Ribonucleic acid-containing freeze-drying protection liquid and application thereof

Abstract

The invention relates to a freeze-drying protection solution containing ribonucleic acid and application of the freeze-drying protection solution. The freeze-drying protection solution is prepared from the following components: wild almond oil polyglycerol-6 esters, polyglycerol-10 oleate, erythritol, trehalose, crithmum maritimum callus culture filtrate and DNA sodium. The freeze-drying protection liquid provided by the invention can prevent the structural integrity of the plant exosome, reduce the osmotic pressure difference inside and outside cells and maintain the cell activity, and also can avoid the influence of stress on the biological activity of the plant exosome in the freeze-drying process, thereby maintaining the stability of the biological activity of the plant exosome.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and in particular relates to a lyophilized protective solution containing ribonucleic acid and an application thereof. Background Art

[0002] Exosomes are the most essential part of stem cells. Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. Nowadays, they specifically refer to disc-shaped vesicles with a diameter of 40-100nm. Exosomes are a type of extracellular vesicle secreted by cells. Their main components are phospholipid bilayers and related contents. Exosomes are divided into plant exosomes and animal exosomes. In the existing technology, animal exosomes are widely used in the medical, beauty and other industries.

[0003] Plant exosomes are a type of membrane vesicles isolated from plant tissues, containing components such as proteins, lipids and nucleic acids. They have a similar structure to animal exosomes. Many common plants in life, such as algae, ginger, Centella asiatica, etc., contain a large number of exosomes. Because of their wide sources, safety and non-toxicity, and their own anti-inflammatory, antibacterial, anti-tumor and cell-targeting effects, plant-derived exosomes have also attracted more and more attention in recent years.

[0004] However, the shelf life of plant exosomes is very short, which greatly limits the scope of their application. Therefore, the preservation of plant exosomes is an urgent problem that needs to be solved. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a freeze-dried protective solution containing ribonucleic acid and its application, which can be used for the long-term preservation of plant exosomes, which can not only maintain the structural integrity of the plant exosomes after preservation, but also maintain their original biological activity.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a freeze-dried protective solution containing ribonucleic acid, wherein the components of the freeze-dried protective solution include wild almond oil polyglycerol-6 esters, polyglycerol-10 oleate, erythritol, trehalose, sea fennel callus culture filtrate and DNA sodium.

[0008] The freeze-dried protective solution provided by the present invention can prevent the structural integrity of plant exosomes, reduce the osmotic pressure difference between the inside and outside of cells, and maintain cell vitality. The freeze-dried protective solution can also avoid the influence of stress on the biological activity of plant exosomes during the freeze-drying process, thereby maintaining the stability of the biological activity of plant exosomes.

[0009] Preferably, the components of the freeze-dried protective solution include, by weight, 0.5-10 parts of wild almond oil polyglycerol-6 esters, 1-10 parts of polyglycerol-10 oleate, 1-10 parts of erythritol, 1-10 parts of trehalose, 0.01-5 parts of sea fennel callus culture filtrate and 1-5 parts of sodium DNA;

[0010] The amount of wild almond oil polyglycerol-6 esters added to the freeze-dried protective solution of the present invention can be 0.8 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or 9 parts, etc.;

[0011] The amount of polyglycerol-10 oleate added can be 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or 9 parts, etc.;

[0012] The amount of erythritol added can be 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or 9 parts, etc.;

[0013] The added amount of trehalose can be 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or 9 parts, etc.;

[0014] The addition amount of the sea fennel callus culture filtrate can be 0.03 parts, 0.05 parts, 0.1 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 parts, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts or 4.5 parts, etc.;

[0015] The added amount of the DNA sodium is 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts or 4.5 parts, etc.

[0016] Preferably, the components of the freeze-dried protective solution include, by weight, 0.5-5 parts of wild almond oil polyglycerol-6 esters, 1-6 parts of polyglycerol-10 oleate, 1-8 parts of erythritol, 1-8 parts of trehalose, 0.01-2 parts of sea fennel callus culture filtrate and 1-3 parts of sodium DNA.

[0017] In a second aspect, the present invention provides a use of the freeze-dried protective solution containing ribonucleic acid as described in the first aspect in the preservation and treatment of plant exosomes.

[0018] In a third aspect, the present invention provides a method for preserving and processing plant exosomes, the method comprising mixing plant exosomes with the freeze-dried protective solution containing ribonucleic acid described in the first aspect in a phosphate buffer to obtain a mixed solution, pre-freezing the mixed solution, and performing a plugging treatment after drying to complete the preservation of the plant exosomes.

[0019] Preferably, the mass ratio of the plant exosomes to the freeze-dried protective solution is 1:(1-5), for example, it can be 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 or 1:4.5, etc.

[0020] Preferably, the concentration of the phosphate buffer is 0.2-0.5 mol / L, for example, it can be 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L or 0.45 mol / L.

[0021] Preferably, the volume ratio of the lyophilization protection solution to the phosphate buffer is 1:(10-20), for example, it can be 1:11, 1:13, 1:15, 1:17 or 1:19.

[0022] Preferably, the plant exosomes include any one of Chlorella exosomes, Centella asiatica exosomes or Camellia exosomes, or a combination of at least two of them.

[0023] Preferably, the plant exosomes are Chlorella exosomes.

[0024] Preferably, the method for preparing Chlorella exosomes comprises:

[0025] (1) treating the cell wall of Chlorella vulgaris, wherein the pH value of the cell wall treatment is 8-10, the temperature is 70-80° C., and the time is 20-60 min;

[0026] (2) subjecting the cell fluid after cell wall disruption to gradient centrifugation, wherein the specific steps of the gradient centrifugation are: centrifugation at 300-400 g for 5-15 min, centrifugation of the supernatant at 1000-2000 g for 10-30 min, centrifugation of the supernatant at 4000-5000 g for 10-30 min, centrifugation of the supernatant at 6000-7000 g for 10-30 min, and then taking the supernatant to obtain a crude exosome extract;

[0027] (3) resuspending the crude exosome extract with Tween 80 aqueous solution at a volume ratio of 1:(0.3-1), and then ultracentrifuging 2-4 times, each time at a speed of 15000-20000g for 30-60 min to obtain purified exosomes;

[0028] (4) The purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution are mixed, and then homogenized by ultra-speed microfluidization to obtain the Chlorella exosomes.

[0029] Preferably, the concentration of the Tween 80 aqueous solution in step (3) is 20-30 mg / mL, for example, 21 mg / mL, 23 mg / mL, 25 mg / mL, 27 mg / mL or 29 mg / mL.

[0030] Preferably, the volume ratio of the purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution in step (4) is (2-5):(0.1-1):(0.1-1).

[0031] Wherein "2-5" can be 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5 or 4.8, etc.;

[0032] The first "0.1-1" can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, etc.;

[0033] The second "0.1-1" can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, etc.

[0034] Preferably, the concentration of the phosphate nanoparticle solution in step (4) is 0.2-0.5%, for example, it can be 0.25%, 0.3%, 0.35%, 0.4% or 0.45%, etc.

[0035] Preferably, the jet velocity of the ultrasonic microjet in step (4) is 5000-7000 m / s. For example, it can be 5200 m / s, 5400 m / s, 5600 m / s, 5800 m / s, 6000 m / s, 6200 m / s, 6400 m / s, 6600 m / s or 6800 m / s.

[0036] Preferably, the pre-freezing step comprises: cooling the mixed solution to 0-3°C (for example, 0.5°C, 1°C, 1.5°C, 2°C or 2.5°C, etc.) and keeping it warm for 1-3h (for example, 1.1h, 1.3h, 1.5h, 1.7h, 2h, 2.5h, 2.5h or 2.8h, etc.), then cooling the mixed solution to -10 to -15°C (for example, -11°C, -12°C, -13°C or -14°C, etc.) and keeping it warm for 1-3h (for example, 1.1h, 1.3h, 1.5h, 1.7h, 2h, 2.5h, 2.5h or 2.8h, etc.). Or 2.8h, etc.), then cool down to -25 ~ -30℃ (for example, it can be -26℃, -27℃, -28℃ or -29℃, etc.) and keep warm for 1-3h (for example, it can be 1.1h, 1.3h, 1.5h, 1.7h, 2h, 2.5h, 2.5h or 2.8h, etc.), and finally cool down to -35 ~ -40℃ (for example, it can be -36℃, -37℃, -38℃ or -39℃, etc.) and keep warm for 1-3h (for example, it can be 1.1h, 1.3h, 1.5h, 1.7h, 2h, 2.5h, 2.5h or 2.8h, etc.).

[0037] Preferably, the cooling rate is 1-1.5°C / min, for example, 1.1°C / min, 1.2°C / min, 1.3°C / min or 1.4°C / min.

[0038] Preferably, the drying step comprises: heating from -35 to -40°C for 40-50 min (for example, 41 min, 43 min, 45 min, 47 min or 49 min, etc.) to -10 to -15°C (for example, -11°C, -12°C, -13°C or -14°C, etc.), and maintaining for 2-4 h (for example, 2.2 h, 2.5 h, 2.8 h, 3 h, 3.2 h, 3.5 h or 3.8 h, etc.); heating from -10 to -15°C for 20-40 min (for example, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min or 38 min, etc.) to -5-0°C (for example, -4°C, -3°C, -2°C or -1°C, etc.), and maintaining for 2-4 h (for example, 2.2 h, 2.5 h, 2.8 h, 3 h, 3.2 h, 3.5 h or 3.8 h, etc.). 3.8h, etc.); from -5-0°C, after 20-40min (for example, it can be 22min, 25min, 28min, 30min, 32min, 35min or 38min, etc.), the temperature is increased to 5-10°C (for example, it can be 6°C, 7°C, 8°C or 9°C, etc.), and maintained for 2-4h (for example, it can be 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h or 3.8h, etc.); from 5-10°C, after 20-40min (for example, it can be 22min, 25min, 28min, 30min, 32min, 35min or 38min, etc.), the temperature is increased to 20-30°C (for example, it can be 21°C, 23°C, 25°C, 27°C or 29°C, etc.), and maintained for 2-4h (for example, it can be 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h or 3.8h, etc.).

[0039] Preferably, the drying process is performed under vacuum.

[0040] Preferably, the vacuum degree is 0.01-0.1 mbar, for example, it can be 0.02 mbar, 0.03 mbar, 0.04 mbar, 0.05 mbar, 0.06 mbar, 0.07 mbar, 0.08 mbar or 0.09 mbar, etc.

[0041] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The freeze-dried protective solution provided by the present invention can prevent the structural integrity of plant exosomes, reduce the osmotic pressure difference between the inside and outside of cells, and maintain cell viability. The freeze-dried protective solution can also avoid the influence of stress on the biological activity of plant exosomes during the freeze-drying process, thereby maintaining the stability of the biological activity of plant exosomes.

[0044] (2) The method provided by the present invention can reduce the aggregation of plant exosomes during the cold drying process by controlling the process parameters, maintain the integrity of the active ingredients of the exosomes, and thus improve the stability of the exosomes during storage. DETAILED DESCRIPTION

[0045] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0046] The polyglycerol-10 laurate mentioned in the raw materials involved in the following examples, comparative examples and preparation examples was purchased from: Guangzhou Jahwa Chemical Co., Ltd.;

[0047] The phosphate nanoparticles were purchased from Suzhou Element Chemical Industry Co., Ltd.

[0048] The wild almond oil polyglycerol-6 esters were purchased from Guangzhou Jahwa Chemical Co., Ltd.

[0049] The polyglycerol-10 oleate was purchased from Guangzhou Jahwa Chemical Co., Ltd.

[0050] The sea fennel callus culture filtrate was purchased from SEPPIC SA;

[0051] The DNA sodium was purchased from Shandong Freda Biotechnology Co., Ltd.

[0052] The rest of the raw materials can be used as long as they are purchased from regular dealers.

[0053] Example 1

[0054] This embodiment provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 4 parts of polyglycerol-10 oleate, 6 parts of erythritol, 4 parts of trehalose, 1 part of sea fennel callus culture filtrate and 2 parts of sodium DNA in parts by weight.

[0055] Example 2

[0056] This embodiment provides a freeze-dried protective solution containing ribonucleic acid, which comprises 5 parts of wild almond oil polyglycerol-6 esters, 3 parts of polyglycerol-10 oleate, 8 parts of erythritol, 3 parts of trehalose, 2 parts of sea fennel callus culture filtrate and 1 part of DNA sodium in parts by weight.

[0057] Example 3

[0058] This embodiment provides a freeze-dried protective solution containing ribonucleic acid, which comprises 2 parts of wild almond oil polyglycerol-6 esters, 6 parts of polyglycerol-10 oleate, 3 parts of erythritol, 8 parts of trehalose, 0.5 parts of sea fennel callus culture filtrate and 3 parts of DNA sodium in parts by weight.

[0059] Comparative Example 1

[0060] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 4 parts of polyglycerol-10 oleate, 6 parts of erythritol, 4 parts of trehalose and 2 parts of sodium DNA in parts by weight.

[0061] Comparative Example 2

[0062] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 4 parts of polyglycerol-10 oleate, 6 parts of erythritol, 1 part of sea fennel callus culture filtrate and 2 parts of sodium DNA in parts by weight.

[0063] Comparative Example 3

[0064] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 4 parts of polyglycerol-10 oleate, 4 parts of trehalose, 1 part of sea fennel callus culture filtrate and 2 parts of DNA sodium in parts by weight.

[0065] Comparative Example 4

[0066] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 6 parts of erythritol, 4 parts of trehalose, 1 part of sea fennel callus culture filtrate and 2 parts of DNA sodium in parts by weight.

[0067] Comparative Example 5

[0068] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 4 parts of polyglycerol-10 oleate, 6 parts of erythritol, 4 parts of trehalose, 1 part of sea fennel callus culture filtrate and 2 parts of sodium DNA in parts by weight.

[0069] Comparative Example 6

[0070] This comparative example provides a freeze-dried protective solution containing ribonucleic acid, which comprises 3 parts of wild almond oil polyglycerol-6 esters, 4 parts of polyglycerol-10 oleate, 6 parts of erythritol, 4 parts of trehalose and 1 part of sea fennel callus culture filtrate in parts by weight.

[0071] Preparation Example 1

[0072] This preparation example provides a Chlorella exosome, and the preparation method of the Chlorella exosome is:

[0073] (1) treating the cell wall of Chlorella vulgaris at a pH of 9.5, a temperature of 73° C., and a time of 55 min;

[0074] (2) subjecting the cell fluid after cell wall disruption to gradient centrifugation, wherein the specific steps of the gradient centrifugation are: centrifugation at 350 g for 10 min, centrifugation of the supernatant at 1500 g for 30 min, centrifugation of the supernatant at 5000 g for 20 min, centrifugation of the supernatant at 6000 g for 20 min, and then taking the supernatant to obtain a crude exosome extract;

[0075] (3) The crude exosome extract was resuspended in a 24 mg / mL Tween 80 aqueous solution at a volume ratio of 1:0.9, and then ultracentrifuged four times, each time at a speed of 18000 g for 45 min to obtain purified exosomes;

[0076] (4) The purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution (concentration 0.4%) were mixed in a volume ratio of 4:0.7:0.7, and then homogenized by ultrafast microfluidization (jet velocity 6000 m / s) to obtain the Chlorella exosomes.

[0077] Application Example 1

[0078] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0079] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 1 to a phosphate buffer having a concentration of 0.4 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer is 1:15) at a mass ratio of 1:3, and mixing to obtain a mixed solution;

[0080] (2) cooling the mixed solution to 2°C and keeping it warm for 2 hours, then cooling it to -12°C and keeping it warm for 2 hours, then cooling it to -28°C and keeping it warm for 2 hours, and finally cooling it to -40°C and keeping it warm for 2 hours; the cooling rate is 1.2°C / min;

[0081] (3) After the mixed solution treated in step (2) is evacuated, the vacuum degree is controlled to be 0.05 mbar, and then a drying treatment is performed, wherein the drying steps are as follows: heating from -40°C to -15°C for 45 minutes and maintaining for 3 hours; heating from -15°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 10°C for 30 minutes and maintaining for 3 hours; heating from 10°C to 25°C for 30 minutes and maintaining for 3 hours; and the preservation of plant exosomes is completed by tamponade.

[0082] Application Example 2

[0083] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0084] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 2 to a phosphate buffer solution having a concentration of 0.5 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer solution is 1:13) at a mass ratio of 1:2, and mixing to obtain a mixed solution;

[0085] (2) cooling the mixed solution to 3°C and keeping it warm for 1 hour, then cooling it to -15°C and keeping it warm for 3 hours, then cooling it to -25°C and keeping it warm for 1 hour, and finally cooling it to -38°C and keeping it warm for 1 hour; the cooling rate is 1.3°C / min;

[0086] (3) After the mixed solution treated in step (2) is evacuated, the vacuum degree is controlled to be 0.03 mbar, and then a drying treatment is performed, wherein the drying steps are as follows: heating from -38°C to -12°C for 40 minutes and maintaining for 4 hours; heating from -12°C to 0°C for 20 minutes and maintaining for 2 hours; heating from 0°C to 8°C for 40 minutes and maintaining for 3 hours; heating from 8°C to 20°C for 40 minutes and maintaining for 4 hours; and the preservation of plant exosomes is completed by tamponade.

[0087] Application Example 3

[0088] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0089] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 3 to a phosphate buffer solution having a concentration of 0.2 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer solution is 1:18) at a mass ratio of 1:5, and mixing to obtain a mixed solution;

[0090] (2) cooling the mixed solution to 0°C and keeping it warm for 3 hours, then cooling it to -10°C and keeping it warm for 1 hour, then cooling it to -30°C and keeping it warm for 2 hours, and finally cooling it to -35°C and keeping it warm for 3 hours; the cooling rate is 1.5°C / min;

[0091] (3) After the mixed solution treated in step (2) is evacuated, the vacuum degree is controlled to be 0.08 mbar, and then a drying treatment is performed, wherein the drying steps are as follows: heating from -35°C to -10°C for 20 minutes and maintaining for 2 hours; heating from -10°C to -5°C for 20 minutes and maintaining for 4 hours; heating from -5°C to 10°C for 20 minutes and maintaining for 2 hours; heating from 10°C to 30°C for 40 minutes and maintaining for 2 hours; and the preservation of plant exosomes is completed by tamponade.

[0092] Application Example 4

[0093] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0094] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 1 to a phosphate buffer having a concentration of 0.4 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer is 1:15) at a mass ratio of 1:3, and mixing to obtain a mixed solution;

[0095] (2) cooling the mixed solution to -40°C and keeping it warm for 2 hours; the cooling rate is 1.2°C / min;

[0096] (3) After the mixed solution treated in step (2) is evacuated, the vacuum degree is controlled to be 0.05 mbar, and then a drying treatment is performed, wherein the drying steps are as follows: heating from -40°C to -15°C for 45 minutes and maintaining for 3 hours; heating from -15°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 10°C for 30 minutes and maintaining for 3 hours; heating from 10°C to 25°C for 30 minutes and maintaining for 3 hours; and the preservation of plant exosomes is completed by tamponade.

[0097] Application Example 5

[0098] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0099] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 1 to a phosphate buffer having a concentration of 0.4 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer is 1:15) at a mass ratio of 1:3, and mixing to obtain a mixed solution;

[0100] (2) cooling the mixed solution to 2°C and keeping it warm for 2 hours, then cooling it to -12°C and keeping it warm for 2 hours, then cooling it to -28°C and keeping it warm for 2 hours, and finally cooling it to -40°C and keeping it warm for 2 hours; the cooling rate is 1.2°C / min;

[0101] (3) The mixed solution treated in step (2) is evacuated to a vacuum degree of 0.05 mbar, and then dried. The drying steps are as follows: heating from -40°C to -15°C for 45 minutes and maintaining for 3 hours; heating from -15°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours; and the preservation of plant exosomes is completed by tamponade.

[0102] Application Example 6

[0103] This application example provides a method for preserving and processing Chlorella exosomes, the method comprising:

[0104] (1) adding the Chlorella exosomes of Preparation Example 1 and the lyophilized protective solution containing RNA of Example 1 to a phosphate buffer having a concentration of 0.4 mol / L (the volume ratio of the lyophilized protective solution to the phosphate buffer is 1:15) at a mass ratio of 1:3, and mixing to obtain a mixed solution;

[0105] (2) cooling the mixed solution to 2°C and keeping it warm for 2 hours, then cooling it to -12°C and keeping it warm for 2 hours, then cooling it to -28°C and keeping it warm for 2 hours, and finally cooling it to -40°C and keeping it warm for 2 hours; the cooling rate is 1.2°C / min;

[0106] (3) After the mixed solution treated in step (2) is evacuated, the vacuum degree is controlled to be 0.05 mbar, and then a drying treatment is performed, wherein the drying steps are as follows: heating from -40°C to -25°C for 45 minutes and maintaining for 3 hours; heating from -25°C to -15°C for 30 minutes and maintaining for 3 hours; heating from -15°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 10°C for 30 minutes and maintaining for 3 hours; heating from 10°C to 25°C for 30 minutes and maintaining for 3 hours; and the preservation of plant exosomes is completed by tamponade.

[0107] Comparative Application Examples 1-6

[0108] This comparative application example provides five methods for preserving and treating Chlorella exosomes. The method differs from Application Example 1 only in that the freeze-dried protective solution of Example 1 is replaced with the freeze-dried protective solution of Comparative Examples 1-6 in equal amounts, and the remaining steps and parameters are the same as those of Application Example 1.

[0109] Test example:

[0110] The Chlorella exosomes (D0) obtained in Preparation Example 1 were subjected to moisturizing, anti-aging and anti-inflammatory tests, and then the exosomes obtained in Preparation Example 1 (control example), Application Examples 1-6, and the samples provided in Comparative Application Examples 1-6 were placed at 25° C. for 6 months and then subjected to moisturizing, anti-aging and anti-inflammatory tests. The samples obtained in Application Examples 1-6 and Comparative Application Examples 1-6 were tested using 0.4 mol / L phosphate buffer for reconstitution (dosage was 0.3 g / mL).

[0111] (1) Moisturizing performance test

[0112] The moisturizing performance of the test samples was tested, and the changes in skin moisture content of the test subjects were compared before and after use (the usage operation was kept consistent). A total of 140 volunteers were included, including 70 males and 70 females, aged 20-40 years old with an average age of 28 years old. They were randomly divided into 14 groups, with 5 females and 5 males in each group.

[0113] The subjects washed their forearms and sat quietly for 30 minutes in a set humidity environment. A 3×3cm2 square experimental area was selected on each of the subjects' left and right forearms. The left arm was used as the test area for the test sample, and the symmetrical area of ​​the right arm was used as a blank control. The moisture content of each experimental part was tested with Corneometer CM825. This was repeated 3 times and the average value was recorded.

[0114] The formula for calculating skin hydration rate is:

[0115] The average skin hydration rate of the subjects after using the samples for 2 hours is shown in Table 1:

[0116] Table 1

[0117]

[0118]

[0119] (2) Evaluation of anti-aging effects

[0120] Test method:

[0121] Determination of the inhibitory efficacy of elastase activity

[0122] Test principle: One of the main causes of skin aging is the change in the structure of the dermis. Due to the action of some external factors, the elastase in the body is activated, which can lead to excessive degradation of collagen and elastin in the dermis that support the skin structure. The anti-aging effect of these samples is evaluated by testing the inhibition rate of elastase on the samples;

[0123] Test method: 200 μL of each test sample and 50 μL of 20 μg / mL elastase-III solution were mixed in a 96-well plate in a 10 mg / L Tris hydrochloride buffer. 250 μM EGCG was used as a positive control group, and distilled water was used as a negative control group. The reaction was carried out at 25°C for 15 minutes. After the reaction, the absorbance at a wavelength of 415 nm was measured.

[0124] The calculation formula is as follows: Elastase activity inhibition rate (%) = 1-(CD) / (AB) × 100%;

[0125] A is the absorbance at 415 nm when no test substance is added but enzyme is added, B is the absorbance at 415 nm when no test substance and enzyme are added, C is the absorbance at 415 nm when test substance and enzyme are added, and D is the absorbance at 415 nm when test substance is added but enzyme is not added;

[0126] The specific test results are shown in Table 2 below:

[0127] Table 2

[0128] Test samples Elastase inhibition rate / % D0(D0) 80.7 Application Example 1 79.5 Application Example 2 78.6 Application Example 3 79.2 Application Example 4 67.8 Application Example 5 69.3 Application Example 6 78.1 Comparative application example 1 52.2 Comparative Application Example 2 50.1 Comparative Application Example 3 53.2 Comparative Application Example 4 49.6 Comparative Application Example 5 48.5 Comparative Application Example 6 48.8 Comparison Example 36.2

[0129] (3) Anti-inflammatory efficacy test

[0130] The test method is as follows:

[0131] (1) Enzyme-linked immunosorbent assay (ELISA): The blood sample was mixed with an equal proportion of blood cell separation solution and centrifuged at 20°C for 40 min. The human peripheral blood mononuclear cell layer was obtained. After the cells were washed with a buffer solution, the cells were suspended in the culture medium RPMI-1640. Then, CD14+ monocytes from human peripheral blood mononuclear cells were purified using CD14+ microspheres (MiniMACS system). The cells were stimulated to differentiate into dendritic cells using cytokine IL-4 and growth hormone GM-CSF, and cultured at 37°C and 5% CO2 for 6 days. The differentiated dendritic cells were collected and seeded into a well plate. The above test samples were diluted to a concentration of 2 μg / mL and added to the well plate, and the wells with an equal volume of culture medium were set as blank controls. The wells were co-cultured at 37°C and 5% CO2 for 48 hours. The cell culture supernatant was collected. The levels of interleukin IL-10 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and the content of anti-inflammatory cytokine IL-10 in the blank control group was defined as 0, and the content of pro-inflammatory cytokine IL-12 was defined as 100%;

[0132] (2) Inhibition of TNF-α expression: Fresh blood was treated with EDTA as anticoagulant, blood cells were separated by Ficoll, and the cells were resuspended in RIMP 1640 medium containing 10% fetal bovine serum. 100 mL of 1×10 5 cells / mL Freshly isolated cells, total number of cells per well: 10 43 wells for each sample; weigh the test sample, add sterile PBS solution to prepare the test sample stock solution (10 mg / mL), and then use sterile PBS to dilute the stock solution to the corresponding working concentration for use; add 10 mL of the test solution of the corresponding working concentration to the well of the culture plate. The culture plate is placed in an incubator at 37°C and 5% CO2 for 15 minutes; add 10 mL of LPS with a concentration of 100 mg / mL and incubate in an incubator at 37°C and 5% CO2 for 16 hours; the culture plate is centrifuged at 1000 rpm for 15 minutes, the supernatant is transferred to a new culture plate, and the TNF-α concentration in the culture supernatant is determined using an enzyme marker; or frozen at -20°C to avoid repeated freezing and thawing, and the inhibition rate is calculated.

[0133] The specific test results are shown in Table 3 below:

[0134] Table 3

[0135]

[0136]

[0137] It can be seen from the test data in Table 1-3 that, compared with the control example, the use of the freeze-dried protective solution provided by the present invention to preserve the Chlorella exosomes in Application Example 1-3 can not reduce the moisturizing, anti-aging and anti-inflammatory effects of the Chlorella exosomes. After the Chlorella exosomes were stored for six months, the skin hydration rate of the volunteers after use was above 41%, the inhibition rate of elastase was above 78%, the content of interleukin IL-10 was above 155%, the level of interleukin IL-12 was below 7%, and the inhibition rate of TNF-a release was above 85%, which was basically consistent with the effect of Chlorella exosomes (D0), indicating that the freeze-dried powder obtained by the method can maintain the biological activity and stability of the exosomes.

[0138] As can be seen from Application Example 4, the pre-freezing temperature control in the method can affect the biological activity of Chlorella exosomes;

[0139] It can be seen from Application Examples 5 and 6 that when the number of drying times in the preservation method provided by the present invention is too small (i.e., the temperature rises too fast), the performance of the Chlorella exosomes will be reduced, and too many drying times will not improve the preservation effect of the Chlorella exosomes, and the process cost is higher;

[0140] It can be seen from comparative application examples 1-6 that the chlorella exosomes, wild almond oil polyglycerol-6 esters, polyglycerol-10 oleate, erythritol, trehalose, sea fennel callus culture filtrate and sodium DNA in the freeze-dried protective solution of the present invention work together to achieve a better preservation effect of Chlorella.

[0141] The applicant declares that the present invention illustrates a freeze-dried protective solution containing ribonucleic acid and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0142] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0143] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A freeze-dried protective solution containing ribonucleic acid, characterized in that: The components of the freeze-dried protective solution include wild almond oil polyglycerol-6 esters, polyglycerol-10 oleate, erythritol, trehalose, sea fennel callus culture filtrate and DNA sodium.

2. The freeze-dried protective solution according to claim 1, characterized in that The components of the freeze-dried protective solution include, by weight, 0.5-10 parts of wild almond oil polyglycerol-6 esters, 1-10 parts of polyglycerol-10 oleate, 1-10 parts of erythritol, 1-10 parts of trehalose, 0.01-5 parts of sea fennel callus culture filtrate and 1-5 parts of sodium DNA; Preferably, the components of the freeze-dried protective solution include, by weight, 0.5-5 parts of wild almond oil polyglycerol-6 esters, 1-6 parts of polyglycerol-10 oleate, 1-8 parts of erythritol, 1-8 parts of trehalose, 0.01-2 parts of sea fennel callus culture filtrate and 1-3 parts of sodium DNA.

3. Use of the freeze-dried protective solution containing ribonucleic acid as claimed in claim 1 or 2 in the preservation and treatment of plant exosomes.

4. A method for preserving and treating plant exosomes, characterized in that: The method comprises mixing plant exosomes with the lyophilized protective solution containing ribonucleic acid according to claim 1 or 2 in a phosphate buffer to obtain a mixed solution, pre-freezing the mixed solution, and performing a plugging process after drying to complete the preservation of the plant exosomes.

5. The method for preserving and treating plant exosomes according to claim 4, characterized in that: The mass ratio of the plant exosomes to the freeze-dried protective solution is 1:(1-5); Preferably, the concentration of the phosphate buffer is 0.2-0.5 mol / L; Preferably, the volume ratio of the lyophilization protection solution to the phosphate buffer is 1:(10-20).

6. The method for preserving and treating plant exosomes according to claim 4 or 5, characterized in that: The plant exosomes include any one of Chlorella exosomes, Centella asiatica exosomes or Camellia exosomes, or a combination of at least two of them.

7. The method for preserving and treating plant exosomes according to any one of claims 4 to 6, characterized in that: The pre-freezing treatment step includes: cooling the mixed solution to 0-3°C and keeping it warm for 1-3 hours, then cooling it to -10--15°C and keeping it warm for 1-3 hours, then cooling it to -25--30°C and keeping it warm for 1-3 hours, and finally cooling it to -35--40°C and keeping it warm for 1-3 hours.

8. The method for preserving and treating plant exosomes according to claim 7, wherein: The cooling rates are independently 1-1.5°C / min.

9. The method for preserving and treating plant exosomes according to any one of claims 4 to 8, characterized in that: The drying steps include: heating from -35 to -40°C to -10 to -15°C after 40 to 50 minutes, and maintaining for 2 to 4 hours; heating from -10 to -15°C to -5 to 0°C after 20 to 40 minutes, and maintaining for 2 to 4 hours; heating from -5 to 0°C to 5 to 10°C after 20 to 40 minutes, and maintaining for 2 to 4 hours; heating from 5 to 10°C to 20 to 30°C after 20 to 40 minutes, and maintaining for 2 to 4 hours.

10. The method for preserving and treating plant exosomes according to claim 9, wherein: The drying process is carried out under vacuum; Preferably, the vacuum degree is 0.01-0.1 mbar.

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