Extraction preparation method and application of bamboo exosome

Abstract

The invention discloses a method for extracting and preparing bamboo exosomes. The method comprises the following steps that S1, fresh and healthy bamboos are selected, cleaned and then cut into fragments to be pretreated; s2, carrying out crude extraction on the pretreated juice; s3, the juice obtained after crude extraction is purified; and S4, carrying out surface modification treatment or load controlled release treatment on the obtained bamboo exosome. The invention further discloses application. The method has the advantages that toxic and harmful organic solvents do not need to be used in the whole process, the method is environmentally friendly, the extraction rate and purity of the bamboo exosomes can be remarkably increased through soaking of the permeation buffer solution and multi-step centrifugal operation, and compared with a traditional method, the extraction rate can be increased by 20-30%, and the purity can reach 90% or above.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biomaterials, and in particular to a method for extracting and preparing bamboo exosomes and their application. Background Technology

[0002] Plant exosomes, membrane-bound vesicles secreted by living cells, are natural nanoscale particles enriched with a large number of plant secondary metabolites and various bioactive substances such as RNA, proteins, and lipids. Compared to traditional chemical extracts, plant exosome extraction technology requires no other media, introduces no impurities, offers higher raw material safety, lower production costs, and retains pharmacological activities similar to those of the original plant, demonstrating significant advantages in cellular uptake and functional synergy. However, current plant exosome extraction methods generally suffer from poor retention and targeting, short in vivo half-life, and difficulty in controlled release, which greatly limits their practical applications.

[0003] In terms of bamboo resource utilization, traditional bamboo leaf extracts suffer from problems such as single components, poor stability, and organic solvent residues. Although bamboo shoots, as a natural renewable resource, exhibit unique advantages in metabolic regulation and health intervention due to their complex component groups, the current processing of bamboo shoots is still mainly focused on primary products, such as dried bamboo shoots and canned goods, with low added value and a resource utilization rate of less than 30%. Research and development of bamboo exosomes are still in the initial stage, lacking efficient extraction and preparation methods and in-depth exploration of their wide application. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing bamboo exosomes and its application.

[0005] The objective of this invention is achieved through the following technical solution: a method for extracting and preparing bamboo exosomes, comprising the following steps:

[0006] S1: Select fresh, healthy bamboo, wash it, and cut it into pieces for pretreatment;

[0007] S2: Perform coarse extraction on the pretreated juice;

[0008] S3: Purify the juice after crude extraction;

[0009] S4: Surface modification or controlled release treatment of bamboo exosomes obtained by loading.

[0010] Preferably, in step S1, the preprocessing further includes the following steps:

[0011] S11: Soak bamboo fragments in osmotic buffer solution for 2-4 hours at a temperature of 4-10℃;

[0012] S12: Remove the soaked bamboo fragments and juice them;

[0013] S13: Centrifuge the juice at a centrifugal force of 2000~3000xg for 20~30 minutes, and collect the supernatant and the precipitate separately.

[0014] Preferably, in step S11, the osmotic buffer contains 0.2-0.5M sucrose, 5-10mM HEPES and 0.5-1mM calcium chloride, and the pH of the osmotic buffer is adjusted to 6.5-7.5.

[0015] Preferably, in step S2, the coarse extraction further includes the following steps:

[0016] S21: Centrifuge the supernatant again for 30-40 minutes at a centrifugal force of 6000-8000xg to remove particulate impurities;

[0017] S22: Filter the supernatant after centrifugation through a filter membrane.

[0018] Preferably, in step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and centrifuged again under the same ultracentrifugation conditions. This process is repeated 2 to 3 times to obtain purified bamboo exosomes.

[0019] Preferably, in step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by tangential flow to obtain purified bamboo exosomes.

[0020] Preferably, in step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by filtration using a membrane to obtain purified bamboo exosomes.

[0021] Preferably, in step S4, the surface modification treatment specifically involves dissolving the phospholipid-polyethylene glycol derivative in an organic solvent, then mixing it with a suspension of bamboo exosomes, stirring and reacting at room temperature for 2-4 hours, and after the reaction is complete, removing the unreacted phospholipid-polyethylene glycol derivative by dialysis to obtain the surface-modified bamboo exosomes.

[0022] The controlled release loading process involves preparing nanoparticles from biodegradable polymer materials, mixing bamboo exosomes with the nanoparticles, and loading the bamboo exosomes onto the inside or surface of the nanoparticles through ultrasonic treatment or high-pressure homogenization.

[0023] An application of bamboo exosomes, including the above-mentioned method for extracting and preparing bamboo exosomes, is specifically applied in the fields of medicine, food, and cosmetics.

[0024] The present invention has the following advantages:

[0025] 1. The entire process of this invention does not require the use of toxic or harmful organic solvents, making it environmentally friendly. Moreover, through soaking in osmotic buffer and multi-step centrifugation, the extraction rate and purity of bamboo exosomes can be significantly improved. Compared with traditional methods, the extraction rate can be increased by 20-30%, and the purity can reach over 90%.

[0026] 2. After surface modification, the bamboo exosomes obtained by this invention can effectively improve their retention and targeting properties and in vivo half-life. After loading and controlled release treatment, the bamboo exosomes can be released in a controlled manner, prolonging their action time and improving the utilization efficiency and therapeutic effect of exosomes. Attached Figure Description

[0027] Figure 1 A schematic diagram of the process flow for extracting and preparing bamboo exosomes;

[0028] Figure 2 This is a schematic diagram illustrating the traditional method of obtaining exosomes.

[0029] Figure 3 This is a schematic diagram illustrating the process of obtaining exosomes according to the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0031] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0033] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0034] In this embodiment, as Figure 1 As shown, a method for extracting and preparing bamboo exosomes includes the following steps:

[0035] S1: Select fresh, healthy bamboo, wash it, and cut it into pieces for pretreatment;

[0036] S2: Perform coarse extraction on the pretreated juice;

[0037] S3: Purify the juice after crude extraction;

[0038] S4: Surface modification or controlled-release loading of the obtained bamboo exosomes is performed. The entire process is environmentally friendly, requiring no toxic or harmful organic solvents. Furthermore, the extraction rate and purity of bamboo exosomes are significantly improved through osmotic buffer soaking and multi-step centrifugation. Compared to traditional methods, the extraction rate can be increased by 20-30%, and the purity can reach over 90%. Figure 2 and Figure 3 As shown. Simultaneously, the obtained bamboo exosomes, after surface modification, can effectively improve their retention, targeting, and in vivo half-life. After controlled-release loading, the bamboo exosomes can be released in a controlled manner, prolonging their duration of action and improving their utilization efficiency and therapeutic effect. In this embodiment, the selected bamboo raw material is bamboo leaves or bamboo shoots.

[0039] Furthermore, in step S1, the preprocessing also includes the following steps:

[0040] S11: Soak bamboo fragments in osmotic buffer for 2-4 hours at a temperature of 4-10°C; furthermore, the osmotic buffer contains 0.2-0.5M sucrose, 5-10mM HEPES and 0.5-1mM calcium chloride, and the pH of the osmotic buffer is adjusted to 6.5-7.5.

[0041] S12: Remove the soaked bamboo fragments and juice them;

[0042] S13: Centrifuge the juice at 2000-3000xg for 20-30 minutes, and collect the supernatant and the precipitate separately. Specifically, when the raw material is bamboo leaves: Select 100g of fresh moso bamboo leaves, wash the leaves clean, cut them into small pieces of about 1 cm, prepare 1L of osmotic buffer solution, with a sucrose concentration of 0.3M, a HEPES concentration of 8mM, a calcium chloride concentration of 0.8mM, and adjust the pH value to 7.0. Soak the cut bamboo leaf pieces in the buffer solution at 4℃ for 3 hours; after soaking, remove the bamboo leaves, extract the juice, centrifuge the juice at 2500xg for 25 minutes, and collect the supernatant and the precipitate separately. When the raw material is bamboo shoots, select 200g of fresh bamboo shoots, clean them, and cut them into thin slices with a thickness of 0.8 cm. Prepare 2L of osmotic buffer with a sucrose concentration of 0.4M, a HEPES concentration of 6mM, a calcium chloride concentration of 0.6mM, and adjust the pH value to 6.8. Soak the bamboo shoot slices at 6℃ for 3.5 hours. After soaking, juice the bamboo shoots and centrifuge the juice at 3000xg for 30 minutes. Collect the supernatant and the precipitate separately.

[0043] In this embodiment, step S2, the coarse extraction further includes the following steps:

[0044] S21: Centrifuge the supernatant again for 30-40 minutes at a centrifugal force of 6000-8000xg to remove particulate impurities;

[0045] S22: Filter the supernatant after centrifugation through a filter membrane. Specifically, when the raw material is bamboo leaves, centrifuge the supernatant at 7000xg for 35 minutes to remove larger particulate impurities, and then filter it through a 0.45 filter membrane. The filter membrane further removes tiny particles. When the raw material is bamboo shoots, the supernatant is centrifuged at 8000xg for 40 minutes to remove larger impurities, and then filtered through a 0.45 filter membrane. Filtration using a filter membrane.

[0046] Further, in step S3, the filtered supernatant is centrifuged at 100,000-120,000 x g for 70-90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and centrifuged again under the same ultracentrifugation conditions. This process is repeated 2-3 times to obtain purified bamboo exosomes. Specifically, when the raw material is bamboo leaves, the filtered supernatant is ultracentrifuged at 110,000 x g for 80 minutes, the precipitate is collected, and the supernatant is resuspended in 1 mL of PBS buffer. The resuspended solution is then centrifuged again under the same ultracentrifugation conditions. This process is repeated twice to obtain high-purity bamboo exosomes. The extraction rate was measured to be 25%, and the purity was 92%. When the raw material is bamboo shoots, the filtered supernatant is centrifuged at 120,000 x g for 90 minutes, the precipitate is collected, resuspended in 2 mL of PBS buffer, and centrifuged again under the same ultracentrifugation conditions. This process is repeated 3 times to obtain high-purity bamboo exosomes. The extraction rate was 28% and the purity was 93%.

[0047] In another embodiment, in step S3, the filtered supernatant is centrifuged at 100,000-120,000 x g for 70-90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by tangential flow to obtain purified bamboo exosomes. The fiber column used is 50 KD-750 KD. In yet another embodiment, in step S3, the filtered supernatant is centrifuged at 100,000-120,000 x g for 70-90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by filtration using a membrane pack to obtain purified bamboo exosomes. The membrane pack used is 50 KD-750 KD.

[0048] In this embodiment, in step S4, the surface modification treatment specifically involves dissolving the phospholipid-polyethylene glycol derivative in an organic solvent, then mixing it with a bamboo exosome suspension, stirring and reacting at room temperature for 2-4 hours, and after the reaction is completed, removing the unreacted phospholipid-polyethylene glycol derivative by dialysis to obtain the surface-modified bamboo exosomes.

[0049] The controlled-release loading process involves preparing nanoparticles from biodegradable polymer materials, mixing bamboo exosomes with the nanoparticles, and then loading the bamboo exosomes onto the interior or surface of the nanoparticles using ultrasonic treatment or high-pressure homogenization. Specifically, for surface modification, 1 mL of bamboo exosomes prepared from bamboo leaves and 5 mg of a phospholipid-polyethylene glycol derivative are taken. The phospholipid-polyethylene glycol derivative is dissolved in 5 mL of chloroform and then mixed with the bamboo exosome resuspension. The mixture is stirred and reacted at room temperature for 3 hours. After the reaction, the mixture is placed in a dialysis bag (molecular weight cutoff of 3000) and dialyzed in PBS buffer for 24 hours, with the buffer being changed every 4 hours to remove unreacted phospholipid-polyethylene glycol derivatives, resulting in surface-modified bamboo exosomes, which can effectively improve their retention, targeting, and in vivo half-life. When performing controlled-release loading, 2 mL of bamboo exosomes prepared from bamboo shoots and 10 mg of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles with an average particle size of 100 nm were mixed. The mixture was then subjected to ultrasonic treatment at a power of 200 W for 10 minutes to load the bamboo exosomes onto the surface of the PLGA nanoparticles. This process enabled the controlled release of the bamboo exosomes, prolonging their duration of action and improving the utilization efficiency and therapeutic effect of the exosomes.

[0050] An application of bamboo exosomes, including the above-mentioned method for extracting and preparing bamboo exosomes, is specifically applied in the pharmaceutical, food, and cosmetic fields. Specifically, in the pharmaceutical field, bamboo exosomes prepared from bamboo shoots can be mixed with physiological saline to prepare an injection with a concentration of 1 mg / mL. The efficacy experiment is as follows: A rat model of oxidative stress injury was selected, and the rats were randomly divided into an experimental group and a control group. The experimental group rats received a tail vein injection of the bamboo exosome injection prepared according to this invention, once daily, 1 mL each time; the control group rats received an equal volume of physiological saline. After 14 days of continuous administration, the activity of antioxidant enzymes in the experimental group rats was significantly increased, and oxidative stress indicators were significantly reduced, indicating that the bamboo exosome injection can effectively alleviate oxidative stress injury.

[0051] In the food industry, for functional beverages, 0.3g of bamboo exosomes prepared from bamboo leaves is added to 1L of the beverage, along with appropriate amounts of fruit juice, sweeteners, and acidulants. For yogurt, 0.5g of bamboo exosomes prepared from bamboo shoots is added to 1kg of raw milk, and then fermentation is carried out according to conventional yogurt production processes.

[0052] In the cosmetics industry, for face creams, the formula includes 1% (by weight) bamboo exosomes prepared from bamboo leaves, along with other commonly used skincare ingredients such as glycerin, hyaluronic acid, and plant oils. For face masks, the formula includes 2% (by weight) bamboo exosomes prepared from bamboo shoots, along with other commonly used moisturizing and whitening ingredients.

[0053] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting and preparing bamboo exosomes, characterized in that: Includes the following steps: S1: Select fresh, healthy bamboo, wash it, and cut it into pieces for pretreatment; S2: Perform coarse extraction on the pretreated juice; S3: Purify the juice after crude extraction; S4: Surface modification or controlled release treatment of bamboo exosomes obtained by loading.

2. The method for extracting and preparing bamboo exosomes according to claim 1, characterized in that: In step S1, the preprocessing also includes the following steps: S11: Soak bamboo fragments in osmotic buffer solution for 2-4 hours at a temperature of 4-10℃; S12: Remove the soaked bamboo fragments and juice them; S13: Centrifuge the juice at a centrifugal force of 2000~3000xg for 20~30 minutes, and collect the supernatant and the precipitate separately.

3. The method for extracting and preparing bamboo exosomes according to claim 2, characterized in that: In step S11, the osmotic buffer contains 0.2-0.5M sucrose, 5-10mM HEPES and 0.5-1mM calcium chloride, and the pH of the osmotic buffer is adjusted to 6.5-7.

5.

4. The method for extracting and preparing bamboo exosomes according to claim 3, characterized in that: In step S2, the coarse extraction also includes the following steps: S21: Centrifuge the supernatant again for 30-40 minutes at a centrifugal force of 6000-8000xg to remove particulate impurities; S22: Filter the supernatant after centrifugation through a filter membrane.

5. The method for extracting and preparing bamboo exosomes according to claim 4, characterized in that: In step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and centrifuged again under the same ultracentrifugation conditions. This process is repeated 2 to 3 times to obtain purified bamboo exosomes.

6. The method for extracting and preparing bamboo exosomes according to claim 4, characterized in that: In step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by tangential flow to obtain purified bamboo exosomes.

7. The method for extracting and preparing bamboo exosomes according to claim 4, characterized in that: In step S3, the filtered supernatant is centrifuged at 100,000 to 120,000 x g for 70 to 90 minutes, and the precipitate is collected to obtain crude bamboo exosomes. The crude bamboo exosomes are then resuspended in PBS buffer and purified by filtration using a membrane to obtain purified bamboo exosomes.

8. The method for extracting and preparing bamboo exosomes according to any one of claims 5 to 7, characterized in that: In step S4, the surface modification treatment specifically involves dissolving a phospholipid-polyethylene glycol derivative in an organic solvent, then mixing it with a bamboo exosome suspension, stirring and reacting at room temperature for 2-4 hours, and after the reaction is complete, removing unreacted phospholipid-polyethylene glycol derivatives by dialysis to obtain surface-modified bamboo exosomes. The controlled release loading process involves preparing nanoparticles from biodegradable polymer materials, mixing bamboo exosomes with the nanoparticles, and loading the bamboo exosomes onto the inside or surface of the nanoparticles through ultrasonic treatment or high-pressure homogenization.

9. An application of bamboo exosomes, comprising the bamboo exosome extraction and preparation method as described in claim 8, characterized in that: Specifically, it is used in the fields of medicine, food, and cosmetics.

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