Extraction optimization method of cactus exosome

Abstract

The invention discloses an extraction optimization method of cactus exosomes, which comprises the following steps: selecting cactus pomace, and carrying out vacuum freeze-drying treatment to obtain a freeze-dried cactus pomace raw material; performing reflux extraction twice; combining the two supernates, concentrating under reduced pressure, and drying under reduced pressure to obtain a water extract; adding an ethanol solution as an extraction solvent to obtain an alcohol extraction sample; performing first-stage centrifugal impurity removal, second-stage centrifugal impurity removal, third-stage centrifugal impurity removal, fourth-stage centrifugal enrichment, refining purification and storage to obtain a cactus exosome sample; and performing sample injection detection. The extraction concentration and purity of the cactus exosomes are improved through a pretreatment-multistage centrifugation combined method. The detection result shows that the concentration of the exosome in the water extract reaches 4.3 * 10 parts / ml, the concentration of the exosome in the alcohol extract reaches 9.2 * 10 parts / ml, and the concentration of the exosome in the water extract and the concentration of the exosome in the alcohol extract are obviously higher than those in puree and fruit juice. The method is clear in parameter, high in extraction efficiency and suitable for large-scale preparation of the cactus exosome.

Description

Technical Field

[0001] This invention relates to the field of natural plant exosome extraction and purification technology, and in particular to an optimized method for extracting cactus exosomes. Background Technology

[0002] Exosomes, as nanoscale vesicles rich in bioactive substances (such as proteins, nucleic acids, and lipids), have important applications in cell communication, delivery of active ingredients, and other fields. While cactus pomace naturally contains exosomes, direct extraction from pomace or juice presents challenges such as extremely low exosome concentrations (only 155.8 particles / ml in pomace and 140.9 particles / ml in juice), high impurity content, and difficulty in separation and purification, failing to meet the requirements for subsequent applications.

[0003] Currently, plant exosome extraction mostly employs simple centrifugation or filtration processes, and a dedicated extraction process for cactus exosomes has not yet been established.

[0004] On the one hand, there is a lack of effective pretreatment methods (such as enriching exosome precursors through water extraction / alcohol extraction), resulting in low initial exosome content;

[0005] On the other hand, unclear centrifugation parameters (speed, time, temperature) can easily lead to exosome rupture or impurity residue.

[0006] Therefore, in order to solve the problems of low concentration and insufficient purity of existing cactus exosome extraction, it is an urgent technical problem to be solved by providing an optimized method combining "pretreatment enrichment-multi-stage purification" to significantly improve the concentration and purity of exosomes and obtain cactus exosome samples with uniform particle size (150-200nm). Summary of the Invention

[0007] In view of this, the present invention provides an optimized method for extracting cactus exosomes.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] An optimized method for extracting cactus exosomes includes the following steps:

[0010] Step 1: Raw material pretreatment

[0011] Select cactus pomace and process it in a vacuum freeze-drying device to obtain freeze-dried cactus pomace raw material for later use;

[0012] Step 2: Water extraction pretreatment

[0013] Weigh the freeze-dried raw material, add distilled water for the first reflux extraction; filter and collect the supernatant, then reflux extract again; combine the two supernatants, concentrate under reduced pressure and dry under reduced pressure to obtain the water extract;

[0014] Step 3: 60% alcohol extraction pretreatment

[0015] Weigh the freeze-dried raw material and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol extract sample;

[0016] Step 4: Multistage centrifugation purification of exosomes

[0017] Take the above-mentioned water extract or 60% alcohol extract, quickly dissolve it in a water bath, and transfer it to a 50ml centrifuge tube for later use; first perform a single centrifugation to remove impurities, and collect the supernatant;

[0018] Then perform a second centrifugation to remove impurities and collect the supernatant;

[0019] Then perform three-stage centrifugation to remove impurities, collect the supernatant, filter it through a membrane to remove submicron impurities, and set it aside for later use;

[0020] Then perform four stages of centrifugation for enrichment, discard the supernatant, and collect the precipitate;

[0021] Further purification was performed by resuspending the precipitate in fresh, pre-cooled 1×PBS buffer and removing small molecule impurities by filtration.

[0022] The precipitate was collected by centrifugation again, and the sample was preserved to obtain cactus exosomes.

[0023] Step 5: Testing and Calibration

[0024] During the extraction process, a nanoparticle tracking analyzer (NTA) was used for detection. Before detection, the sample cell was cleaned with deionized water, the instrument was calibrated with polystyrene microspheres, and then the sample cell was cleaned with 1×PBS buffer. Finally, the exosome sample was diluted with 1×PBS buffer before injection for detection.

[0025] Preferably, in step 1, the vacuum freeze-drying conditions are: temperature -40 to -35°C, vacuum degree -0.08 to 0.10 MPa, and drying time 8 to 10 hours.

[0026] Preferably, in step 2, the first reflux extraction conditions are as follows: distilled water is added at a ratio of 12 times the mass of the raw material, and reflux extraction is performed for 1 hour.

[0027] Preferably, in step 2, the conditions for refluxing and extracting are as follows: add 10 times the mass of distilled water to the filter residue and reflux for 1 hour.

[0028] Preferably, in step 2, the vacuum concentration conditions are: temperature 50-60℃, vacuum degree -0.07 to -0.09MPa, concentrated until no obvious moisture is present; the vacuum drying conditions are: temperature 50-60℃, vacuum degree -0.07 to -0.09MPa.

[0029] Preferably, in step 4, the water bath temperature is 37°C.

[0030] Preferably, in step 4, the conditions for primary centrifugation to remove impurities are 300g, 4℃, and centrifugation for 30min;

[0031] The method for secondary centrifugation to remove impurities is as follows: place the supernatant from the primary centrifugation into a centrifuge; the conditions for secondary centrifugation to remove impurities are: 2000g, 4℃, centrifugation for 30min;

[0032] The method for removing impurities by three-stage centrifugation is as follows: place the supernatant from the second-stage centrifugation in a centrifuge; the conditions for removing impurities by three-stage centrifugation are: 10000g, 4℃, centrifugation for 30min, and filter the supernatant through a 0.45um filter membrane;

[0033] The conditions for fourth-stage centrifugation enrichment are as follows: centrifuge 100,000g of the filtrate at 4℃ for 70 minutes.

[0034] Preferably, in step 4, the purification is performed by resuspending the sample in fresh, pre-cooled 1×PBS buffer at 4°C and filtering it through a 0.22µm filter membrane; the purification conditions are: 100,000g, 4°C, centrifugation for 70 min.

[0035] Preferably, in step 4, after the precipitate is resuspended in 100-200 μL of 1×PBS buffer, it is transferred to a cryogenic storage tube and stored at -80°C.

[0036] Preferably, in step 5, the polystyrene microspheres have a particle size of 100 nm.

[0037] The present invention achieves the following technical effects compared to the prior art:

[0038] (1) Significantly increased exosome concentration: After treatment with this invention, the concentration of exosomes in the water extract reached 4.3 × 10¹. 0 The particle / ml concentration of the alcohol extract reached 9.2 × 10⁻⁶. 9 particles / ml, which are 2.76 × 10⁻⁶ particles of fruit puree. 8 times, 5.9×10 7 3.05 × 10 times, the juice 8 times, 6.53×10 7 It has a multiplier effect and an extremely strong enrichment effect;

[0039] (2) High purity and uniform particle size of exosomes: Through four-stage centrifugation + double filtration (0.45um→0.22um), large particles, cell debris and small molecule impurities are effectively removed, and the exosome particle size is concentrated in 150-200nm with good structural integrity;

[0040] (3) Clear and controllable process parameters: This invention strictly limits the extraction parameters (12 times / 10 times solvent, 1h extraction), centrifugation parameters (speed 300g→2000g→10000g→100000g, temperature 4℃, time 30min / 70min), and filter membrane pore size (0.45um / 0.22um) to ensure that the extraction effect of exosomes in different batches is stable and has good repeatability;

[0041] (4) Flexible pretreatment methods: This invention provides two pretreatment schemes: water extraction and 60% alcohol extraction. Among them, water extraction has the best effect and can be selected according to actual production needs, making it highly applicable.

[0042] (5) Strong operational compatibility: The equipment used in this invention (reflux extractor, centrifuge, NTA analyzer) are all conventional instruments, requiring no special customized equipment, which facilitates rapid production for enterprises. Attached Figure Description

[0043] Figure 1 This is a particle size distribution diagram of the fruit juice according to the present invention;

[0044] Where a is the particle size distribution of cactus juice (particles / mL); b is the particle size distribution of cactus juice (Volume / nm). 3 )

[0045] Figure 2 Electron microscope images of this invention;

[0046] Wherein, a is a photograph of region 1 at 500nm; b is a photograph of region 1 at 200nm; c is a photograph of region 2 at 100nm; d is a photograph of region 2 at 200nm;

[0047] Figure 3 This is a particle size distribution diagram of the present invention;

[0048] Where a is the particle size distribution of cactus juice (particles / mL); b is the particle size distribution of cactus juice (Volume / nm). 3 )

[0049] Figure 4 Electron microscope images of this invention;

[0050] Where a is a photo taken at 500nm; b is a photo taken at 200nm. Detailed Implementation

[0051] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] This invention discloses an optimized method for extracting cactus exosomes, comprising the following steps:

[0053] Step 1: Raw material pretreatment

[0054] Select cactus pomace and process it in a vacuum freeze-drying device to obtain freeze-dried cactus pomace raw material for later use;

[0055] Step 2: Water extraction pretreatment

[0056] Weigh the freeze-dried raw material, add distilled water for the first reflux extraction; filter and collect the supernatant, then reflux extract again; combine the two supernatants, concentrate under reduced pressure and dry under reduced pressure to obtain the water extract;

[0057] Step 3: 60% alcohol extraction pretreatment

[0058] Weigh the freeze-dried raw material and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol extract sample;

[0059] Step 4: Multistage centrifugation purification of exosomes

[0060] Take the above-mentioned water extract or 60% alcohol extract, quickly dissolve it in a water bath, and transfer it to a 50ml centrifuge tube for later use; first perform a single centrifugation to remove impurities, and collect the supernatant;

[0061] Then perform a second centrifugation to remove impurities and collect the supernatant;

[0062] Then perform three-stage centrifugation to remove impurities, collect the supernatant, filter it through a membrane to remove submicron impurities, and set it aside for later use;

[0063] Then perform four stages of centrifugation for enrichment, discard the supernatant, and collect the precipitate;

[0064] Further purification was performed by resuspending the precipitate in fresh, pre-cooled 1×PBS buffer and removing small molecule impurities by filtration.

[0065] The precipitate was collected by centrifugation again, and the sample was preserved to obtain cactus exosomes.

[0066] Step 5: Testing and Calibration

[0067] During the extraction process, a nanoparticle tracking analyzer (NTA) was used for detection. Before detection, the sample cell was cleaned with deionized water, the instrument was calibrated with polystyrene microspheres, and then the sample cell was cleaned with 1×PBS buffer. Finally, the exosome sample was diluted with 1×PBS buffer before injection for detection.

[0068] In step 1, the vacuum freeze-drying conditions are: temperature -40 to -35℃, vacuum degree -0.08 to 0.10MPa, and drying time 8-10 hours.

[0069] In step 2, the conditions for the first reflux extraction are as follows: add distilled water at a ratio of 12 times the mass of the raw material and reflux for 1 hour.

[0070] In step 2, the conditions for refluxing and extraction are as follows: add 10 times the mass of distilled water to the filter residue and reflux for 1 hour.

[0071] In step 2, the vacuum concentration conditions are: temperature 50-60℃, vacuum degree -0.07-0.09MPa, concentrated until there is no obvious moisture; the vacuum drying conditions are: temperature 50-60℃, vacuum degree -0.07-0.09MPa.

[0072] In step 4, the water bath temperature is 37℃.

[0073] In step 4, the conditions for primary centrifugation to remove impurities are 300g, 4℃, and centrifugation for 30 minutes;

[0074] The method for secondary centrifugation to remove impurities is as follows: place the supernatant from the primary centrifugation into a centrifuge; the conditions for secondary centrifugation to remove impurities are: 2000g, 4℃, centrifugation for 30min;

[0075] The method for removing impurities by three-stage centrifugation is as follows: place the supernatant from the second-stage centrifugation in a centrifuge; the conditions for removing impurities by three-stage centrifugation are: 10000g, 4℃, centrifugation for 30min, and filter the supernatant through a 0.45um filter membrane;

[0076] The conditions for fourth-stage centrifugation enrichment are as follows: centrifuge 100,000g of the filtrate at 4℃ for 70 minutes.

[0077] In step 4, purification involves resuspending the sample in fresh, pre-cooled 1×PBS buffer at 4°C and filtering it through a 0.22µm filter membrane. The purification conditions are: 100,000g, 4°C, centrifugation for 70 min.

[0078] In step 4, after resuspending the precipitate in 100-200 μL of 1×PBS buffer, transfer it to a cryogenic storage tube and store it at -80°C.

[0079] In step 5, the polystyrene microspheres have a particle size of 100 nm.

[0080] Example 1:

[0081] 1. Experimental Materials

[0082] Raw materials: cactus pomace (processing byproduct), freeze-dried cactus pomace raw material;

[0083] Reagents: distilled water, 60% ethanol solution, 1×PBS buffer (Biological Industries, Israel), polystyrene microspheres (particle size 100nm, catalog number 3100A, ThermoFisher).

[0084] Instruments and equipment: reflux extraction device, vacuum concentrator, vacuum drying oven, high-speed refrigerated centrifuge (supports 100,000 g speed), 37℃ water bath, 0.45um / 0.22um filter membrane, nanoparticle tracking analyzer (NTA), transmission electron microscope (for exosome morphology observation), -80℃ low temperature freezer.

[0085] 2. Experimental Procedure

[0086] Extract preparation:

[0087] Preparation of water extract: Weigh 100g of freeze-dried cactus pomace, add 1200mL of distilled water, and reflux for 1h; filter and collect the first extract; add 1000mL of distilled water to the filter residue and reflux for 1h again; combine the extracts, concentrate under reduced pressure to 50mL, and dry under reduced pressure to obtain 12.5g of water extract.

[0088] Preparation of 60% alcohol extract: Weigh 100g of freeze-dried raw material, add 1200mL of 60% ethanol solution, reflux extract, concentrate and dry as described above, to obtain 10.8g of 60% alcohol extract.

[0089] Preparation of exosomes (taking water extract as an example):

[0090] Take 5g of water extract, quickly melt it in a 37℃ water bath, transfer it to a 50ml centrifuge tube, add 1×PBS buffer to 20mL, and mix well.

[0091] Centrifuge at 300g and 4℃ for 30 min, and take 18 mL of the supernatant; centrifuge at 2000g and 4℃ for 30 min, and take 17 mL of the supernatant; centrifuge at 10000g and 4℃ for 30 min, and take 16 mL of the supernatant. Filter through a 0.45 μm filter membrane to obtain 15 mL of the filtrate.

[0092] Centrifuge at 100,000g, 4℃ for 70 min, discard the supernatant, resuspend the precipitate in 5 mL of pre-cooled 1×PBS, and filter through a 0.22 μm filter membrane; centrifuge again at 100,000g, 4℃ for 70 min, resuspend the precipitate in 200 μL of 1×PBS, and store at -80℃ to obtain the water-extracted exosome sample.

[0093] Preparation of exosomes by alcohol extraction: Take 5g of 60% alcohol extract and repeat the above steps of centrifugation, filtration and resuspension to obtain alcohol-extracted exosome samples.

[0094] Control sample processing: Take 20 mL each of cactus pulp and juice directly, and process them according to the above "multi-stage centrifugation purification" steps (skipping extract preparation) to obtain exosome control samples of pulp and juice.

[0095] 3. Testing and Results:

[0096] The test results are shown in Table 1.

[0097] Table 1:

[0098]

[0099] Transmission electron microscopy revealed that both water-extracted and alcohol-extracted exosomes exhibited typical vesicular structures with no obvious impurities attached, and their particle size was consistent with NTA detection.

[0100] As shown in Table 1, the present invention can significantly enrich cactus exosomes through "water extraction / alcohol extraction pretreatment", among which water extraction pretreatment has the best effect, with an exosome concentration of 4.3×10¹. 0 The exosomes were obtained in particles / ml and, after four-stage centrifugation and double filtration, the particle size was concentrated in the range of 150-200nm. They were highly pure and structurally intact, fully meeting the requirements of subsequent applications for raw materials.

[0101] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for optimizing the extraction of cactus exosomes, characterized in that, Includes the following steps: Step 1: Raw material pretreatment Select cactus pomace and process it in a vacuum freeze-drying device to obtain freeze-dried cactus pomace raw material for later use; Step 2: Water extraction pretreatment Weigh the freeze-dried raw material, add distilled water for the first reflux extraction; filter and collect the supernatant, then reflux extract again. The supernatants from both extractions were combined, concentrated under reduced pressure, and dried under reduced pressure to obtain the aqueous extract. Step 3: 60% alcohol extraction pretreatment Weigh the freeze-dried raw material and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol extract sample; Step 4: Multistage centrifugation purification of exosomes Take the above-mentioned water extract or 60% alcohol extract, quickly dissolve it in a water bath, and transfer it to a 50ml centrifuge tube for later use; first perform a single centrifugation to remove impurities, and collect the supernatant; Then perform a second centrifugation to remove impurities and collect the supernatant; Then perform three-stage centrifugation to remove impurities, collect the supernatant, filter it through a membrane to remove submicron impurities, and set it aside for later use; Then perform four stages of centrifugation for enrichment, discard the supernatant, and collect the precipitate; Further purification was performed by resuspending the precipitate in fresh, pre-cooled 1×PBS buffer and removing small molecule impurities by filtration. The precipitate was collected by centrifugation again, and the sample was preserved to obtain cactus exosomes. Step 5: Testing and Calibration During the extraction process, a nanoparticle tracking analyzer (NTA) was used for detection. Before detection, the sample cell was cleaned with deionized water, the instrument was calibrated with polystyrene microspheres, and then the sample cell was cleaned with 1×PBS buffer. Finally, the exosome sample was diluted with 1×PBS buffer before injection for detection.

2. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 1, the vacuum freeze-drying conditions are: temperature -40 to -35℃, vacuum degree -0.08 to 0.10MPa, and drying time 8-10 hours.

3. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 2, the conditions for the first reflux extraction are as follows: add distilled water at a ratio of 12 times the mass of the raw material, and reflux for 1 hour.

4. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 2, the conditions for refluxing and extraction are as follows: add 10 times the mass of distilled water to the filter residue and reflux for 1 hour.

5. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 2, the vacuum concentration conditions are: temperature 50-60℃, vacuum degree -0.07--0.09MPa, concentrated until there is no obvious moisture; the vacuum drying conditions are: temperature 50-60℃, vacuum degree -0.07-0.09MPa.

6. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 4, the water bath temperature is 37°C.

7. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 4, the conditions for primary centrifugation to remove impurities are 300g, 4℃, and centrifugation for 30min; The method for secondary centrifugation to remove impurities is as follows: place the supernatant from the primary centrifugation into a centrifuge; the conditions for secondary centrifugation to remove impurities are: 2000g, 4℃, centrifugation for 30min; The method for removing impurities by three-stage centrifugation is as follows: place the supernatant from the second-stage centrifugation in a centrifuge; the conditions for removing impurities by three-stage centrifugation are: 10000g, 4℃, centrifugation for 30min, and filter the supernatant through a 0.45um filter membrane; The conditions for fourth-stage centrifugation enrichment are as follows: centrifuge 100,000g of the filtrate at 4℃ for 70 minutes.

8. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 4, purification involves resuspending the sample in fresh, pre-cooled 1×PBS buffer at 4°C and filtering it through a 0.22µm filter membrane. The purification conditions are: 100,000g, 4°C, centrifugation for 70 minutes.

9. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 4, the precipitate is resuspended in 100-200 μL of 1×PBS buffer and then transferred to a cryogenic storage tube for storage at -80°C.

10. The method for extracting and optimizing cactus exosomes according to claim 1, characterized in that, In step 5, the polystyrene microspheres have a particle size of 100 nm.

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