Preparation method for plant-derived vesicle-like nanoparticles, product and use thereof

Abstract

The present invention relates to the field of plant extraction, and in particular to a preparation method for plant-derived vesicle-like nanoparticles, a product and a use thereof. The preparation method for plant-derived vesicle-like nanoparticles provided herein comprises: (1) juicing a sample, performing primary filtration, sterile filtration and tangential flow filtration, and collecting the filtrate; and (2) concentrating and diafiltering the filtrate to obtain vesicle-like nanoparticles, wherein the sterile filtration is performed by means of a membrane filter selected from those having pore sizes of 5 μm, 0.85 μm, 0.45 μm, and 0.2 μm. The preparation method not only yields vesicle-like nanoparticles having relatively high concentration and purity, but also produces cosmetic that exhibit excellent anti-wrinkle, firming, and repair effects.

Description

A method for preparing plant-derived vesicle-like nanoparticles, the product, and its application. Technical Field

[0001] This invention belongs to the field of plant extraction, specifically relating to a method for preparing plant-derived vesicle-like nanoparticles, the product, and its application. Background Technology

[0002] Vesicle-like nanoparticles (PDVLNs) are nanoscale microvesicles secreted by various cells, containing proteins, lipids, mRNA, and miRNA. They act as important mediators of intercellular communication by acting on the endocrine system. Current research indicates that PDVLNs isolated from edible plants are non-toxic and non-immunogenic compared to those secreted by mammalian cells, and exhibit very high in vivo stability and biocompatibility. Furthermore, while PDVLNs isolated from mammalian cells have been reported to contain approximately 20% cholesterol, edible plant-derived PDVLNs are cholesterol-free, and the raw materials are readily available in large quantities.

[0003] In most studies, plant-derived vesicle-like nanoparticles (PDVLNs) are characterized by the size or shape of exosomes, which are cell-released nanoparticles encapsulated in a lipid bilayer (similar to cell membrane structures). To isolate plant-derived vesicle-like nanoparticles (PDVLNs), a method is needed to select appropriately sized exosomes for isolation. Current techniques for extracting plant-derived exosomes primarily involve homogenization of plant tissues and ultracentrifugation. For example, in extracting ginger exosomes, coarsely filtered ginger juice is centrifuged at 10,000g for 1 hour at 4°C, the supernatant is repeated 2-3 times to remove cell debris, and then centrifuged at 100,000g for 80 minutes to precipitate the precipitate, yielding ginger exosomes. In addition, another method is to obtain materials with smaller pore sizes than the filter by filtering and separating extracellular vesicles. That is, when separating exosomes from a large amount of plant juice, the filtration efficiency decreases rapidly because impurities or exosomes are adsorbed and accumulated in the pores of the filter, resulting in low purity and concentration of the obtained exosomes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing plant-derived vesicle-like nanoparticles, the product thereof, and its application, in order to solve the problems existing in the prior art.

[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0006] On one hand, the present invention provides a method for preparing plant-derived vesicle-like nanoparticles, the method comprising the following steps:

[0007] (1) After the sample is juiced, it is subjected to primary filtration, sterile filtration and tangential flow filtration, and the filtrate is collected;

[0008] (2) The filtrate was concentrated and washed to obtain vesicle-like nanoparticles;

[0009] The sterilization filter is selected from membrane filters of 5μm, 0.85μm, 0.45μm and 0.2μm.

[0010] In this invention, "plant-derived vesicle-like nanoparticles (PDVLNs)" and "exosomes" have the same meaning.

[0011] Specifically, 1×PBS buffer is added to the sample before juicing in step (1).

[0012] Specifically, the initial filter described in step (1) uses a 100-mesh nylon mesh.

[0013] Specifically, the tangential flow filtration described in step (1) includes three parameters: shear rate, transmembrane pressure (TMP), and molecular weight cutoff (MWCO).

[0014] Specifically, the shear rate is 4000-8000 m / s, the transmembrane pressure is <0.5 MPa, and the molecular weight cutoff is 100-1000 kDa.

[0015] More specifically, the shear rate is 6000-8000 m / s, the transmembrane pressure is <0.3 MPa, and the molecular weight cutoff is 500-1000 kDa.

[0016] Preferably, the shear rate is 6000 m / s, the transmembrane pressure is 0.2 MPa, and the molecular weight cutoff is 800 kDa.

[0017] Specifically, the concentration described in step (2) is to obtain a concentrated solution by 4-20 times.

[0018] More specifically, the concentration described in step (2) is increased by 10-20 times to obtain a concentrated solution.

[0019] Preferably, the concentration is achieved by multiplying the concentrate by 15 times.

[0020] In some implementations, the sample is a cucumber, but the type of sample is not limited to this.

[0021] On the other hand, the present invention provides the application of vesicle-like nanoparticles prepared by the aforementioned preparation method in the preparation of products with anti-wrinkle, firming, and repairing effects.

[0022] Specifically, the products include cosmetics or pharmaceuticals.

[0023] Specifically, the product is a cosmetic, and its dosage form includes aqueous solution, powder, or emulsion.

[0024] Specifically, the product also includes excipients, including antioxidants.

[0025] In another aspect, the present invention provides a freeze-dried powder composed of vesicle-like nanoparticles prepared by the aforementioned preparation method, trehalose, and mannitol.

[0026] Specifically, the lyophilized powder is formulated from a vesicle-like nanoparticle solution of 90% (w / w), trehalose of 2.5% (w / w), and mannitol of 7.5% (w / w).

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention discloses a method for preparing plant-derived vesicle-like nanoparticles, the product, and its application. This preparation method can not only be used for large-scale production of plant-derived vesicle-like nanoparticles, but also the vesicle-like nanoparticles obtained by this method have high purity and concentration, and the cosmetics prepared by this method have anti-wrinkle, firming, and repairing effects. Attached Figure Description

[0029] Figure 1 shows the TEM characterization results of cucumber vesicle-like nanoparticles.

[0030] Figure 2 shows the change rate of transepidermal water loss (TEWL) value after 30 minutes of testing. "**" indicates a highly significant difference compared with the negative control group, P<0.010. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional methods and conditions or as selected in the product instructions.

[0032] Laboratory consumables:

[0033] Table 1 Experimental Consumables

[0034] Experimental apparatus:

[0035] Table 2 Sources of Experimental Instruments

[0036] Preparation of basic experimental lyophilized powder:

[0037] 1. Preparation of freeze-dried powder:

[0038] (1) Prepare a lyophilization solution according to the following concentrations: 90% (w / w) cucumber vesicle-like nanoparticle solution, 2.5% (w / w) trehalose and 7.5% (w / w) mannitol;

[0039] (2) Dispense the prepared lyophilized solution into vials at a rate of 1.2 mL / vial;

[0040] (3) Then, freeze-drying process is carried out to prepare freeze-dried powder. The freeze-drying process is shown in Table 3.

[0041] Table 3 Freeze-drying process conditions

[0042] 2. Preparation of serum: Add 0.1g of freeze-dried powder to 1.2mL of water and mix well to obtain the serum.

[0043] Example 1: A method for preparing cucumber vesicle-like nanoparticles

[0044] S1. Take about 1kg of cucumber, wash it twice with tap water and pure water, cut it into pieces, put it into a juicer to extract juice, add 500mL of 1×PBS, juice in juice mode for 2min50s;

[0045] S2. Perform preliminary filtration using a 100-mesh nylon mesh. Gently squeeze the gauze during the filtration process and collect the filtrate.

[0046] S3. Sterilization Filtration: Connect the 5μm, 0.85μm, 0.45μm, and 0.2μm filters and the 800kDa tangential flow filter sequentially using tubing. Insert the sample inlet tube into the slot of the peristaltic pump. Rinse the tubing with 500mL of pure water, then rinse with approximately 100mL of 1×PBS. Pour the filtered fruit juice into the peristaltic pump at a speed of 30rpm.

[0047] S4. Concentration and Filtration:

[0048] S4.1 Cleaning: Clean with 0.5M NaOH at a shear rate of 6000 m / s. The inlet pressure is 0.5 bar. Adjust the reflux shut-off valve to ensure the permeate and reflux flow rates are approximately 1:1. Circulate for 60 minutes, then end CIP and drain the system of alkali. Rinse the system and filter column with water at 6000 m / s, adjusting the reflux pressure to 5 Psi, for 5 minutes. Use twice the system volume of water. After draining, repeat this step twice. Continue cleaning the reflux and permeate ends with clean water. Check the pH of the permeate and reflux ends with pH paper; if neutral, rinsing is complete, and the system is drained.

[0049] S4.2 Rinse: Rinse three system volumes with process buffer to drain the solution from the system.

[0050] S4.3 Sample Concentration: Pour the sample solution into a collection bottle. First, close the permeate end with hemostats, adjust the appropriate pump speed, and circulate the sample within the system for 10 minutes. Then, open the permeate end, adjust the transmembrane pressure to 0.2 MPa, start timing, and record the weight of the liquid at the permeate end. During the process, the TMP may gradually increase; adjust the reflux shut-off valve as needed to maintain a constant TMP. Calculate the concentration factor based on the weight at the permeate end.

[0051] S4.4 Sample Washing and Filtration: After concentrating to 15 times, begin washing and filtration. Continue to concentrate the sample over a period of time (system volume; subsequent top washing will dilute the feed solution, so over-concentration is necessary).

[0052] S5 yielded enriched vesicle-like nanoparticles.

[0053] Example 2: A method for preparing cucumber vesicle-like nanoparticles

[0054] The difference from Example 1 is that: "Step S4.1 shear rate is 4000 m / s, S4.3 transmembrane pressure is 0.4 MPa and concentration is 20 times", while the other steps are the same as in Example 1, and vesicle-like nanoparticles are prepared.

[0055] Example 3: A method for preparing cucumber vesicle-like nanoparticles

[0056] The difference from Example 1 is that "step S4.1 shear rate is 8000 m / s, S4.3 transmembrane pressure is 0.3 MPa and concentration is 10 times", while the other steps are the same as in Example 1, to prepare vesicle-like nanoparticles.

[0057] Comparative Example 1: A method for preparing cucumber vesicle-like nanoparticles

[0058] The difference from Example 1 is that: "the sterilization filtration pore size in step S3 is 5μm, 0.85μm and 0.2μm, and the concentration process in step S4.3 and the washing process in step S4.4 are omitted", while the other steps are the same as in Example 1, and the exosomes are prepared.

[0059] Comparative Example 2: A method for preparing cucumber vesicle-like nanoparticles

[0060] The difference from Example 1 is that: "the sterilization filtration pore size in step S3 is 5μm, 0.85μm, 0.45μm, and 0.22μm, and the concentration process in step S4.3 and the washing process in step S4.4 are omitted". The other steps are the same as in Example 1, and the exosomes are prepared.

[0061] Comparative Example 3: A method for preparing cucumber vesicle-like nanoparticles

[0062] The difference from Example 1 is that "the S4.3 concentration and S4.4 washing and filtration processes are omitted", while the other steps are the same as in Example 1, and the exosomes are prepared.

[0063] Effect Experiment:

[0064] The concentration and purity of the enriched vesicle-like nanoparticles were determined using a Rockchip Coulter nanoparticle size analyzer.

[0065] 1. Concentration determination: Dilute the sample to an appropriate concentration to determine the concentration of vesicle-like nanoparticles;

[0066] 2. Purity determination: After membrane rupture reaction and treatment with 1% Tritonx-100 for 15 min, particle size concentration was measured.

[0067] 3. TEM Detection of Vesicular Nanoparticles: 10 μL of vesicular nanoparticle sample was dropped onto a copper grid and allowed to adsorb at room temperature for about 10 min. Excess liquid was then absorbed with a small piece of filter paper. Next, 10 μL of 2% uranium acetate was dropped onto the copper grid and stained at room temperature for 1-3 min. Excess staining liquid was carefully absorbed with filter paper. After drying for 10 min, the copper grid was allowed to air dry naturally. Electron microscopy was performed at 1.5-100 kV, and the results are shown in Figure 1, clearly showing the vesicular nanoparticles.

[0068] The experimental results are shown in Table 4 below. It can be seen that the concentration and purity of the vesicle-like nanoparticles obtained by Example 1 of the preparation method of the present invention are relatively high, while the other examples and comparative examples do not achieve the effect of the technical solution of the present invention.

[0069] Table 4. Results of yield, concentration, and purity determination of vesicle-like nanoparticles.

[0070] Test Example 1: Detection of the anti-wrinkle and firming effects of cucumber vesicle-like nanoparticles

[0071] The elastase inhibition rate assay is used to evaluate the ability of cosmetic products or ingredients to inhibit elastase activity. The results can serve as a reference indicator for assessing whether a cosmetic product has anti-wrinkle and firming effects. This experiment uses absorbance values ​​to reflect the inhibitory effect of exosomes on elastase activity.

[0072] Experimental sample: 0.1g lyophilized powder + 3mL liquid, 2mg / mL epigallocatechin gallate solution (EGCG) (positive control) (from Merck);

[0073] Sample preparation: Add 3 mL of liquid to a 0.1 g lyophilized powder bottle, mix thoroughly, and prepare sample concentrations of 100% (v / v), 50% (v / v), and 20% (v / v) for testing.

[0074] Experimental procedure: Set up sample wells (T), sample background wells (T0), enzyme reaction wells (C), solvent background wells (C0), positive control wells (P), and positive control background wells (P0) in a 96-well microplate, and add samples in sequence according to the steps in Table 5.

[0075] Table 5 Experimental Procedures

[0076] The ELISA plate was transferred into the microplate reader and incubated at 25°C for 15 min. The absorbance was measured at 410 nm. The average absorbance and average inhibition rate of the samples and positive controls are shown in Table 6.

[0077] Table 6. Average absorbance and average inhibition rate of samples and positive controls.

[0078] Wherein, C represents the enzyme reaction group; T represents the sample groups with different concentrations; P represents the positive control group; * indicates that the difference between the sample group and the enzyme reaction group is statistically significant, P<0.05.

[0079] Table 6 shows that, under the experimental conditions, the inhibition rate of the positive control EGCG was 68.59% (≥50%), indicating the effectiveness of the experimental system. Furthermore, the CV values ​​of the OD values ​​of all parallel tubes in this experiment were ≤10%, demonstrating the parallelism of the experiment. When the sample concentration was 100%, it exhibited significant inhibitory function against elastase, with an inhibition rate of 7.10%. This concentration of sample demonstrated anti-wrinkle and firming effects, revealing that the vesicle-like nanoparticles of this invention possess anti-wrinkle and firming efficacy.

[0080] Test Example 2: Determination of the Repairing Efficacy of Cucumber Vesicle-like Nanoparticles

[0081] This test verifies the repair efficacy of the submitted samples by measuring the transepidermal water loss (TEWL) values ​​of the skin in the sample area and the negative control area before and after use in 33 subjects.

[0082] Experimental sample: 0.1g lyophilized powder + 3mL liquid, the same as in test example 1;

[0083] Subject information: Subject gender (male:female = 1:32); Subject age (20-60 years);

[0084] Subject selection criteria: age 18-60 years; normal skin type; regular work and rest schedule during the testing period; able to voluntarily sign informed consent form; must not use products with the same efficacy as the test sample during the testing period; must not undergo any medical aesthetic procedures during the testing period.

[0085] Experimental instrument: Skin moisture loss test probe (CTI Certification & Testing Group Co., Ltd.);

[0086] Experimental steps:

[0087] (1) Subjects cleaned the inner forearms of both hands uniformly by wiping them clean with a dry tissue. The test area was the inner forearm, and one sample area and one negative control area were randomly selected from each subject (each area was 3×3cm). 2 At least 1cm apart, use 3M tape to peel off the areas, peeling 10-40 times per area until the TWEL value reaches 1.25 times that before peeling.

[0088] (2) Apply the test sample (the essence and lyophilized powder are thoroughly mixed) to the sample area at a concentration of (2.0 ± 0.1) mg / cm³. 2 Apply the sample in a single coat until the sample is absorbed; do not use the sample in the negative control area.

[0089] (3) Sit quietly for 30 minutes in a laboratory with a temperature of 21±1℃ and a humidity of 50±10%RH, and the instrument will detect the TWEL value.

[0090] The transepidermal water loss (TEWL) values ​​of the skin are shown in Table 7 and Figure 2. The results indicate that after 30 minutes of use, the TEWL values ​​of the skin in the sample areas of the 33 subjects decreased by 36.72%, while the TEWL values ​​of the skin in the negative control area decreased by 16.02%. The difference between the two groups was highly significant, indicating that the test sample had a repairing effect 30 minutes after use.

[0091] The above examples not only demonstrate that vesicle-like nanoparticles have anti-wrinkle and firming effects, but also repair effects.

[0092] Table 7. Transepidermal water loss (TEWL) values ​​of the skin

Claims

1. A method for preparing plant-derived vesicle-like nanoparticles, characterized in that, The method includes the following steps: (1) After the sample was juiced, it was subjected to primary filtration, sterile filtration and tangential flow filtration, and the filtrate was collected; (2) The filtrate was concentrated and washed to obtain vesicle-like nanoparticles; The sterilization filter is selected from membrane filters of 5μm, 0.85μm, 0.45μm and 0.2μm.

2. The preparation method according to claim 1, characterized in that, The tangential flow filtration described in step (1) includes three parameters: shear rate, transmembrane pressure, and molecular weight cutoff. The shear rate is 4000-8000 m / s, the transmembrane pressure is <0.5 MPa, and the molecular weight cutoff is 100-1000 kDa.

3. The preparation method according to claim 2, characterized in that, The shear rate is 6000-8000 m / s, the transmembrane pressure is <0.3 MPa, and the molecular weight cutoff is 500-1000 kDa.

4. The preparation method according to claim 1, characterized in that, The concentration described in step (2) is increased by 4-20 times to obtain a concentrated solution.

5. The preparation method according to claim 4, characterized in that, The concentration described in step (2) is increased by 10-20 times to obtain a concentrated solution.

6. The application of the vesicle-like nanoparticles prepared by the preparation method according to any one of claims 1-5 in the preparation of products with anti-wrinkle, firming, and repairing effects.

7. The application according to claim 6, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

8. The application according to claim 7, characterized in that, The product is a cosmetic, and its dosage form includes aqueous solution, powder or emulsion.

9. The application according to claim 6, characterized in that, The product also includes excipients, including antioxidants.

10. A freeze-dried powder, characterized in that, It consists of vesicle-like nanoparticles prepared by the preparation method according to any one of claims 1-5, trehalose, and mannitol.

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