Method for efficiently extracting rose exosomes

Abstract

The invention discloses a method for efficiently extracting rose exosomes, which comprises the following steps: (1) pretreatment of rose petals: refrigerating and pre-cooling fresh rose petals, and then alternately irradiating with red light and blue light; (2) preparing rose homogenate: mixing rose petals with an antioxidant solvent, and homogenizing to obtain the rose homogenate; (3) exosome extraction: performing ultrasonic treatment on the rose homogenate; (4) exosome purification: filtering the rose homogenate to remove residues so as to obtain a rose exosome purified solution; and (5) exosome preservation: adding a solid membrane component into the exosome purified liquid, and freeze-drying to obtain exosome powder. Red light and blue light are alternately irradiated to promote release of the exosome in cells, and the composite antioxidant reagent is adopted to protect the exosome, so that the extraction efficiency and the integrity of active ingredients in the exosome are remarkably improved, the extraction cost is reduced, and powerful support is provided for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to a method for extracting plant exosomes, and in particular to a method for efficiently extracting rose exosomes. Background Art

[0002] Rose exosomes refer to extracellular vesicles extracted from rose tissues or cells. They carry bioactive components including nucleic acids, proteins, lipids and metabolites, and have potential application value in related fields such as skin care, medical care and agriculture.

[0003] Currently, rose exosomes are primarily extracted using multiple centrifugation cycles. For example, patent CN109568414A discloses a method for extracting rose essence and exosomes from rose flowers. The specific steps are: soaking the rose flowers in physiological saline or PBS, homogenizing them, and centrifuging them at 500-5000g, 10,000-15,000g, 80,000-130,000g for 20-40 min, 80-120 min, and 80-120 min, respectively. The corresponding supernatants are collected to obtain rose exosomes. This method utilizes the differences in density and size between exosomes and other impurity particles, achieving initial separation through stepwise centrifugation. It is relatively simple to operate and does not require complex instrumentation, making it feasible and reliable for small-scale laboratory studies. However, this method requires multiple, prolonged ultracentrifugation cycles, resulting in low extraction efficiency and a lengthy extraction process. This makes it difficult to meet the requirements for efficient extraction for large-scale industrial production. In addition, during ultracentrifugation, excessively high centrifugal force may cause certain damage to the structure and function of rose exosomes, affecting the biological activity and quality of exosomes. Patent CN 117257714A discloses a cosmetic composition containing exosomes and a preparation method thereof. The exosome extraction method comprises the following steps: using fresh samples of Panax notoginseng, black red rose, geranium, short-stemmed Erigeron breviscapus, ground lotus and polygonatum as raw materials, taking 225g of the raw materials, crushing them at -40°C, centrifuging them at 1000 rpm for 35 minutes and then at 8000 rpm for 45 minutes, collecting the supernatant, and centrifuging the supernatant at 30,000 rpm for 2.25 hours. The precipitate obtained by centrifugation is suspended in PBS, and 3.5% glycerol and 48% sucrose are added to the suspension, followed by centrifugation at 30,000 rpm for 3.75 hours. The sucrose is washed off with PBS, and the suspension is centrifuged again at 30,000 rpm for 3.75 hours. The resulting precipitate is freeze-dried to prepare the exosomes corresponding to the raw materials. This patent adopts complex processes such as multi-step centrifugation and freeze-drying, including tissue fragmentation, multiple centrifugation, suspension, washing and other steps. The operation is cumbersome and has high requirements for equipment and operating conditions, making it difficult to achieve large-scale and efficient production.

[0004] In summary, the existing technology mainly extracts rose exosomes through multi-step centrifugation, which has low extraction efficiency. Summary of the Invention

[0005] Purpose of the invention: The present invention aims to provide a method for efficiently extracting rose exosomes, which improves the extraction efficiency of rose exosomes and the integrity of the active ingredients in the exosomes.

[0006] Technical solution: The method for efficiently extracting rose exosomes according to the present invention comprises the following steps:

[0007] (1) Rose petal pretreatment

[0008] Fresh rose petals were pre-cooled in the refrigerator and then irradiated with red light and blue light alternately;

[0009] (2) Preparation of rose homogenate

[0010] Mixing rose petals with an antioxidant solvent and then homogenizing the mixture to obtain a rose homogenate;

[0011] (3) Exosome extraction

[0012] Rose homogenate was treated by ultrasonication;

[0013] (4) Exosome purification

[0014] The rose homogenate is filtered to remove the residue to obtain the rose exosome purification solution;

[0015] (5) Exosome preservation

[0016] The solid film component is added to the exosomes, and then dried to obtain exosome powder.

[0017] Preferably, in step (1), the pre-cooling temperature of the low-temperature refrigeration is 4-8° C. Low temperature can reduce the physiological metabolic rate of the petals and reduce the risk of exosome degradation.

[0018] Preferably, in step (1), the wavelength of the red light is precisely 620-700 nm, and the intensity is 100-150 μmol / (m 2 ·s); the wavelength of the blue light is 450 to 490 nm, and the intensity is 80 to 120 μmol / (m 2·s). Preferably, the alternating irradiation of red light and blue light is as follows: red light irradiation for 10 to 12 hours and blue light irradiation for 6 to 8 hours for light treatment, and the duration is 1 to 3 days. Alternating irradiation with red and blue light can effectively stimulate the physiological activities in petal cells, thereby facilitating the release of exosomes in cells. Light is crucial to the physiological activities of plant cells. Plant cells contain specific photoreceptors, such as phytochromes that sense red light and cryptochromes that sense blue light. When these photoreceptors are alternately activated by red and blue light, they work synergistically to activate complex downstream signaling pathways, thereby significantly enhancing the overall metabolic activity of the cells and further promoting the release of cellular exosomes.

[0019] Preferably, in step (2), the solvent in the antioxidant solvent is normal saline, and the antioxidant components are 0.1-0.3 g / L vitamin C, 0.05-0.15 g / L vitamin E, and 0.1-0.3 g / L tea polyphenols. Preferably, the weight ratio of the rose petals to the antioxidant solvent is 1:10-1:20. Vitamin C, vitamin E, and tea polyphenols act synergistically in the extraction system, effectively capturing free radicals and blocking the oxidation reaction chain, thereby providing all-round protection for the active ingredients in the exosomes, preventing them from oxidative denaturation or degradation during the extraction process, and ensuring that the biological activity of the exosomes is fully preserved.

[0020] Preferably, in step (2), the homogenization is performed by grinding the mixed solution in a tissue homogenizer to obtain rose homogenate.

[0021] Preferably, in step (3), the ultrasonic treatment power is 50 to 150 W and the duration is 10 to 30 minutes. The mechanical vibration of low-power ultrasound can gently destroy the cell wall and cell membrane structure of the petals, allowing the contents within the cells to be fully released, thereby significantly promoting the release of exosomes from the petal cells and improving the extraction efficiency.

[0022] Preferably, in step (4), the method for filtering and removing residues is: first filtering with gauze to remove rose residues, and then filtering and purifying with tangential flow filtration technology, and the exosomes are trapped in the filter membrane. Further preferably, first filtering with 100 mesh and 400 mesh gauze to remove rose residues. The exosomes are further purified by tangential flow filtration technology, and the rose infiltration liquid is filtered and purified using a fully automatic exosome extraction and purification instrument. The exosomes are trapped in the filter membrane, and finally the filter membrane is rinsed with physiological saline to obtain a rose exosome purified liquid. Tangential flow filtration can effectively retain exosomes while allowing small molecular impurities to pass through the filter membrane, thereby obtaining a filtrate rich in exosomes and high purity, which is ready for subsequent storage and application.

[0023] Preferably, in step (5), the film-forming components are mannitol, trehalose, and maltodextrin. Further preferably, the amount of mannitol added is 1-3% of the volume of the purified exosome solution, the amount of trehalose added is 2-4% of the volume of the purified exosome solution, and the amount of maltodextrin added is 3-5% of the volume of the purified exosome solution.

[0024] Preferably, the drying step is freeze-drying. The uniformly mixed exosomes and solid film components are placed in a freeze-drying apparatus and processed according to a preset freeze-drying procedure to obtain a stable and easily storable freeze-dried exosome powder. These solid film components can be tightly adsorbed to the surface of the exosomes during the freeze-drying process, forming a protective membrane structure that enhances the exosomes' resistance to external environmental factors. During long-term storage of this freeze-dried powder, the active ingredients of the exosomes are well protected and can be readily reconstituted for subsequent applications in cosmetics, biopharmaceuticals, and other fields.

[0025] Invention mechanism:

[0026] The present invention activates metabolic activities in petal cells in advance through light regulation in the rose pre-treatment stage, putting exosomes in an "easy to release" physiological state and promoting the release of more exosomes.

[0027] Low-power ultrasonic treatment during the extraction process further destroys the cell structure and opens channels for the release of exosomes.

[0028] During the extraction stage, antioxidants such as vitamin C, vitamin E and tea polyphenols scavenge free radicals in real time during the extraction process, blocking the oxidation reaction pathway and ensuring the integrity of exosomes.

[0029] During the storage stage, the solid film structure formed by mannitol, trehalose and maltodextrin can effectively prevent the exosomes from undergoing adverse phenomena such as oxidation, aggregation and degradation during long-term storage, ensuring the quality and effectiveness of exosomes in practical applications.

[0030] During the extraction and preservation process, the constructed composite antioxidant system and solid film preservation strategy are like a layer of "double armor" for the exosomes, ensuring the integrity of the exosomes.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The exosome yield of the present invention is significantly improved, the extraction efficiency is significantly increased, the extraction cost is reduced, and strong support is provided for large-scale industrial production; (2) The integrity of the active ingredients in the exosomes extracted by the method of the present invention is significantly improved; (3) The process of the present invention is simple and efficient, easy to operate, and easy to replicate and promote in industrial production environments of different scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Flow chart of the preparation process of rose exosomes;

[0033] Figure 2 This is a morphological diagram of rose exosomes prepared in Example 1;

[0034] Figure 3 This is the morphology of rose exosomes in comparative example 2;

[0035] Figure 4 This is a graph showing the particle concentration and particle size of rose exosomes in Example 1;

[0036] Figure 5 This is a graph showing the particle concentration and particle size of rose exosomes in comparative example 1;

[0037] Figure 6 This is a graph showing the particle concentration and particle size of rose exosomes in comparative example 2. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0039] Example 1

[0040] The method for efficiently extracting rose exosomes of the present invention comprises the following steps:

[0041] (1) Rose petal pretreatment

[0042] 200g of fresh Pingyin roses were selected and quickly stored at 4-8℃ for 2h to reduce the physiological metabolic rate of the roses. Then, they were placed in an intelligent light incubator with a red light wavelength of 630nm and an intensity of 100μmol / (m 2 ·s), blue light wavelength 450nm, intensity 120μmol / (m 2 ·s), and the light treatment was carried out according to the cycle of red light irradiation for 12 hours and blue light irradiation for 7 hours, which lasted for 2 days.

[0043] (2) Preparation of rose homogenate

[0044] Preparation of composite antioxidant solvent: 0.2 g of vitamin C, 0.1 g of vitamin E and 0.2 g of tea polyphenols were dissolved in 1 L of normal saline to prepare a composite antioxidant solvent.

[0045] The treated rose petals were mixed with the composite antioxidant solvent at a weight ratio of 1:15 and ground with a tissue homogenizer for 10 minutes to obtain rose homogenate.

[0046] (3) Exosome extraction

[0047] The rose homogenate was placed in an ultrasonic instrument, the ultrasonic power was set to 100 W, and the treatment was performed for 20 min.

[0048] (4) Exosome purification

[0049] The homogenate was first filtered through 100-mesh gauze to remove large debris, and then filtered through 400-mesh gauze to obtain a relatively clear filtrate. The filtrate was introduced into a fully automated exosome extraction and purification instrument using tangential flow filtration technology. Filtration was performed at a pressure of 0.2-0.3 MPa and a temperature of 4-8°C for 30 minutes. The filter membrane was rinsed with 5 mL of physiological saline to obtain a purified rose exosome solution rich in rose exosomes.

[0050] (5) Exosome preservation

[0051] Mannitol 2% (volume fraction), trehalose 3% (volume fraction) and maltodextrin 4% (volume fraction) were added to the rose exosome purified solution, mixed evenly and poured into a freeze drying tray. The freeze drying equipment was placed at -40°C and a vacuum degree of 10 Pa. The solution was dried for 48 hours to obtain exosome freeze-dried powder.

[0052] Example 2

[0053] The method for efficiently extracting rose exosomes of the present invention comprises the following steps:

[0054] (1) Rose petal pretreatment

[0055] 200g of fresh Pingyin roses were selected and quickly stored at 4℃ for 3h to reduce the physiological metabolic rate of the roses. Then they were placed in an intelligent light incubator with a red light wavelength of 625nm and an intensity of 120μmol / (m 2 ·s), blue light wavelength 455nm, intensity 100μmol / (m 2 ·s), and the light treatment was carried out according to the cycle of red light irradiation for 11 hours and blue light irradiation for 7 hours, which lasted for 3 days.

[0056] (2) Preparation of rose homogenate

[0057] Preparation of composite antioxidant solvent: 0.25 g of vitamin C, 0.12 g of vitamin E and 0.25 g of tea polyphenols were dissolved in 1 L of normal saline to prepare a composite antioxidant solvent.

[0058] The treated rose petals were mixed with the composite antioxidant solvent at a weight ratio of 1:18 and ground with a tissue homogenizer for 12 minutes to obtain rose homogenate.

[0059] (3) Exosome extraction

[0060] The rose homogenate was placed in an ultrasonic instrument, the ultrasonic power was set to 120W, and the treatment was carried out for 25 minutes.

[0061] (4) Exosome purification

[0062] The homogenate was first filtered through 100-mesh gauze to remove large debris, then filtered through 400-mesh gauze to obtain a relatively clear filtrate. The filtrate was then introduced into a fully automated exosome extraction and purification instrument using tangential flow filtration technology. Filtration was performed at a pressure of 0.25 MPa and a temperature of 5°C for 35 minutes. The filter membrane was rinsed with 5 mL of saline to obtain a purified rose exosome solution rich in rose exosomes.

[0063] (5) Exosome preservation

[0064] To the purified rose exosomes solution, add 2.5% mannitol, 3.5% trehalose, and 4.5% maltodextrin, mix well, pour into a freeze-drying tray, place in a freeze-drying equipment, set the freezing temperature to -45°C and the vacuum degree to 8 Pa, and dry for 50 hours to obtain exosome freeze-dried powder.

[0065] Example 3

[0066] The method for efficiently extracting rose exosomes of the present invention comprises the following steps:

[0067] (1) Rose petal pretreatment

[0068] 200g of fresh Pingyin rose petals were selected and quickly stored at 8℃ for 1h to reduce the physiological metabolic rate of roses. Then they were placed in an intelligent light incubator with red light wavelength of 630nm and intensity of 150μmol / (m 2 ·s), blue light wavelength 460nm, intensity 120μmol / (m 2 ·s), and the light treatment was carried out according to the cycle of red light irradiation for 10 hours and blue light irradiation for 8 hours, which lasted for 1 day.

[0069] (2) Preparation of rose homogenate

[0070] Preparation of composite antioxidant solvent: 0.15 g of vitamin C, 0.08 g of vitamin E and 0.15 g of tea polyphenols were dissolved in 1 L of normal saline to prepare a composite antioxidant solvent.

[0071] The treated rose petals were mixed with the composite antioxidant solvent at a weight ratio of 1:12 and ground with a tissue homogenizer for 8 minutes to obtain rose homogenate.

[0072] (3) Exosome extraction

[0073] The rose homogenate was placed in an ultrasonic instrument, the ultrasonic power was set to 80W, and the treatment was carried out for 15 minutes.

[0074] (4) Exosome purification

[0075] The homogenate was first filtered through 100-mesh gauze to remove large debris, then filtered through 400-mesh gauze to obtain a relatively clear filtrate. The filtrate was then introduced into a fully automated exosome extraction and purification instrument using tangential flow filtration technology. Filtration was performed at a pressure of 0.22 MPa and a temperature of 6°C for 25 minutes. The filter membrane was rinsed with 5 mL of saline to obtain a purified rose exosome solution rich in rose exosomes.

[0076] (5) Exosome preservation

[0077] 1.5% mannitol, 2.5% trehalose, and 3.5% maltodextrin were added to the rose exosome purified solution, mixed evenly, and poured into a freeze-drying tray. The solution was placed in a freeze-drying apparatus, set at a freezing temperature of -35°C and a vacuum degree of 12 Pa, and dried for 40 h to obtain exosome freeze-dried powder.

[0078] Comparative Example 1

[0079] On the basis of Example 1, red and blue light irradiation was not performed in step (1), and other conditions remained unchanged to obtain rose exosome freeze-dried powder.

[0080] Comparative Example 2

[0081] On the basis of Example 1, in step (2), physiological saline was used to replace the composite antioxidant solvent, and other conditions remained unchanged to obtain rose exosome lyophilized powder.

[0082] Structural characterization

[0083] 1. Morphological characterization of rose exosomes

[0084] The lyophilized powders of Example 1 and Comparative Example 2 were dissolved in 5 mL of normal saline and gently pipetted evenly. Then, about 10 μL of the suspension was adsorbed on a copper mesh, and 10 μL of 2% phosphotungstic acid solution was added for staining. After drying, the microscopic morphology of the exosomes was observed using a transmission electron microscope. Figure 2 and 3 shown.

[0085] Depend on Figure 2 The exosomes prepared in Example 1 exhibited a typical saucer-shaped exosome morphology, with uniform size, smooth surface, no obvious damage, and a complete vesicle structure. This morphological feature is a typical hallmark of exosomes, indicating that the structure of the exosomes was well preserved during the extraction process, with high integrity.

[0086] Depend on Figure 3 It can be seen that the exosomes prepared in Comparative Example 2 were incomplete in morphology, showing characteristics of deformation, damage or granular aggregation. Some exosomes had blurred structures, and even ruptured and leaked contents.

[0087] The exosomes prepared in Example 1 have higher integrity than those in Comparative Example 2. This is because Example 1 uses alternating red and blue light irradiation and a composite antioxidant system during the extraction process. Alternating red and blue light irradiation can effectively stimulate physiological activities in petal cells and promote the release of exosomes. At the same time, combined with low-power ultrasonic treatment, it gently destroys the cell structure and fully releases the exosomes. The composite antioxidants (vitamin C, vitamin E and tea polyphenols) scavenge free radicals in real time during the extraction process, block the oxidation reaction path, and form all-round protection for the active ingredients in the exosomes, preventing them from oxidative denaturation or degradation during the extraction process, thereby ensuring the integrity of the exosomes. In contrast, Comparative Example 2 did not use an antioxidant solvent when preparing the rose homogenate, lacking antioxidant protection, making the exosomes more susceptible to oxidative damage during the extraction process, further affecting their integrity.

[0088] 2. Rose exosome particle concentration and particle size test

[0089] The lyophilized powders of Example 1 and Comparative Examples 1-2 were dissolved in 5 mL of normal saline, and gently pipetted evenly. The particle concentration and particle size of the exosomes were detected using a nanoparticle tracking analyzer (NTA). The results were as follows: Figures 4-6 shown.

[0090] Depend on Figure 4 It can be seen that the particle size of the exosomes prepared in Example 1 is mainly concentrated at 135 nm, and the concentration is 1.1×10 12 particles / mL.

[0091] Depend on Figure 5 It can be seen that the particle size of the exosomes prepared in Comparative Example 1 is mainly concentrated at 148 nm, and the concentration is 1.8×10 11 The concentration of Comparative Example 1 is lower than that of Example 1 because Comparative Example 1 is not irradiated with red and blue light, and the release of exosomes is small.

[0092] Depend on Figure 6 It can be seen that the particle size of the exosomes prepared in Comparative Example 2 is mainly concentrated at 156 nm, and the concentration is 1.3×10 11 In Comparative Example 2, the concentration was lower and the particle size distribution was wider than in Example 1. This was because no antioxidant solvent was used in the preparation of the rose homogenate, lacking antioxidant protection. Some exosomes underwent oxidative aggregation during the extraction process, resulting in larger particle size and wider distribution, and also affecting the extraction efficiency.

Claims

1. A method for efficiently extracting rose exosomes, characterized in that: The following steps are involved: (1) Rose petal pretreatment Fresh rose petals were pre-cooled in the refrigerator and then irradiated with red light and blue light alternately; (2) Preparation of rose homogenate Mixing rose petals with an antioxidant solvent and then homogenizing the mixture to obtain a rose homogenate; (3) Exosome extraction Rose homogenate was treated by ultrasonication; (4) Exosome purification The rose homogenate is filtered to remove the residue to obtain the rose exosome purification solution; (5) Exosome preservation The solid film component is added to the exosome purified solution, and then dried to obtain exosome powder.

2. The method for efficiently extracting rose exosomes according to claim 1, characterized in that In step (1), the wavelength of the red light is precisely 620-700 nm, and the intensity is 100-150 μmol / (m 2 ·s); the wavelength of the blue light is 450 to 490 nm, and the intensity is 80 to 120 μmol / (m 2 ·s).

3. The method for efficiently extracting rose exosomes according to claim 2, characterized in that: In step (1), the alternating irradiation of red light and blue light is as follows: the irradiation cycle is 10 to 12 hours of red light and 6 to 8 hours of blue light, and the duration is 1 to 3 days.

4. The method for efficiently extracting rose exosomes according to claim 1, characterized in that In step (2), the solvent in the antioxidant solvent is physiological saline, and the antioxidant components are vitamin C 0.1-0.3 g / L, vitamin E 0.05-0.15 g / L and tea polyphenols 0.1-0.3 g / L.

5. The method for efficiently extracting rose exosomes according to claim 4, characterized in that: The weight ratio of the rose petals to the antioxidant solvent is 1:10 to 1:

20.

6. The method for efficiently extracting rose exosomes according to claim 1, characterized in that: In step (3), the power of the ultrasonic treatment is 50 to 150 W, and the time is 10 to 30 minutes.

7. The method for efficiently extracting rose exosomes according to claim 1, characterized in that: In step (4), the method for filtering and removing the residue is: first filtering with gauze to remove the rose residue, and then using tangential flow filtration technology to filter and purify, and the exosomes are trapped in the filter membrane.

8. The method for efficiently extracting rose exosomes according to claim 1, characterized in that: In step (5), the film-forming components are mannitol, trehalose and maltodextrin.

9. The method for efficiently extracting rose exosomes according to claim 8, characterized in that: The amount of mannitol added is 1-3% of the volume of the exosome purified solution, the amount of trehalose added is 2-4% of the volume of the exosome purified solution, and the amount of maltodextrin added is 3-5% of the volume of the exosome purified solution.

10. The method for efficiently extracting rose exosomes according to claim 1, characterized in that: The drying is freeze-drying.

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