Preparation method of transdermal absorption plant exosome

Abstract

The invention relates to a preparation method of a transdermal absorption plant exosome. The preparation method comprises the following steps: 1) grinding a fresh plant, then mixing with a TBS buffer solution, standing and filtering; (2) centrifuging the filtrate, and harvesting supernate; (3) filtering, concentrating and removing impurities from the supernate through a solid-phase extraction device to obtain a collected solution; according to the solid-phase extraction device, a filling material is AB-8 macroporous resin; the sample loading volume is 100 to 500 mL; the filling volume is 600 to 1500 mL; the volume of the eluent is 1500 to 3000 mL; an eluent is a TBS buffer solution; 4) filtering the collected liquid, mixing with polyacrylic acid and gelatin, carrying out polymerization precipitation, and centrifuging the obtained suspension to obtain a precipitate; and re-suspending the precipitate by using a TBS buffer solution, centrifuging, and taking the supernate, namely the exosome. According to the method, high-quality plant exosomes are efficiently extracted from plant tissues through a specific extraction method and prepared into a transdermal absorption system, deep permeation and absorption of nutrient substances are achieved, and therefore the effect of a skin care product is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome preparation, and specifically relates to a method for preparing transdermal plant exosomes. Background Art

[0002] In the field of skin care, traditional methods of delivering skin care ingredients face many challenges, the most prominent of which is the problem of poor transdermal absorption. Transdermal absorption refers to the process by which skin care ingredients pass through the skin barrier, enter the deep layers of the skin, and even reach the blood circulation. However, due to the existence of the skin barrier, many skin care ingredients are difficult to effectively penetrate the skin, resulting in a significant reduction in skin care effects.

[0003] In order to improve this situation, researchers are constantly exploring new transdermal absorption technologies. In recent years, exosomes have gradually attracted attention as a new type of transdermal delivery system. Exosomes are nanoscale vesicles released by cells, with a unique lipid bilayer structure, which can carry a variety of bioactive substances, such as proteins, nucleic acids, lipids, etc. This structural characteristic makes exosomes have excellent biocompatibility and stability, can penetrate into skin cells, participate in information exchange between cells, and thus improve skin quality.

[0004] Among the many sources of exosomes, plant exosomes are favored due to their unique advantages. Compared with animal exosomes, plant exosomes have higher stability, lower immunogenicity and are easier to mass produce. Plant exosomes not only contain a variety of nutrients that are beneficial to the human body, such as antioxidants, anti-inflammatory factors, etc., but also have a relatively simple extraction process, low cost, and are suitable for industrial production.

[0005] However, despite the many advantages of plant exosomes, they still face some challenges in practical applications. For example, how to efficiently extract high-quality plant exosomes, how to prepare a plant exosome system with high transdermal absorption, etc. These problems limit the application and development of plant exosomes in the field of skin care.

[0006] Therefore, the present invention proposes a plant exosome transdermal absorption technology to solve the above problems. The present invention efficiently extracts high-quality plant exosomes from plant tissues through a specific extraction method and prepares them into a transdermal absorption system. The system can utilize the lipid bilayer structure of exosomes and the effect of transdermal absorption enhancers to achieve deep penetration and absorption of nutrients, thereby improving the efficacy of skin care products. The introduction of this technology provides new ideas and methods for the application of plant exosomes in the field of skin care, and has broad market prospects and application value. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing transdermal plant exosomes, which efficiently extracts high-quality plant exosomes from plant tissues through a specific extraction method and prepares them into a transdermal absorption system. The system can utilize the lipid bilayer structure of exosomes and the effect of transdermal absorption enhancers to achieve deep penetration and absorption of nutrients, thereby improving the efficacy of skin care products. The introduction of this technology provides new ideas and methods for the application of plant exosomes in the field of skin care, and has broad market prospects and application value.

[0008] To achieve the above object, the present invention adopts the following technical solution: A method for preparing transdermal plant exosomes, comprising the following steps: 1) Grind the fresh plant, mix it with TBS buffer, let it stand, and filter it; 2) Centrifuge the filtrate and harvest the supernatant; 3) The supernatant is filtered, concentrated and impurity-removed through a solid phase extraction device to obtain a collection solution; the solid phase extraction device: filling material: AB-8 macroporous resin; sample volume: 100~500 mL; filling volume: 600~1500 mL; eluent volume: 1500~3000 mL; eluent: TBS buffer; 4) After the collected liquid is filtered, it is mixed with polyacrylic acid and gelatin for polymerization precipitation (polymerization precipitation is performed at a temperature of 3-5°C for 10-14 hours). The resulting suspension is centrifuged to harvest the precipitate; the precipitate is resuspended in TBS buffer, centrifuged, and the supernatant is the exosome solution; 5) The exosome solution is mixed with eucalyptol and poloxamer 184 to obtain transdermal plant exosomes.

[0009] Specifically, in step 1), the plant material can be selected from one or more of the following: peony, sweet orange, sour orange, lemon, grape, water chestnut, ginger, etc.

[0010] Furthermore, in step 1), the plant can be mixed with TBS buffer having a pH of 7.0-7.3 (preferably pH 7.2) at a solid-liquid ratio of 1 g: 1-5 mL. Preferably, the solid-liquid ratio of the fresh plant to the TBS buffer is 1 g: 2 mL.

[0011] Specifically, in step 2), centrifugation can be performed at 6000-12000 rpm for 30-60 min. The preferred centrifugation condition is: 12000 rpm, 40 min.

[0012] Preferably, in step 3), the filling material of the solid phase extraction device is AB-8 macroporous resin; 200-300 mL is used as one interception branch, and 4-6 interception branches are collected; and the eluent is TBS buffer (pH 7.2).

[0013] Specifically, in step 4), the mass ratio of the polyacrylic acid to gelatin may be 1.5-2.5:1, and the mass ratio is preferably 2:1.

[0014] Further, in step 4), the ratio of the collected liquid to the sum of the mass of polyacrylic acid and gelatin can be 8-15:1, and the mass ratio is preferably 10:1. In step 4), the filtrate is centrifuged at 8000-18000 rpm for 40-80 min (preferably at 12000 rpm for 50 min), and the precipitate is harvested. The precipitate is resuspended in TBS buffer and centrifuged at 10000-15000 rpm for 10-30 min (preferably at 12500 rpm for 25 min). The mass ratio of the precipitate to the TBS buffer is 1:20-50, preferably 1:30.

[0015] Furthermore, in step 5), the mass ratio of the exosome solution to eucalyptol and poloxamer 184 can be 1:1%-2%:0.1%-0.5%, preferably 1:2%:0.2%.

[0016] The present invention provides transdermal absorption plant exosomes prepared by the above preparation method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a novel method for preparing transdermal plant exosomes, which efficiently extracts high-quality plant exosomes from plant tissues through a specific extraction method and prepares them into a transdermal absorption system. The system can utilize the lipid bilayer structure of exosomes and the effect of transdermal absorption enhancers to achieve deep penetration and absorption of nutrients, thereby improving the efficacy of skin care products. The introduction of this technology provides new ideas and methods for the application of plant exosomes in the field of skin care, and has broad market prospects and application value. The method of the present invention prepares different plant exosomes, which have the advantages of good transdermal effect and the like. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0019] In the following examples, unless otherwise specified, the raw materials used are all common commercial products that can be directly purchased, and the processes not described in detail can be adopted by conventional techniques in the art. The composition of the TBS buffer used is: 100 mM Tris, 100 mM NaCl, 10 mM EDTA, 10% HCl, and the pH is adjusted to 7.2.

[0020] Example 1 A method for preparing transdermal plant exosomes, which specifically comprises the following steps: 1) Grind the sweet orange, then mix it with TBS buffer (pH 7.2) at a solid-liquid ratio of 1 g: 2 mL, let it stand, and filter; 2) Centrifuge the filtrate (12000 rpm, 40 min) and harvest the supernatant; 3) The supernatant is filtered, concentrated and impurity-removed through a solid phase extraction device to obtain a collection liquid. The solid phase extraction device: filling material: AB-8 macroporous resin; sample volume: 200 mL; filling volume: 1000 mL; eluent volume: 2000 mL; eluent: TBS buffer (pH 7.2); collection 4-6 interception branches; 4) After filtering, the collected liquid was mixed with polyacrylic acid and gelatin (mass ratio 2:1) for polymerization precipitation (polymerization precipitation was performed at 4°C for 12 hours), and the ratio of the collected liquid to the sum of the mass of polyacrylic acid and gelatin was 10:1. The resulting suspension was centrifuged (12000 rpm, 50 min) to harvest the precipitate. The precipitate was resuspended in TBS buffer (pH 7.2) at a mass ratio of 1:30, centrifuged (125000 rpm, 25 min), and the supernatant was taken as the exosome solution; 5) The exosome solution is mixed evenly with eucalyptol and poloxamer 184 in a mass ratio of 1:2%:0.2% to obtain transdermal plant exosomes.

[0021] Example 2 A method for preparing transdermal plant exosomes, which specifically comprises the following steps: 1) Grind the grapes, then mix with TBS buffer (pH 7.2) at a solid-liquid ratio of 1 g: 2 mL, let stand, and filter; 2) Centrifuge the filtrate (12000 rpm, 40 min) and harvest the supernatant; 3) The supernatant is filtered, concentrated and impurity-removed through a solid phase extraction device to obtain a collection liquid. The solid phase extraction device: filling material: AB-8 macroporous resin; sample volume: 200 mL; filling volume: 1000 mL; eluent volume: 2000 mL; eluent: TBS buffer (pH 7.2); collection 4-6 interception branches; 4) After filtering, the collected liquid was mixed with polyacrylic acid and gelatin (mass ratio 2:1) for polymerization precipitation (polymerization precipitation was performed at 4°C for 12 hours), and the ratio of the collected liquid to the sum of the mass of polyacrylic acid and gelatin was 10:1. The resulting suspension was centrifuged (12000 rpm, 50 min) to harvest the precipitate. The precipitate was resuspended in TBS buffer (pH 7.2) at a mass ratio of 1:30, centrifuged (125000 rpm, 25 min), and the supernatant was taken as the exosome solution; 5) The exosome solution is mixed evenly with eucalyptol and poloxamer 184 in a mass ratio of 1:2%:0.2% to obtain transdermal plant exosomes.

[0022] Example 3 A method for preparing transdermal plant exosomes, which specifically comprises the following steps: 1) Grind water chestnuts, mix with TBS buffer (pH 7.2) at a solid-liquid ratio of 1 g: 2 mL, let stand, and filter; 2) Centrifuge the filtrate (12000 rpm, 40 min) and harvest the supernatant; 3) The supernatant is filtered, concentrated and impurity-removed through a solid phase extraction device to obtain a collection liquid. The solid phase extraction device: filling material: AB-8 macroporous resin; sample volume: 200 mL; filling volume: 1000 mL; eluent volume: 2000 mL; eluent: TBS buffer (pH 7.2); collection 4-6 interception branches; 4) After filtering, the collected liquid was mixed with polyacrylic acid and gelatin (mass ratio 2:1) for polymerization precipitation (polymerization precipitation was performed at 4°C for 12 hours), and the ratio of the collected liquid to the sum of the mass of polyacrylic acid and gelatin was 10:1. The resulting suspension was centrifuged (12000 rpm, 50 min) to harvest the precipitate. The precipitate was resuspended in TBS buffer (pH 7.2) at a mass ratio of 1:30, centrifuged (125000 rpm, 25 min), and the supernatant was taken as the exosome solution; 5) The exosome solution is mixed evenly with eucalyptol and poloxamer 184 in a mass ratio of 1:2%:0.2% to obtain transdermal plant exosomes.

[0023] Comparative Example 1 1) Take fresh sweet oranges, add them to the PBS solution, immerse them, crush and squeeze the juice, and filter through gauze to obtain the first supernatant; 2) Centrifuge the first supernatant at 3000 rpm and 4°C for 30 min, then take the supernatant, centrifuge at 10000 rpm and 4°C for 30 min, take the supernatant, centrifuge again at 12000 rpm and 4°C for 30 min, take the centrifuged supernatant, and discard the precipitate; 3) Filter using 0.45 μm and 0.22 μm microporous membranes in sequence to obtain the preliminary filtrate of exosomes; 4) Add a 16% polyethylene glycol solution with a molecular weight of 8000 to the preliminary filtrate of exosomes, and the volume ratio of the preliminary filtrate to the polyethylene glycol solution is 1:1. Invert and mix thoroughly, and incubate at 4°C for 12 hours to obtain a reaction solution; 5) Centrifuge the reaction solution at 12000 rpm and 4°C for 60 min, collect the precipitate containing sweet orange exosomes, add 1 mL 1× PBS to resuspend the precipitate. Transfer the resuspended solution to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4°C for 10 min, and take the supernatant to obtain sweet orange exosomes.

[0024] Comparative Example 2 1) Take fresh grapes, add them to PBS solution, immerse them, crush and squeeze the juice, and filter with gauze to obtain the first supernatant; 2) Centrifuge the first supernatant at 3000 rpm and 4°C for 30 min, then take the supernatant, centrifuge at 10000 rpm and 4°C for 30 min, take the supernatant, centrifuge again at 12000 rpm and 4°C for 30 min, take the centrifuged supernatant, and discard the precipitate; 3) Filter using 0.45 μm and 0.22 μm microporous membranes in sequence to obtain the preliminary filtrate of exosomes; 4) Add a 16% polyethylene glycol solution with a molecular weight of 8000 to the preliminary filtrate of exosomes, and the volume ratio of the preliminary filtrate to the polyethylene glycol solution is 1:1. Invert and mix thoroughly, and incubate at 4°C for 12 hours to obtain a reaction solution; 5) Centrifuge the reaction solution at 12000 rpm and 4°C for 60 min, collect the precipitate containing grape exosomes, add 1 mL 1× PBS to resuspend the precipitate. Transfer the resuspended solution to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4°C for 10 min, and take the supernatant to obtain grape exosomes.

[0025] Comparative Example 3 1) Take fresh water chestnuts, add them to PBS solution, immerse them, crush and squeeze out the juice, and filter with gauze to obtain the first supernatant; 2) Centrifuge the first supernatant at 3000 rpm and 4°C for 30 min, then take the supernatant, centrifuge at 10000 rpm and 4°C for 30 min, take the supernatant, centrifuge again at 12000 rpm and 4°C for 30 min, take the centrifuged supernatant, and discard the precipitate; 3) Filter using 0.45 μm and 0.22 μm microporous membranes in sequence to obtain the preliminary filtrate of exosomes; 4) Add a 16% polyethylene glycol solution with a molecular weight of 8000 to the preliminary filtrate of exosomes, and the volume ratio of the preliminary filtrate to the polyethylene glycol solution is 1:1. Invert and mix thoroughly, and incubate at 4°C for 12 hours to obtain a reaction solution; 5) Centrifuge the reaction solution at 12000rpm and 4℃ for 60min, collect the precipitate containing water chestnut exosomes, add 1mL 1×PBS to resuspend the precipitate. Transfer the resuspended solution to a 1.5mL centrifuge tube, centrifuge at 12000rpm and 4℃ for 10min, and take the supernatant to obtain water chestnut exosomes.

[0026] Transdermal absorption test Take 10 mL of the transdermal plant exosomes prepared in the above examples and comparative examples, add 10 mg of allantoin to each group and perform ultrasonic oscillation for 30 min; take 10 ml of pure water in the blank group and add an equal amount of allantoin, add 1 g of lyophilization protectant to each example and the blank group for lyophilization (lyophilization procedure: -40°C, 2h; -20°C, 1h; vacuum 1Pa; 0°C, 1h; 20°C, 1h), and the lyophilization protectant is an aqueous solution containing 5% trehalose and 5% sorbitol. Take 1 g of sample and dissolve it in 1 ml of pure water for in vitro skin permeation study, using fresh pig skin (abdominal skin, hair and subcutaneous fat removed). The pig skin is placed and sandwiched between the donor and receptor compartments of the Franz trough assembly (RYJ-6B drug transdermal diffusion tester, Shanghai Kehuai Instrument Co., Ltd.), with the stratum corneum of the skin piece facing the donor compartment. The receiving compartment is filled with 0.1 mmol / L PBS solution to avoid bubbles between the lower side of the skin and the PBS solution. 1 g sample + 1 mL pure water was evenly applied on the stratum corneum of the skin. A magnetic stirrer was placed at the bottom of the Franz tank and stirred continuously at 400 rpm. Samples of PBS solution (100 µl) were taken at 12 and 24 hours. After each sampling, the same volume of PBS solution was re-injected into the Franz cell. Detection was performed by high performance liquid chromatography (Agilent 1100 HPLC), column: C 18 Column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile: 0.01 mol / L potassium dihydrogen phosphate (10:90); flow rate: 1.0 ml / min, column temperature: 25 °C, detection wavelength: 224 nm to detect allantoin and measure the allantoin peak area. The results are shown in Table 1 below.

[0027] Table 1 Transdermal absorption performance of exosomes from different examples (allantoin peak area) The experimental results in Table 1 above prove that compared with the blank group, the allantoin peak areas of the plant exosomes prepared by this preparation method at 6h and 12h were significantly increased, indicating that they all had good transdermal absorption performance, and the transdermal performance of water chestnut exosomes was the best.

Claims

1. A method for preparing plant exosomes for transdermal absorption, characterized in that: The steps include: 1) Grind the fresh plant, mix it with TBS buffer, let it stand, and filter it; 2) Centrifuge the filtrate and harvest the supernatant; 3) filtering, concentrating and removing impurities from the supernatant through a solid phase extraction device to obtain a collected liquid; the solid phase extraction device: filling material: AB-8 macroporous resin; Sample volume: 100~500 mL; Filling volume: 600~1500 mL; Eluent volume: 1500~3000 mL; Eluent: TBS buffer; 4) After the collected liquid is filtered, it is mixed with polyacrylic acid and gelatin for polymerization precipitation. The resulting suspension is centrifuged to harvest the precipitate; the precipitate is resuspended in TBS buffer, centrifuged, and the supernatant is the exosome solution; 5) The exosome solution is mixed with eucalyptol and poloxamer 184 to obtain transdermal plant exosomes.

2. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 1), the plant material is selected from one or more of the following: peony, sweet orange, sour orange, lemon, grape, water chestnut, and ginger.

3. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 1), the mixture is mixed with TBS buffer having a pH of 7.0-7.3 at a solid-liquid ratio of 1 g: 1-5 mL.

4. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 2), centrifuge at 6000-12000 rpm for 30-60 min.

5. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 4), the mass ratio of polyacrylic acid to gelatin is 1.5-2.5:

1.

6. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 4), the ratio of the collected liquid to the sum of the mass of polyacrylic acid and gelatin is 8-15:

1.

7. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 4), the filtrate is centrifuged at 8000-18000 rpm for 40-80 min to harvest the precipitate.

8. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 4), the precipitate is resuspended in TBS buffer and centrifuged at 10,000-15,000 rpm for 10-30 min.

9. The method for preparing transdermal plant exosomes according to claim 1, characterized in that: In step 5), the mass ratio of the exosome solution to eucalyptol and poloxamer 184 is 1:1%-2%:0.1%-0.5%.

10. Transdermal plant exosomes prepared by the preparation method according to any one of claims 1 to 9.

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