchumauriculatum* Royleex Wight, and its use in Jiangsu has a history of several hundred years, making it a major source of the traditional Chinese medicine white fleeceflower root. The C21 steroidal skeleton in *Polygonum multiflorum* is a typical pregnane derivative. The β-OH groups at C-12 and C-20 readily form glycosides with sugars, mostly 2,6-deoxyglucoses, such as digitoxose (digitox), diginose, digin, oleandrose (ole), cymarose (cym), and glucose (glc). The sugars are linked by a 1→4 linkage. C21 steroidal esters are the most abundant and the most important active ingredient found in *Polygonum multiflorum*. Numerous studies have reported that *Polygonum multiflorum* possesses anti-tumor, immune-enhancing, liver-protective, anti-aging, and neuroprotective effects.

[0004] Exosomes are small membrane vesicles (30-150 nm) secreted by cells containing complex microRNAs (miRNAs) and proteins. Exosome-like nanoparticles containing proteins and miRNAs have been found in many plants. These plant exosomes, due to their lack of toxicity, have the potential for large-scale production, and their inherent properties suggest the possibility of carrying drugs or other compounds such as miRNA molecules. Plant exosomes are approximately 40-150 nm in diameter, possess a phospholipid bilayer structure, and are cup-shaped or saucer-shaped, composed of abundant lipids, including miRNAs and proteins. Studies have shown that plant exosomes have anti-inflammatory, antiviral, anti-fibrotic, and antitumor effects, and can also participate in the defense response against pathogen invasion. However, there are currently no reports of using plant exosomes, especially those from *Polygonum multiflorum* (white shou wu), to prevent or treat Alzheimer's disease.

[0005] In summary, to address the aforementioned issues, we propose a method for extracting exosomes from Polygonum multiflorum and their application in neuroprotection. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting and applying *Polygonum multiflorum* exosomes, in order to solve the problems existing in the prior art. To address the shortcomings of current Alzheimer's disease treatments, such as slow onset of action, weak efficacy, short duration of action, and inability to cross the blood-brain barrier, this invention provides a method for extracting *Polygonum multiflorum* exosomes and their application in reducing Aβ-induced neuronal damage and improving cognitive impairment in Alzheimer's disease.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of this invention is a method for extracting exosome extracts from Polygonum multiflorum, comprising the following steps:

[0009] Remove the roots from fresh white fleeceflower root, cut it into pieces, and extract the juice. Take the filtrate and centrifuge it continuously and then centrifuge it at a differential speed. Take the supernatant, centrifuge the supernatant at an ultra-high speed, and then take the precipitate. Resuspend the precipitate to obtain the white fleeceflower root exudate extract.

[0010] The continuous centrifugation method is as follows: centrifuge continuously 3 times, each time for 30-60 minutes;

[0011] The ultra-high speed centrifugation method is as follows: centrifuge 1000g for 12-15min for the first time, and then take the supernatant after centrifugation;

[0012] Centrifuge a second time at 3000g for 25-30 minutes, and collect the supernatant after centrifugation;

[0013] Centrifuge for the third time at 10000g for 50-40 minutes, and collect the supernatant after centrifugation;

[0014] Centrifuge for the fourth time at 150,000g for 70-80 minutes, and collect the precipitate after centrifugation.

[0015] Preferably, the juicing time is 20-30 minutes, and the juice is filtered through gauze after juicing.

[0016] The second technical solution of the present invention is the *Polygonum multiflorum* exosomes extracted by the above extraction method.

[0017] The third technical solution of this invention is the application of the above-mentioned Polygonum multiflorum exosomes in the preparation of neuroprotective drugs.

[0018] The fourth technical solution of the present invention is the application of the above-mentioned Polygonum multiflorum exosome extract in the preparation of drugs for preventing Alzheimer's disease.

[0019] The fifth technical solution of the present invention is a pharmaceutical composition for treating Alzheimer's disease, using the above-mentioned Polygonum multiflorum exosomes as the active ingredient.

[0020] Based on the above technical solution, the present invention has the following technical effects:

[0021] The extraction steps of *Polygonum multiflorum* exosomes in this invention are simple to operate, have a high recovery rate, and can obtain a high-purity, high-concentration *Polygonum multiflorum* exosome solution without intermediate contamination.

[0022] The extraction method of this invention utilizes ultra-high-speed centrifugation, which can greatly improve the purity and yield of Polygonum multiflorum exosomes, increasing the purity from 50% to 100%.

[0023] The application of the white Polygonum multiflorum exosomes obtained in this invention in the preparation of drugs for the prevention and treatment of Alzheimer's disease was demonstrated by using 3×TgAD transgenic homozygous mice and injecting the extracted white Polygonum multiflorum exosomes into AD mice via tail vein injection. Small animal behavioral methods, such as open field, new object recognition, and water maze, were used to observe changes in the cognitive behavior of the mice and verify that the white Polygonum multiflorum exosomes improve cognitive impairment in AD mice. Detailed Implementation

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] Example 1

[0026] A method for extracting exosomes from Polygonum multiflorum includes the following steps:

[0027] S1. Take fresh white Polygonum multiflorum fruit, remove the roots and wash it clean. First, wash it with deionized water 3-5 times, 3-5 minutes each time; then wash it with ultrapure water 2-3 times, 2-3 minutes each time.

[0028] S2. Cut the washed white fleeceflower root fruit from step S1 into chunks, use a juicer to continuously crush and extract the juice for 20-30 minutes, filter it through gauze, and centrifuge the juice four times consecutively.

[0029] Centrifuge at 1000g for 12-15 minutes. After centrifugation, collect the supernatant and transfer it into a new 50ml centrifuge tube.

[0030] Centrifuge a second time at 3000g for 25-30 minutes. After centrifugation, collect the supernatant and transfer it into a new 50ml centrifuge tube.

[0031] Centrifuge for the third time at 10000g for 50-40 minutes. After centrifugation, collect the supernatant and transfer it into a new 50ml centrifuge tube.

[0032] Centrifuge for the fourth time at 150,000g for 70-80 min, remove the supernatant to obtain the crude extract, and resuspend the precipitate in 1-2 ml of PBS.

[0033] Take the precipitate, dilute the supernatant with sterile PBS, and dispense the filtered liquid into centrifuge tubes to obtain a precipitate suspension;

[0034] S3. Take the precipitate suspension from step S2, remove the supernatant, retain the precipitate, add an appropriate amount of sterile PBS to the centrifuge tube to resuspend the precipitate, collect the exudate extract of Polygonum multiflorum and put it into an EP tube, store it in the refrigerator for later use, wherein the refrigerator temperature is controlled at -75 to -95℃.

[0035] The purity and yield of the obtained Polygonum multiflorum exosomes were tested:

[0036] Working principle: The particle size distribution and density of exosomes are determined by a nanoparticle tracking analyzer (Particle Metrix GmbH, Germany). The purity is initially judged based on the peak distribution, with fewer peaks indicating higher purity. The yield of exosomes is determined by the number of exosomes per ml, with a higher yield indicating a higher number of exosomes.

[0037] Detection method:

[0038] (1) Improve the purity and yield of exosomes after ultracentrifugation: Prepare sucrose gradient solutions (8%, 30%, 45%, 60%) using 20 mmol / L Tris-HCl solution with pH 7.2, add the pre-extracted exosomes, and perform gradient centrifugation;

[0039] (2) The particle size distribution and purity were analyzed using a nanoparticle tracking analyzer (Particle Metrix GmbH, Germany): 5 microliters of exosomes were added to the instrument, and the nanoparticle suspension was irradiated with a laser light source. The Brownian motion and electrophoresis of individual nanoparticles could be observed against a completely black background. This instrument can track individual nanoparticles, measure particle size, and measure concentration.

[0040] The results showed that the size of *Polygonum multiflorum* was 130.7 nm in this study, and the number of exosomes per ml was 6.2E+10. Transmission electron microscopy showed that the exosomes of *Polygonum multiflorum* exhibited a typical invaginated double membrane structure.

[0041] Example 2

[0042] Application of *Polygonum multiflorum* exosomes in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

[0043] In this invention, 3×Tg AD transgenic (Psen1, APPSwe, TauP301L allele mutation) homozygous mice were used, with PBS as the negative control group. Exosomes of Polygonum multiflorum were injected intravenously. The animal experimental groups were: AD+PBS group, AD+CbD-EXO (10mg / kg), and AD+CbD-EXO (20mg / kg); the cell experimental groups were: NC group, APP group, APP+CbD-EXO (1ug / ml) group, APP+CbD-EXO (10ug / ml) group, and APP+CbD-EXO (100ug / ml) group. Through different experiments, its preventive and therapeutic effects on Alzheimer's disease were observed and compared.

[0044] (1) Research materials

[0045] (11) Animals: Clean-grade 3×Tg AD transgenic AD mice, 10-11 months old, 25-30g, normally fed, room temperature and humidity, and sterile. All experiments were conducted in accordance with the experimental animal ethics guidelines of Jiangsu Medical Vocational College.

[0046] (12) Cells: SY5Y, NC, APPOE;

[0047] (13) Reagents: Exosomes of Polygonum multiflorum (obtained using the method in Example 1), PBS;

[0048] (14) Instruments: water maze instrument, new object recognition instrument, open field instrument, elevated cross maze instrument, tail vein injection instrument, centrifuge, particle microscope, small animal imaging instrument, Leica microscope.

[0049] (2) Research Methods

[0050] (21) Establishment of Alzheimer's disease model (AD)

[0051] The 3×Tg AD transgenic (Psen1,APPSwe, TauP301L allele mutation) homozygous mice were used (these mice showed gradually increasing Aβ deposition at 3 months of age, impaired synaptic transmission function at 6 months of age, and hyperphosphorylated tau protein and neurofibrillary tangles at 12-15 months of age, which are similar to the pathological features in AD).

[0052] (22) Mouse behavioral evaluation experiment

[0053] (221) Open field experiment

[0054] The open field test is a commonly used behavioral assessment to determine an animal's general motor skills, anxiety level, and exploratory drive. The test area measures 96cm x 96cm x 96cm, with four wooden boards 50cm high on each side. After the animals acclimatized to the test room for one hour, the mice were removed from their cages and placed in the center of the open field. They were allowed to freely explore the open field for five minutes, and the distance traveled was recorded. After each mouse's experiment, the entire open field was cleaned with 75% alcohol. The next rat was tested only after the alcohol odor had completely dissipated. The distance traveled by the rats in the open field experiment was recorded and analyzed using the AnyMaze video tracking system.

[0055] (222) New Object Recognition Experiment

[0056] Three days before the experiment, the mice were petted for 5 minutes each day. On the first day, the mice were placed in the test room and apparatus for 10 minutes to acclimatize. At the start of the experiment, two identical objects were placed at the far end of the apparatus, with the mice facing away from both objects (at the same distance). The number of times and duration of contact between the mice and the objects were recorded within 10 minutes (feet resting on the objects, sniffing the objects, and licking the objects were all considered exploration activities; climbing on the objects was not counted). After 1 hour (short-term) and 24 hours (long-term), one of the two objects was replaced with a new object (different in shape, color, and size), and the number of times and duration of contact between the mice and the objects within 10 minutes were recorded again using the same criteria. The recognition index was defined as: time spent in contact with the new object / total exploration time.

[0057] (23) Water Maze

[0058] The Morris water maze (MWM) is an experiment that forces laboratory animals to swim, observes and records the time and trajectory of the animals searching for hidden platforms in the water after entry, and analyzes the data to infer the animals' learning, memory, and spatial cognition abilities. In this study, the Morris water maze test was used to measure the memory ability of mice on day 5 after drug administration. Acquisition training was conducted from day 1 to day 5, and the learning time of the mice was recorded over the 5 days. In the water, mice were placed in the water facing the pool wall in one quadrant and searched for the underwater platform. On day 6, the platform was removed, and all mice were placed in the water facing the pool wall from the entry point in the first quadrant. The monitoring system automatically tracked and recorded their movements over 600 seconds.

[0059] (3) Research Results

[0060] (31) Neuroprotective effects of Polygonum multiflorum exosomes in vitro

[0061] The in vitro cell experiments were divided into: NC group, APP group, APP+CbD-EXO (1ug / ml) group, APP+CbD-EXO (10ug / ml) group, and APP+CbD-EXO (100ug / ml) group.

[0062] The results showed that, compared with the APP group, the cell viability of the APP+CbD-EXO (1ug / ml) group and the APP+CbD-EXO (10ug / ml) group was significantly improved, indicating that the exosomes of Polygonum multiflorum have a neuroprotective effect in vitro.

[0063] (32) Behavioral detection of AD mice after drug administration

[0064] (321) Performance of *Polygonum multiflorum* exosomes in open field experiments in mice

[0065] The experiment was divided into 3 groups, with the same grouping method as above. The open field experiment was conducted on mice. The results showed that, compared with AD mice, AD mice injected with CbD-EXO spent a longer time in the central area of ​​the open field, indicating that the exosomes of Polygonum multiflorum can improve anxiety-like behavior in mice.

[0066] (322) Performance of exosomes from Polygonum multiflorum in new object recognition experiment

[0067] The experimental groups were the same as above, and a new object recognition experiment was conducted. The results showed that, compared with AD mice, AD mice injected with CbD-EXO had significantly higher new object recognition index and spatial memory ability, indicating that Polygonum multiflorum exosomes have the effect of improving cognitive impairment in AD mice.

[0068] (323) The performance of *Polygonum multiflorum* exosomes in the water maze experiment

[0069] The experimental groups were the same as above, and the water maze test was performed. The results showed that, compared with AD mice, AD mice injected with CbD-EXO had a significantly shorter escape latency in the Morris water maze, and a significantly increased time spent in the target quadrant and the number of times they crossed the platform. This indicates that Polygonum multiflorum exosomes have the effect of improving learning and memory abilities and cognitive impairment in AD mice.

[0070] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for extracting exosome extract from Polygonum multiflorum, characterized in that, Includes the following steps: Remove the roots from fresh white fleeceflower root, cut it into pieces, and extract the juice. Take the filtrate and centrifuge it continuously and then centrifuge it at a differential speed. Take the supernatant, centrifuge the supernatant at an ultra-high speed, and then take the precipitate. Resuspend the precipitate to obtain the white fleeceflower root exudate extract. The continuous centrifugation method is as follows: centrifuge continuously 3 times, each time for 30-60 minutes; The ultra-high speed centrifugation method is as follows: centrifuge 1000g for 12-15min for the first time, and then take the supernatant after centrifugation; Centrifuge a second time at 3000g for 25-30 minutes, and collect the supernatant after centrifugation; Centrifuge for the third time at 10000g for 50-40 minutes, and collect the supernatant after centrifugation; Centrifuge for the fourth time at 150,000g for 70-80 minutes, and collect the precipitate after centrifugation.

2. The extraction method according to claim 1, characterized in that, The juicing time is 20-30 minutes, and the juice is filtered through gauze after juicing.

3. The *Polygonum multiflorum* exosomes extracted by the extraction method described in claim 1 or 2.

4. The use of the white fleeceflower root exosomes as described in claim 3 in the preparation of neuroprotective drugs.

5. The use of the Polygonum multiflorum exosome extract as described in claim 3 in the preparation of a medicament for the prevention of Alzheimer's disease.

6. A pharmaceutical composition for treating Alzheimer's disease, characterized in that, The exosomes of Polygonum multiflorum as described in claim 3 are used as the active ingredient.