Preparation method and application of blood orange exosome

Abstract

The invention discloses a preparation method and application of a blood orange exosome. The method comprises the following steps: cleaning, squeezing, preliminary filtering, enzymolysis, high-speed refrigerated centrifugation, tangential flow concentration and two times of ultracentrifugation, so as to obtain the blood orange exosome. By adopting a mode of combining single squeezing with short-time enzymolysis, damage to the exosomes caused by human factors and pollution caused by obtained non-exosome vesicles can be avoided, so that the blood orange exosomes can be better collected.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a preparation method of blood orange exosomes and application thereof. Background Art

[0002] In recent years, plant-derived extracellular vesicles have attracted great attention from the biological and medical communities. They have a phospholipid bilayer structure similar to that of animal-derived exosomes and carry abundant proteins, lipids, nucleic acids, and plant-specific secondary metabolites. The extraction and efficacy verification of various types of plant exosomes have been carried out at home and abroad. Plant exosomes have been proven to have good biosafety and oral safety and can cross biological barriers.

[0003] At present, the extraction process of plant exosomes mainly includes pretreatment process and separation and extraction process. The pretreatment generally includes screening and cleaning of plant raw materials, crushing and juicing, low-speed centrifugation, etc., to obtain a supernatant solution rich in plant exosomes; the separation and extraction methods mainly include ultracentrifugation and sucrose density gradient centrifugation. These two methods are the most commonly used methods for separation and purification of plant exosomes reported in the literature. In addition, polyethylene glycol precipitation method is used to separate plant exosomes. There are more diverse ways to separate and extract exosomes from animal cells. In addition to ultracentrifugation, sucrose density gradient centrifugation, and precipitation, there are also immunomagnetic beads, ultrafiltration centrifugation, size exclusion chromatography, and microfluidics. Compared with animal exosomes, plant exosomes are relatively simple in extraction methods, and there are fewer studies on the optimization of extraction processes. In addition, unlike animal exosomes, plant cells have natural cell wall components. The plant cell wall is mainly composed of three parts, namely the middle lamella, primary wall, and secondary wall. The middle lamella is also called the middle lamella, and its main component is pectin, which constitutes the adhesive substance of the middle layer of adjacent cells, so that plant tissue cells are tightly bonded together and can buffer the squeezing between cells. The main components of the primary wall and the secondary wall are cellulose, which has a supporting and protective effect on plant cells. Natural pectin substances are mainly present in the form of protopectin, pectin and pectic acid in the fruits, roots, stems and leaves of plants. Pectin has certain thickening properties. All plants contain a certain amount of pectin. Citrus fruits, apples and apricots are the richest in pectin.

[0004] In the research on the extraction process of plant exosomes, the current focus is on the separation and purification of plant exosomes. The pretreatment process of plants is crushing and juicing, or squeezing to obtain juice. However, there are many types of plants, and this unified method is not applicable to all plant types. Especially for blood oranges, the samples directly separated by crushing the wall and juicing contain pectin, which will form a jelly state, affecting the subsequent characterization, efficacy detection and application of exosomes. Based on this, the present invention explores the exosome extraction process of this type of special plants, adds enzymatic hydrolysis treatment to the plants with rich pectin content in the pretreatment process, verifies the enzymatic hydrolysis time and temperature, explores the enzymatic hydrolysis process with the maximum juice yield, and the treatment method does not affect the structure and function of the exosomes, and is committed to improving the extraction content of such plant exosomes after pretreatment. In the prior art, there are also means for extracting plant exosomes containing pectin or fiber, but directly using these schemes or making reasonable adjustments cannot effectively extract blood orange exosomes. Therefore, it is necessary to develop a separate method for extracting blood orange exosomes, which can remove pectin without affecting the extraction of exosomes. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting blood orange exosomes.

[0006] In order to achieve the above object, the technical solution adopted by the present invention comprises the following steps:

[0007] 1) Wash the surface of blood orange pulp with deionized water, then squeeze and filter, add 0.01-0.03g / L pectinase, fully dissolve, place at 35-40℃, and perform enzymolysis for 30-90min to obtain enzymolysis solution;

[0008] 2) The enzymatic hydrolysate is subjected to high-speed refrigerated centrifugation to collect the supernatant, which is then concentrated and filtered to obtain a concentrate;

[0009] 3) The concentrated solution is resuspended after ultracentrifugation, and then ultracentrifuged again and resuspended to obtain blood orange exosomes.

[0010] Furthermore, the specific method of squeezing is: take fresh blood oranges, wash them, and peel them; place the peeled blood oranges in a squeezer, slowly and evenly rotate the upper handle ball, collect the blood orange juice squeezed once, and discard the residue.

[0011] Furthermore, the sieve used for the filtration is 80-120 mesh.

[0012] Furthermore, the parameters of the high-speed refrigerated centrifugation are: 4°C, 10000-16000g, centrifugation for 40-60min.

[0013] Furthermore, the concentration and filtration specifically includes: collecting the supernatant, and concentrating and filtering it through a tangential flow ultrafiltration system, and the ultrafiltration membrane used has a molecular weight cutoff of 100 kDa.

[0014] Furthermore, the ultracentrifugation parameters are: 4°C, 100,000-150,000 g for 60-80 min.

[0015] Based on the same inventive concept, the present invention also provides blood orange exosomes prepared by the above method.

[0016] Based on the same inventive concept, the present invention also provides the use of blood orange exosomes prepared by the above method in the preparation of cosmetics.

[0017] The beneficial effects of the present invention are:

[0018] 1) The present invention adopts a single squeezing combined with a short-time enzymatic hydrolysis method to avoid the damage to exosomes caused by human factors and the contamination caused by the obtained non-exosome vesicles, thereby better collecting blood orange exosomes.

[0019] 2) The present invention pre-treats the blood orange juice by short-time enzymatic hydrolysis, improves the conventional enzymatic hydrolysis conditions and methods, and innovates an enzymatic hydrolysis condition that does not affect the structure and function of blood orange exosomes. The squeezing and enzymatic hydrolysis pre-treatment process used in the present invention can greatly improve the extraction efficiency and extraction content of blood orange exosomes, and the obtained blood orange exosomes have high purity and high content. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention is further described below in conjunction with the accompanying drawings.

[0021] Figure 1 This is a schematic diagram of the extraction process of blood orange exosomes of the present invention.

[0022] Figure 2 These are actual photos of blood orange exosomes, from left to right they are blood orange exosomes collected from Comparative Example 1, Comparative Example 2, and Example 1.

[0023] Figure 3 This is a transmission electron micrograph of blood orange exosomes obtained in Example 1 of the present invention.

[0024] Figure 4 This is the transmission electron microscopy image of blood orange exosomes collected in Comparative Example 2.

[0025] Figure 5 This is a particle size distribution diagram of blood orange exosomes obtained in Example 1 of the present invention.

[0026] Figure 6 This is the particle size distribution diagram of blood orange exosomes collected in Comparative Example 2.

[0027] Figure 7This is the in vitro clearance standard curve used in effect verification 2. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the present invention is explained in detail below through the description of preferred specific embodiments of the present invention, but the present invention is not limited in any form.

[0029] Unless otherwise specified, the raw materials or chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels.

[0030] instrument:

[0031] Press: Yijie screw press. 2L specification;

[0032] Reagents:

[0033] Pectinase: Biotech grade, McLean, 25g;

[0034] Embodiment 1:

[0035] This example provides a method for preparing blood orange-derived exosomes:

[0036] (1) Take fresh blood oranges, in this embodiment, the Chinese red blood oranges are used, wash them clean, and peel them;

[0037] (2) Place the peeled blood orange in the squeezer, slowly and evenly rotate the handle ball on the top, collect the blood orange juice squeezed out in a single time, and discard the residue;

[0038] (3) collecting juice, and filtering the obtained blood orange juice using an 80-mesh sieve;

[0039] (4) Add 0.02 g / L pectinase to the filtered blood orange juice, fully dissolve it, and place it at 37°C for enzymatic hydrolysis for 70 min;

[0040] (5) subjecting the blood orange enzymatic hydrolysate to high-speed refrigerated centrifugation at 4°C and 10,000 g for 60 min;

[0041] (6) After centrifugation, the supernatant was collected and concentrated and filtered using a tangential flow ultrafiltration system with an ultrafiltration membrane having a molecular weight cutoff of 100 KDa;

[0042] (7) Ultracentrifuge the concentrate at 4°C and 150,000 g for 70 min. Resuspend the concentrate in sterile saline and repeat twice.

[0043] After two ultracentrifugations, the precipitate was resuspended in sterile saline to obtain blood orange exosomes.

[0044] Comparative Example 1:

[0045] Each step and the reagents, processes and parameters used in each step are the same as those in Example 1, except that in step (2), blood orange juice is obtained by squeezing the juice, and in step (4), enzymatic hydrolysis is not performed, and finally a jelly-like sample is collected, and no exosomes are collected;

[0046] Comparative Example 2:

[0047] Each step and the reagents, processes and parameters used in each step are the same as those in Example 1, except that step (4) is not enzymatically hydrolyzed, and blood orange exosomes are finally collected, but the sample begins to become viscous after being left for 4 hours.

[0048] Comparative Example 3:

[0049] Each step and the reagents, processes and parameters used in each step were the same as those in Example 1, except that in step (4), the amount of pectinase added was 0.02 g / L, the enzymatic hydrolysis temperature was 37° C., the enzymatic hydrolysis time was 12 h, and no exosomes were collected.

[0050] In order to prove the effect of blood orange exosomes prepared by the present invention, the following verification experiments were conducted:

[0051] Effect verification 1:

[0052] Characterization of blood orange exosomes:

[0053] (1) Structural identification of blood orange exosomes: adjust the sample concentration, take 10uL sample and drop it on the copper mesh, let it stand at room temperature for 10min, wash it with deionized water three times, negatively stain the copper mesh with phosphotungstic acid for 30s, wash it with deionized water three times, dry it naturally at room temperature, observe the vesicle morphology and structure under a transmission electron microscope, and take pictures and record them.

[0054] (2) Blood orange exosome particle size detection: exosomes were collected for NTA testing.

[0055] Results: The isolated blood orange exosomes ( Figure 2 ) were characterized and tested respectively, and it can be seen that the blood orange exosome vesicles obtained in Example 1 are enriched and have a typical structure ( Figure 3 ), uniform particle size distribution ( Figure 5 ), the exosome morphology and particle size distribution obtained in comparative example 2 are shown in Figure 4 , Figure 6 After adjusting the two to the same protein concentration of 1 mg / mL, the particle concentration was detected. The number of exosome particles obtained in the embodiment of the present invention was 1.0E+12 Particles / mL, and the number of exosome particles in the control group was 5.3E+11 Particles / mL, which was 1.87 times that of the control group. The specific data are shown in Table 1.

[0056] Table 1 Comparison of exosome characterization

[0057]

[0058] Effect verification 2:

[0059] Detection of DPPH free radical scavenging ability of blood orange exosomes in vitro

[0060] (1) Solution preparation: DPPH solution: weigh 5 mg DPPH, dissolve it in anhydrous ethanol by ultrasonication and make up to 100 mL. Protect from light and prepare it before use. Sample solution: use physiological saline to gradually dilute the sample solution to different concentrations. Positive control: use physiological saline to prepare 100 ug / mL VC solution.

[0061] (2) Add reagents to the 96-well plate as shown in Table 2:

[0062] Table 2

[0063]

[0064] (3) Experimental data processing:

[0065] (4) DPPH free radical scavenging rate P% = (1-(As-Ac) / (Ab-A empty))*100%

[0066] P%——clearance rate;

[0067] As —— absorbance of the mixture of the test solution and DPPH solution;

[0068] Ac ——absorbance of the mixture of the test solution and anhydrous ethanol solution;

[0069] Ab ——absorbance of the mixture of DPPH solution and sample solvent solution;

[0070] A Blank - absorbance of blank tube

[0071] Results: The blood orange exosomes obtained by the present invention have the effect of scavenging DPPH in vitro, and its IC50 is 2.1209ug / mL. Figure 7 is the in vitro clearance rate standard curve, and Table 3 is the blood orange exosome clearance rate results.

[0072] Table 3 Results of blood orange exosome clearance rate

[0073]

[0074] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing blood orange exosomes, characterized in that: The following steps are involved: 1) Wash the surface of blood orange pulp with deionized water, then squeeze and filter, add 0.01-0.03g / L pectinase, fully dissolve, place at 35-40℃, and perform enzymolysis for 30-90min to obtain enzymolysis solution; 2) The enzymatic hydrolysate is subjected to high-speed refrigerated centrifugation to collect the supernatant, which is then concentrated and filtered to obtain a concentrate; 3) The concentrated solution is resuspended after ultracentrifugation, and then ultracentrifuged again and resuspended to obtain blood orange exosomes.

2. The method according to claim 1, characterized in that In step 1), the specific method of squeezing is: take fresh blood oranges, wash them, and peel them; place the peeled blood oranges in a squeezer, slowly and evenly rotate the upper handle ball, collect the blood orange juice squeezed once, and discard the residue.

3. The method according to claim 1, characterized in that In step 1), the sieve used for the filtration is 80-120 mesh.

4. The method according to claim 1, characterized in that: In step 2), the parameters of the high-speed refrigerated centrifugation are: 4°C, 10000-16000g, centrifugation for 40-60min.

5. The method according to claim 1, characterized in that In step 2), the concentration and filtration specifically includes: collecting the supernatant, and concentrating and filtering it through a tangential flow ultrafiltration system, and the ultrafiltration membrane used has a molecular weight cutoff of 100 kDa.

6. The method according to claim 1, characterized in that In step 3), the ultracentrifugation parameters are: 4°C, 100,000-150,000 g, 60-80 min.

7. Blood orange exosomes prepared by the method according to any one of claims 1 to 6.

8. Use of the blood orange exosomes according to claim 7 in the preparation of cosmetics.

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