KR20230161719A - Method for extraction of plant-derived exosomes with high purity - Google Patents

Abstract

The present invention relates to a method for extracting plant-derived exosomes and plant-derived exosomes extracted by the method. The extraction method of the present invention involves first ultracentrifugation of a plant sample to obtain a pellet containing exosomes. steps; Subjecting the pellet to sucrose density gradient ultracentrifugation to obtain a fraction containing exosomes from a layer with a sucrose concentration of 30% (w/v) to 45% (w/v); and separating exosomes from the fraction using a polymer-based precipitation method, thereby making it possible to extract high-purity exosomes from plant samples.

Description

{Method for extraction of plant-derived exosomes with high purity}

The present invention relates to a method for extracting high purity plant-derived exosomes.

Exosomes are nano-sized spherical lipid bilayer vesicles with a diameter of approximately 50-150 nm, and are known to play a central role in signaling between eukaryotic cells by transporting proteins and small RNA. Exosome research has been extensively studied in animal cells such as mammals and bacteria, including function, physical properties, components, production and secretion, etc. Recent studies have reported that not only animals but also plants secrete exosomes, and research is underway showing that plant exosomes are important components in various pathophysiological situations, including the regulation of plant immune responses to pathogen attacks. In addition, plant exosomes are relatively easier to extract than animal cell-derived exosomes, have less immune response when administered to animals, and show therapeutic effects on mammalian intestinal diseases, colitis tumors, and alcoholic liver disease. For this reason, plant exosomes are attracting attention for their potential use as a material for new drugs or useful health foods that can alleviate human diseases.

Ginseng ( Panax ginseng ) has been used as a useful medicinal plant in East Asian countries for more than 2,000 years due to its various beneficial effects on human health, such as regulating immune responses, protecting against nervous and heart diseases, and anticancer. Additionally, ginseng extract has been used as a herbal medicine to improve immunity and memory, and to improve anti-cortical and anti-aging abilities. The pharmacological action of ginseng is due to its bioactive components such as ginsenoside and other proteins, nucleic acids, and lipids, and is known to be effective for human health. Furthermore, a study on the extraction of ginseng-derived exosomes has recently been reported, and has anti-aging and anti-pigmentation effects in human skin cells, suggesting that ginseng-derived exosomes can potentially be used as a sustainable bioactive material and functional carrier. there is.

However, despite the potential importance of plant-derived exosomes, including ginseng, their biological effects, including their components and surface proteins, are not fully known. This is because the technology to extract plant exosomes with high purity is not sufficiently secured. Therefore, establishing an effective plant exosome isolation method is a key task that must be preceded in research on plant extracellular vesicle utilization, including clinical research.

Korean Patent No. 2125567

The purpose of the present invention is to provide a method for extracting plant-derived exosomes with high purity.

1. Obtaining a pellet containing exosomes by first ultracentrifuging the plant sample;

Subjecting the pellet to sucrose density gradient ultracentrifugation to obtain a fraction containing exosomes from a layer with a sucrose concentration of 30% (w/v) to 45% (w/v); and

A plant-derived exosome extraction method comprising the step of isolating exosomes from the fraction using a polymer-based precipitation method.

2. The method of extracting plant-derived exosomes according to 1 above, wherein the plant is a medicinal plant.

3. In 1 above, wherein the plant is ginseng, plant-derived exosome extraction method.

4. The method of extracting plant-derived exosomes according to 1 above, wherein the density of the layer with a sucrose concentration of 30% (w/v) to 45% (w/v) is 1.12 g/cm ³ to 1.20 g/cm ³ .

5. In 1 above, the step of separating the exosomes is to separate the exosomes from the fraction by treating the sample containing PEG (polyethylene glycol) to the fraction.

6. Plant-derived exosomes extracted by any of the methods 1 to 5 above.

Using the method of the present invention, high purity exosomes can be extracted from plant samples, and exosomes can be efficiently separated and extracted for impurities such as cell debris, waste, proteins, and large particles. In particular, high purity exosomes can be obtained from medicinal plants, so they can be widely used in the pharmaceutical industry.

Figure 1 shows the results of separating Arabidopsis and ginseng -derived exosomes by the sucrose density gradient ultracentrifugation method after performing the ultracentrifugation method and analyzing them using an electron microscope. It represents.
Figure 2 shows the results of Nanoparticle Tracking Analysis (NTA) quantitative analysis of Arabidopsis and ginseng-derived exosomes separated by sucrose concentration gradient ultracentrifugation after ultracentrifugation.
Figure 3 shows the results of separating Arabidopsis and ginseng-derived exosomes by a polymer-based precipitation method (ExoQuick reagent treatment method) and analyzing them through an electron microscope.
Figure 4 shows the results of Nanoparticle Tracking Analysis (NTA) quantitative analysis of exosomes derived from Arabidopsis thaliana and ginseng isolated by a polymer-based precipitation method (ExoQuick reagent treatment method).
Figure 5 shows the results of Nanoparticle Tracking Analysis (NTA) quantitative analysis of Arabidopsis and ginseng-derived exosomes isolated by a method combining ultracentrifugation, sucrose gradient ultracentrifugation, and polymer-based precipitation (ExoQuick reagent treatment method). .
Figure 6 shows the results of quantitative comparison of concentration and purity of exosomes derived from Arabidopsis thaliana and ginseng by extraction method.

Hereinafter, the present invention will be described in detail.

The present invention includes the steps of first ultracentrifuging a plant sample to obtain a pellet containing exosomes; Subjecting the pellet to sucrose density gradient ultracentrifugation to obtain a fraction containing exosomes from a layer with a sucrose concentration of 30% (w/v) to 45% (w/v); and isolating exosomes from the fraction using a polymer-based precipitation method.

When extracting exosomes from plants using all of the ultracentrifugation method, sucrose density gradient ultracentrifugation, and polymer-based precipitation method, the present inventors used ultracentrifugation After performing the separation method, it was found that exosomes of high purity could be obtained compared to the case of using sucrose concentration gradient ultracentrifugation or polymer-based precipitation alone.

Before performing the step of obtaining the pellet, a step of sequential centrifugation of the plant sample may be further performed. Before performing the step of obtaining the pellet, a step of sequentially centrifuging the plant sample to obtain a supernatant may be further performed. Cell debris and large endoplasmic reticulum can be removed from the plant sample by performing the sequential centrifugation steps. In this case, the step of obtaining the pellet may be to obtain a pellet containing exosomes by first ultracentrifuging the obtained supernatant.

The plant may be a medicinal plant or an edible plant. The medicinal plants may be ginseng, red ginseng, angelica root, peony, euphorbia, honeysuckle, motherwort, Morus chinensis, sophora ginseng, goji berry, Houttuynia cordata, rhodiola rosea, etc.

The plant sample includes all leaves, stems, roots, flowers, and plants of the plant. The plant sample may be a ground plant. The plant sample may be an extract obtained by grinding a plant.

The plant sample may be a supernatant obtained by pulverizing the plant and then performing sequential centrifugation to remove cell debris and large endoplasmic reticulum. The plant sample may be a supernatant obtained by pulverizing the plant and sequentially centrifuging the extract to remove cell debris and large endoplasmic reticulum.

The centrifugation is a method of separating each substance by rotating the sample to create a difference in the sedimentation speed of the substances contained in the sample according to the density difference, viscosity, mass, shape, viscosity, etc. The sequential centrifugation is a method of performing centrifugation multiple times by increasing the rotation speed, and can separate substances with relatively low density contained in the sample.

The plant sample is subjected to first ultracentrifugation to obtain a pellet containing exosomes.

The term “first ultracentrifugation” is intended to distinguish it from the sucrose concentration gradient ultracentrifugation described later, and refers to general ultracentrifugation.

The ultracentrifugation is a separation method based on the same principle as the centrifugation, but the rotation speed is different from centrifugation. The rotation speed of the ultracentrifugation may be, for example, 100,000 g or more, 120,000 g or more, or 150,000 g or more, but is not limited thereto.

The exosome refers to a nano-sized endoplasmic reticulum that cells secrete into the external environment for information exchange between cells. In the present specification, the exosome may be used interchangeably with extracellular endoplasmic reticulum.

Thereafter, the obtained pellet is subjected to sucrose density gradient ultracentrifugation to obtain a fraction containing exosomes from a layer with a sucrose concentration of 30% (w/v) to 45% (w/v). .

Before performing the step of obtaining the fraction containing the exosomes, a step of suspending the pellet in a suspension solution may be further performed. The suspension solution may be, for example, a PBS solution, but is not limited thereto.

The sucrose density gradient ultracentrifugation is ultracentrifugation performed after layering a sample in a centrifuge tube in which a sucrose concentration (density) gradient is formed. For example, the sucrose concentration (density) gradient may be a sucrose concentration gradient of 8% (w/v) to 60% (w/v).

The density of the layer having a sucrose concentration of 30% (w/v) to 45% (w/v) may be 1.12 g/cm ³ to 1.20 g/cm ³ . The density of the layer corresponds to a density in a similar range to that of exosomes, so when the layer is fractionated and used, exosomes can be obtained.

The obtained fraction containing exosomes may be an exosome band formed in a layer with a sucrose concentration of 30% (w/v) to 45% (w/v).

Afterwards, exosomes are separated from the fraction using a polymer-based precipitation method.

Before performing the step of separating the exosomes, a step of suspending the fraction in a suspension solution may be further performed. The suspension solution may be, for example, a PBS solution, but is not limited thereto.

The polymer-based precipitation method is a method of separating exosomes with high purity by mixing a sample with a precipitation solution containing a polymer to precipitate exosomes. The polymer-based precipitation method may be to separate exosomes from a pellet formed by centrifuging a mixture of a sample and a precipitation solution containing a polymer.

The step of separating the exosomes may involve treating the sample containing the polymer to the fraction and separating the exosomes from the fraction.

In one embodiment, the step of separating exosomes from the fraction using the polymer-based precipitation method may be separating exosomes from the fraction by treating the fraction with a sample containing PEG (polyethylene glycol). .

In one embodiment, the step of isolating exosomes from the fraction using the polymer-based precipitation method may be separating exosomes from the fraction by treating the fraction with ExoQuick-TC reagent from System Biosciences.

The present invention provides plant-derived exosomes extracted by the above-described plant-derived exosome extraction method.

As the plant-derived exosome extraction method has been described above, it will be omitted.

Hereinafter, the present invention will be described in detail with reference to examples.

Example

1. Isolation of extracellular endoplasmic reticulum from Arabidopsis thaliana and ginseng through ultracentrifugation isolation

(1) Experimental materials and methods

The plants used in the experiment were wild-type Arabidopsis thaliana (Col-0) and ginseng, a Korean native variety. To prepare an extract for isolation of extracellular endoplasmic reticulum, lg of 4-week-old Arabidopsis thaliana and 1-year-old ginseng root were used as samples, respectively. Each plant was washed repeatedly in running water, and after washing, it was pulverized in a homogenizer with 10 mL of Phosphate-buffered Saline (PBS, pH 7.2) to obtain the extract. Then, to purify the Arabidopsis thaliana and ginseng extracts, each extract was sequentially centrifuged at 4°C for 10 minutes at 1,000 g, 20 minutes at 3,000 g, and 60 minutes at 10,000 g to remove cell debris.

The obtained extracts of Arabidopsis thaliana and ginseng were first ultracentrifuged at 150,000 g for 90 minutes, and the precipitated pellet was then diluted in 100 μL of PBS and separated according to the sucrose concentration gradient. The diluted pellet was transferred to an ultracentrifuge tube divided into layers by concentration (8%/15%/30%/45%/60%) for sucrose concentration gradient, and then ultracentrifuged for 90 minutes at 4°C. Centrifugation was performed at 150,000 g (Optima XE-100, Beckman Coulter, USA). A band of 30-45% (w/v) layer corresponding to a density of about 1.12-1.20 g/cm ³ was collected, diluted in 10 mL of PBS, and centrifuged under the same centrifugation conditions (150,000 g, 90 minutes, 4°C). Centrifugation was performed, and the final remaining extracellular vesicle pellet was diluted with 100 μL of PBS (pH 7.2) and stored in cryogenic conditions at -80°C until use. The isolated extracellular vesicles were morphologically characterized through microscopic image analysis using cryogenic transmission electron microscopy (Cyro-TEM, JEM3200FS, JEOL, USA). In addition, extracellular vesicles were characterized by measuring the concentration, size distribution, and purity of extracellular vesicles through NTA (Nanoparticle tracking analysis) analysis. NTA analysis was performed using NTA analysis equipment (NanosightNS300™).

(2) Experiment results

Ultracentrifugation, a conventional extracellular vesicle separation method, has been used and verified in various biological fluids such as human serum and urine, and in the case of plants, it has been used to separate extracellular vesicles from ginger and grapefruit. It was confirmed whether the ultracentrifugation method is effective in separating extracellular endoplasmic reticulum from Arabidopsis thaliana, a model for plant research, and ginseng, a representative medicinal plant.

As described in the experimental method above, cell debris was removed from each plant extract, extracellular vesicles were precipitated through centrifugation at 150,000 g, and each extracellular vesicle was purified through a sucrose concentration gradient process. The isolated Arabidopsis and ginseng-derived extracellular vesicles were morphologically characterized using cryogenic transmission electron microscopy, and were confirmed to be spherical double-membrane vesicles (Figure 1).

Figure 1A is an image of 4-week-old Arabidopsis thaliana (top) and 1-year ginseng (bottom) used in this example, and white arrowheads indicate ginseng roots used to produce ginseng extract (Scale bar = 3 cm). . Figure 1B is an image of an Arabidopsis thaliana extract (top) and a ginseng extract (bottom) separated by layer after sucrose gradient ultracentrifugation, and each black arrowhead represents the formed extracellular endoplasmic reticulum band. Figures 1C and 1D are cryogenic transmission electron microscopy representative images (Figure 1C) and enlarged images (Figure 1D) of extracellular vesicles derived from Arabidopsis thaliana and ginseng isolated in this example, respectively, and each arrow represents isolated extracellular vesicles. represents. (Scale bar=100 nm).

Arabidopsis and ginseng-derived extracellular vesicles isolated through ultracentrifugation were characterized not only in terms of morphology but also in terms of concentration and size distribution through NTA analysis (Figure 2). Extracellular vesicles separated from each 1 g of plant through ultracentrifugation were subjected to NTA analysis by measuring images taken three times per sample using the NTA analysis equipment, as described in the experimental method above, and the calculated values were used. The concentration and purity of extracellular endoplasmic reticulum of Arabidopsis thaliana and ginseng were compared. The concentration of extracellular vesicles ^derived from Arabidopsis thaliana isolated through ^{ultracentrifugation} is about 3.96 It was measured. In addition, the purity of extraction of Arabidopsis and ginseng-derived cytoplasmic vesicles isolated through ultracentrifugation was compared. The purity of extracellular vesicle extraction was calculated by measuring the ratio of vesicles with a size of 50 to 150 nm among all extracted particles. The concentrations of endoplasmic reticulum corresponding to the size of 50~100 nm were 7.14×10 ¹² particles/mL and 2.39×10 ¹² particles/mL, respectively. This corresponds to 75.9% and 34.1%, respectively, of the total endoplasmic reticulum, and each percentage figure represents the extraction purity of extracellular endoplasmic reticulum.

Figure 2A shows NTA video frame images of Arabidopsis-derived extracellular vesicles (top) and ginseng-derived extracellular vesicles (bottom) isolated in this example. Figure 2B shows a linear graph of the size distribution and concentration of the endoplasmic reticulum particles isolated in this example. The green box in the graph of Figure 2B represents the range corresponding to the endoplasmic reticulum size of 50-150 nm, and the red area represents the standard error based on three repeated measurements. Figure 2C shows a graph comparing the quantitative concentration (top) and purity (bottom) of extracellular endoplasmic reticulum derived from Arabidopsis thaliana and ginseng isolated in this example. The error bar in Figure 2C represents the standard error based on three repeated measurements, and the asterisk (*) represents a statistically significant difference between the sample and the control group (p value < 0.01, t-test).

2. Isolation of extracellular endoplasmic reticulum from Arabidopsis and ginseng using the ExoQuick-TC™ method

(1) Experimental materials and methods

Extracellular vesicles were isolated from Arabidopsis and ginseng-derived extracts using ExoQuick (ExoQuick-TC™, System Biosciences, USA), a representative extracellular vesicle isolation reagent. Among various extracellular vesicle isolation kits, ExoQuick is a representative precipitation-based extracellular vesicle isolation reagent. Each plant extract was filled with PBS to 10 mL, then treated with 2 mL of ExoQuick reagent and thoroughly mixed. The mixed solution was kept refrigerated at 4°C for more than 12 hours to precipitate extracellular vesicles, then centrifuged at 1,500 g for 30 minutes to remove the supernatant, and then the precipitated extracellular vesicle pellet was obtained. The extracted extracellular vesicle pellet was diluted with 100 μL of PBS and morphologically characterized through microscopic image analysis using cryogenic transmission electron microscopy. In addition, the concentration, size distribution, and purity of extracellular vesicles isolated through the ExoQuick treatment method were calculated through NTA analysis. NTA analysis was performed using NTA analysis equipment (Nanosight NS300 ^TM ) and calculated through the average value of images measured three times per sample.

(2) Experiment results

Recently, a number of commercial kits that can isolate and study extracellular vesicles for various purposes have been released, and a representative method for separating extracellular vesicles using these is the ExoQuick treatment method, a polymer-based exosome precipitation kit. Using Arabidopsis thaliana, a model for plant research, and ginseng extract, a representative medicinal plant, the ExoQuick treatment was confirmed to be effective in extracting plant extracellular endoplasmic reticulum, and its concentration and purity were confirmed.

Arabidopsis thaliana and ginseng extracts, from which cell debris was removed by sequential centrifugation, were treated with ExoQuick reagent as in the above experimental method to obtain precipitated extracellular vesicle pellets. The isolated extracellular vesicles of Arabidopsis thaliana and ginseng were morphologically characterized through cryogenic transmission electron microscopy, and were confirmed to be spherical double-membrane vesicles (Figure 3).

Figure 3A is an image of 4-week-old Arabidopsis thaliana (top) and 1-year ginseng (bottom) used in this example, where white arrowheads indicate the roots of ginseng used to produce ginseng extract (Scale bar = 3 cm). . Figure 3B is an image of an extracellular vesicle pellet (Arabidopsis (top) and ginseng (bottom)) obtained through ExoQuick reagent treatment, and each black arrowhead represents a precipitated extracellular vesicle pellet. Figures 3C and 3D are cryogenic transmission electron microscopy representative images (Figure 3C) and enlarged images (Figure 3D) of extracellular vesicles derived from Arabidopsis thaliana and ginseng isolated in this example, respectively, and each arrow represents isolated extracellular vesicles. represents. (Scale bar=100 nm).

In addition, plant-derived extracellular vesicles isolated through the ExoQuick treatment method were characterized in terms of concentration and size distribution through NTA analysis using NTA analysis equipment (Nanosight NS300) (Figure 4).

Figure 4A shows NTA video frame images of Arabidopsis-derived extracellular vesicles (top) and ginseng-derived extracellular vesicles (bottom) isolated in this example. Figure 4B shows a linear graph of the size distribution and concentration of the endoplasmic reticulum particles isolated in this example. The green box in the graph of Figure 4B represents the range corresponding to the endoplasmic reticulum size of 50-150 nm, and the red area represents the standard error based on three repeated measurements. Figure 4C shows a graph comparing the quantitative concentration (top) and purity (bottom) of extracellular endoplasmic reticulum derived from Arabidopsis thaliana and ginseng isolated in this example. The error bar in Figure 4C represents the standard error based on three repeated measurements, and the asterisk (*) represents a statistically significant difference between the sample and the control group (p value < 0.01, t-test).

3. Isolation of extracellular endoplasmic reticulum through Combined Ultracentrifugation-ExoQuick method

(1) Experimental materials and methods

In order to extract high purity extracellular vesicles, extracellular vesicles were extracted from Arabidopsis thaliana and ginseng using the Combined Ultracentrifugation-ExoQuick method, which combines ultracentrifugation and purification through ExoQuick treatment. 1 g each of Arabidopsis thaliana and ginseng extracts were obtained, and sequential centrifugation was performed as described in 1-(1) above (1,000 g for 10 minutes, 3,000 g for 20 minutes, and 10,000 g for 60 minutes at 4°C). Cell debris and large endoplasmic reticulum were removed from each extract. First, to separate extracellular vesicles through ultracentrifugation, centrifugation was performed at 150,000 g for 90 minutes at 4°C. The pellet obtained through this was diluted with 100 μL of PBS and then placed in a sucrose concentration gradient tube (8%/15%/30%/45%/60%) divided by concentration under the same conditions (150,000 g, 90 minutes, 4 After centrifugation at ℃), the extracellular endoplasmic reticulum band generated in the 30-45% (w/v) sucrose layer was collected. The sucrose layer (30-45%) corresponds to a density of about 1.12-1.20 g/cm ³ , which is the density corresponding to extracellular endoplasmic reticulum, and the collected extracellular endoplasmic reticulum bands were filled with PBS and diluted to 10 mL. 2 mL of ExoQuick reagent was used for secondary purification of 10 mL of a solution of extracellular endoplasmic reticulum and PBS. To induce precipitation, the tube was left standing and refrigerated at 4°C for 12 hours, then centrifuged at 1,5000 g for 30 minutes to remove the supernatant, and an extracellular vesicle pellet was obtained. The extracellular vesicle pellet was diluted with 100 μL of PBS and morphologically characterized through microscopic image analysis using cryogenic transmission electron microscopy. For more specific characterization, as with extracellular vesicles isolated through other separation methods, the concentration, size distribution, and purity of extracellular vesicles were calculated through NTA analysis. NTA analysis was performed using NT analysis equipment (Nanosight NS300 ^TM ) and calculated through the average value of images measured three times per sample.

(2) Experiment results

Research on the use and application of extracellular vesicles requires high-purity extracellular vesicle isolation technology targeting substances other than endoplasmic reticulum or extracellular vesicles rather than large endoplasmic reticulum. In order to isolate extracellular endoplasmic reticulum from plants with high purity, the Combined Ultracentrifugation-ExoQuick method, which combines ultracentrifugation and purification through ExoQuick treatment, was applied to Arabidopsis thaliana and ginseng and its function was confirmed. . Arabidopsis and ginseng-derived extracellular endoplasmic reticulum, which was firstly extracted through ultracentrifugation and then sucrose concentration gradient ultracentrifugation, was secondarily purified by treatment with ExoQuick reagent. Arabidopsis and ginseng-derived extracellular vesicles extracted through the Combined Ultracentrifugation-ExoQuick method were morphologically characterized using cryogenic transmission electron microscopy. As a result, spherical double-membrane vesicles were identified, thereby verifying the function of the extracellular vesicle isolation method. Arabidopsis and ginseng-derived extracellular vesicles isolated through the Combined Ultracentrifugation-ExoQuick method were characterized not only by morphological characterization but also by concentration and size distribution analysis through NTA analysis (Figure 5).

Figure 5A shows NTA video frame images of Arabidopsis-derived extracellular vesicles (top) and ginseng-derived extracellular vesicles (bottom) isolated in this example. Figure 5B shows a linear graph of the size distribution and concentration of the endoplasmic reticulum particles isolated in this example. The green box in the graph of Figure 5B represents the range corresponding to the endoplasmic reticulum size of 50-150 nm, and the red area represents the standard error based on three repeated measurements. Figure 5C shows a graph comparing the quantitative concentration (top) and purity (bottom) of extracellular endoplasmic reticulum derived from Arabidopsis thaliana and ginseng isolated in this example. The error bar in Figure 5C represents the standard error based on three repeated measurements, and the asterisk (*) represents a statistically significant difference between the sample and the control group (p value < 0.01, t-test).

Furthermore, to compare and analyze the efficiency of the Combined Ultracentrifugation-ExoQuick method (UC+EQ), the concentration, size distribution, and purity of Arabidopsis and ginseng-derived extracellular vesicles isolated through NTA analysis were analyzed, followed by ultracentrifugation (UC), The concentration and purity of extracellular endoplasmic reticulum derived from Arabidopsis thaliana and ginseng isolated through ExoQuick treatment (EQ) were quantitatively compared (Figure 6). In Figure 6, the error bar indicates the standard error based on three repeated measurements, and the asterisk (*) indicates a statistically significant difference between the sample and the control group (p value < 0.01, t-test).

The concentration of endoplasmic reticulum extracted through the combined Ultracentrifugation-ExoQuick method was measured to be 9.23×10 ¹⁰ particles/mL for Arabidopsis-derived extracellular vesicles and 1.87×10 ¹² particles/mL for ginseng-derived extracellular vesicles. This amount was reduced by approximately 19 and 8 times, respectively, compared to the endoplasmic reticulum isolated through ultracentrifugation. However, in the case of purity (the amount of endoplasmic reticulum (50-150 nm in size compared to all endoplasmic reticulum)), it was 99.9 ± 0.1% and 83.3 ± 5.7%, respectively, 1.3- and 2.4-fold increases compared to extracellular endoplasmic reticulum through ultracentrifugation.

These results show that the Combined Ultracentrifugation-ExoQuick method is a method that can uniformly separate plant extracellular endoplasmic reticulum with high purity compared to the two methods.

The present invention proposes a method for extracting extracellular endoplasmic reticulum from plants with high purity, and provides a source technology for isolating extracellular endoplasmic reticulum with high purity from ginseng as well as other edible and medicinal crops. In addition, we expect that this ultra-pure separation technology will be used as an important foundational technology for future research on plant cell body formation and function.

Claims (6)

Obtaining a pellet containing exosomes by first ultracentrifuging the plant sample;
Subjecting the pellet to sucrose density gradient ultracentrifugation to obtain a fraction containing exosomes from a layer with a sucrose concentration of 30% (w/v) to 45% (w/v); and
A plant-derived exosome extraction method comprising the step of isolating exosomes from the fraction using a polymer-based precipitation method.
The method of extracting plant-derived exosomes according to claim 1, wherein the plant is a medicinal plant.
The method of claim 1, wherein the plant is ginseng.
The method of claim 1, wherein the density of the layer having a sucrose concentration of 30% (w/v) to 45% (w/v) is 1.12 g/cm ³ to 1.20 g/cm ³ .
The method of claim 1, wherein the step of isolating the exosomes involves separating the exosomes from the fraction by treating the sample containing PEG (polyethylene glycol) with the fraction.
Plant-derived exosomes extracted by the method of any one of claims 1 to 5.