Preparation method of plant-derived vesicle-like nanoparticles, product and application of plant-derived vesicle-like nanoparticles

Abstract

The invention belongs to the field of plant extraction, and particularly relates to a preparation method of plant-derived vesicle-like nanoparticles, a product and application of the plant-derived vesicle-like nanoparticles. The preparation method of the plant-derived vesicle-like nanoparticles comprises the following steps: (1) juicing a sample, performing primary filtration, sterile filtration and tangential flow filtration, and collecting filtrate; (2) concentrating, washing and filtering the filtrate to obtain vesicle-like nanoparticles; wherein the degerming filtration is selected from membrane filters with the sizes of 5 microns, 0.85 microns, 0.45 microns and 0.2 microns. According to the preparation method disclosed by the invention, the extracted vesicle-like nanoparticles are relatively high in concentration and purity, and the prepared cosmetic has relatively good anti-wrinkle, firming and repairing effects.

Description

Technical Field

[0001] The present invention belongs to the field of plant extraction, and in particular relates to a preparation method, a product and an application of plant-derived vesicle-like nanoparticles. Background Art

[0002] Vesicle-like nanoparticles are nanoscale microvesicles secreted by a variety of cells, containing proteins, lipids, mRNA and miRNA. They act on the endocrine system and are important mediators of intercellular communication. Existing studies have shown that compared with vesicle-like nanoparticles secreted by mammalian cells, vesicle-like nanoparticles isolated from edible plants have no toxicity and immunogenicity, and have very high in vivo stability and biocompatibility. In addition, it is reported that vesicle-like nanoparticles isolated from mammalian cells contain about 20% cholesterol, but edible plant-derived vesicle-like nanoparticles (PDVLNs) do not contain cholesterol, and the raw material plants have the advantage of being available in large quantities.

[0003] In most studies, the characteristics of plant-derived vesicle-like nanoparticles (PDVLNs) are determined by the size or shape of exosomes, which are nanoparticles released by cells and wrapped in a lipid bilayer (similar to a cell membrane structure). In order to isolate plant-derived vesicle-like nanoparticles (PDVLNs), a method of selecting the appropriate size and isolating only exosomes is needed. In the prior art, plant-derived exosomes are extracted mainly by plant tissue homogenization, ultrahigh-speed centrifugation, and the like. For example, when extracting ginger exosomes, the coarsely filtered ginger juice is centrifuged at 10,000 g for 1 hour at 4°C, the supernatant is taken and repeated 2-3 times to remove cell debris, and then centrifuged at 100,000 g for 80 minutes to obtain ginger exosomes. In addition, there is a method of obtaining materials smaller than the pore size of the filtration filter by filtering the separated extracellular vesicles. That is, when isolating 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, and the purity and concentration of the obtained exosomes are not high. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method, product and application of plant-derived vesicle-like nanoparticles to solve the problems existing in the prior art.

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

[0006] In one aspect, 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 squeezed, it is filtered, sterilized and filtered, and then tangentially filtered, and the filtrate is collected;

[0008] (2) the filtrate is concentrated and filtered to obtain vesicle-like nanoparticles;

[0009] Wherein, the sterilizing filtration is selected from 5μm, 0.85μm, 0.45μm and 0.2μm membrane filters.

[0010] In the present 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 primary filtration in step (1) uses a 100-mesh nylon mesh.

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

[0014] Specifically, the shear rate is 4000-8000 m / s, the transmembrane pressure is <0.5 MPa, and the molecular weight cut-off 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 cut-off is 500-1000 KDa.

[0016] Preferably, the shear rate is 6000 m / s, the transmembrane pressure is 0.2 MPa, and the molecular weight cut-off is 800 KDa.

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

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

[0019] Preferably, the concentration is 15 times to obtain a concentrated solution.

[0020] In some embodiments, the sample is a plant fruit, cucumber, but the sample type is not limited thereto.

[0021] On the other hand, the present invention provides the use 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 product includes cosmetics or medicines.

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

[0024] Specifically, the product further comprises auxiliary materials, and the auxiliary materials comprise antioxidants.

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

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

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discloses a preparation method, product and application of plant-derived vesicle-like nanoparticles. The preparation method can not only be used for large-scale production of plant-derived vesicle-like nanoparticles, but also the purity and concentration of the vesicle-like nanoparticles obtained by the preparation method are relatively high, and the prepared cosmetics have anti-wrinkle, firming and repairing effects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 This is the result of the change rate of transepidermal water loss (TEWL) after 30 minutes of testing. “**” indicates that there is a very significant difference compared with the negative control area group, P<0.010. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually selected according to conventional methods and conditions or according to the product specifications.

[0032] Experimental consumables:

[0033] Table 1 Experimental consumables

[0034] name Model requirements Membrane filter 5μm Membrane filter 0.85μm Membrane filter 0.45μm Membrane filter 0.22μm Membrane filter 0.2μm Tangential Flow Filters 800Kda

[0035] Experimental instruments:

[0036] Table 2 Sources of experimental instruments

[0037] name source model Peristaltic Pumps LongerPump BT100-3J Nano Coulter Particle Sizer Rockchip RESUN-S09 ELISA reader Thermo Fisher Multiskan FC Skin water loss test probe Centre Testing International Group Co., Ltd. Tewameter TM Hex

[0038] Preparation of freeze-dried powder for basic experiments:

[0039] 1. Preparation of freeze-dried powder:

[0040] (1) preparing a freeze-dried solution according to 90% (w / w) cucumber vesicle-like nanoparticle solution, 2.5% (w / w) trehalose and 7.5% (w / w) mannitol;

[0041] (2) Dispense the prepared lyophilized solution into vials at a rate of 1.2 mL / vial;

[0042] (3) Then, a freeze-drying process is performed to prepare a freeze-dried powder. The freeze-drying process is shown in Table 3;

[0043] Table 3 Freeze-drying process conditions

[0044] Section Temperature(℃) Slope temperature control (M) Constant temperature time (M) Vacuum(Pa) 1 -45 0 360 - 2 -30 30 1560 10 3 -15 30 120 10 4 0 30 60 10 5 10 30 360 0

[0045] 2. Preparation of essence: Add 1.2 mL of water to 0.1 g of freeze-dried powder and mix well to obtain the essence.

[0046] Example 1 A method for preparing cucumber vesicle-like nanoparticles

[0047] S1. Take about 1 kg of cucumber, wash it twice with tap water and pure water, cut it into pieces, put it into a juicer to extract juice, add 500 mL of 1×PBS, and extract juice for 2 min 50 s in juice mode;

[0048] S2, perform preliminary filtration using a 100-mesh nylon mesh, gently squeeze the gauze during the filtration process, and collect the filtrate;

[0049] S3, sterilization filtration: connect the 5μm, 0.85μm, 0.45μm, 0.2μm filters and the 800KDa tangential flow filter in sequence with hoses, and install the sample inlet tube into the slot of the peristaltic pump. Rinse the pipeline with 500mL of pure water, and then rinse the pipeline with about 100mL of 1×PBS. The peristaltic pump speed is 30rpm, and the filtered fruit juice is poured into the peristaltic pump at a speed of 15rpm;

[0050] S4, concentration and diafiltration:

[0051] S4.1 Cleaning: Clean with 0.5M NaOH at a shear rate of 6000m / s. The inlet pressure is 0.5bar, and the stop valve at the reflux end is adjusted to ensure that the flow rate of the permeate and reflux liquid is basically 1:1. Circulate for 60min, CIP is completed, and the alkali in the system is emptied. Rinse the system and filter column with water at 6000m / s, adjust the reflux end pressure to 5Psi, and circulate and flush for 5min. The water consumption is twice the system volume. After emptying, repeat this step twice. Continue to clean the reflux end and the permeate end with clean water. Use pH test paper to test the permeate end and the reflux end. If it is neutral, the flushing is completed and the system is emptied.

[0052] S4.2 Rinse: Flush 3 system volumes with process buffer to drain the solution in the system.

[0053] S4.3 Sample concentration: Pour the liquid into the collection bottle. First close the permeate end with a hemostatic forceps, adjust the appropriate pump speed, and let the sample circulate in the system for 10 minutes. Then open the permeate end, adjust the transmembrane pressure to 0.2MPa, start timing and record the weight of the liquid at the permeate end. During the process, the TMP may gradually increase. Adjust the stop valve at the reflux end at any time to maintain a constant TMP. Calculate the concentration multiple based on the weight of the permeate end.

[0054]

[0055] S4.4 Sample diafiltration: After concentration to 15 times, start diafiltration. Continue to over-concentrate the sample for a period of time (system volume, subsequent top wash will dilute the feed liquid, so over-concentration is required).

[0056] S5 obtains enriched vesicle-like nanoparticles.

[0057] Example 2 A method for preparing cucumber vesicle-like nanoparticles

[0058] The difference from Example 1 is: "Step S4.1 shear rate is 4000 m / s, S4.3 transmembrane pressure is 0.4 MPa and concentrated to 20 times", and the other steps are the same as those in Example 1 to prepare vesicle-like nanoparticles.

[0059] Example 3 Preparation method of cucumber vesicle-like nanoparticles

[0060] The difference from Example 1 is: "Step S4.1 shear rate is 8000 m / s, S4.3 transmembrane pressure is 0.3 MPa and concentrated to 10 times", and the other steps are the same as Example 1 to prepare vesicle-like nanoparticles.

[0061] Comparative Example 1: A method for preparing cucumber vesicle-like nanoparticles

[0062] The difference from Example 1 is that: "In step S3, the pore sizes of the sterilization filtration are 5 μm, 0.85 μm and 0.2 μm, and the S4.3 concentration and S4.4 diafiltration processes are deleted." The other steps are the same as those in Example 1 to prepare exosomes.

[0063] Comparative Example 2 Preparation method of cucumber vesicle-like nanoparticles

[0064] The difference from Example 1 is that: "In step S3, the pore sizes of the sterilization filtration are 5 μm, 0.85 μm, 0.45 μm, and 0.22 μm, and the S4.3 concentration and S4.4 diafiltration processes are deleted." The other steps are the same as those in Example 1 to prepare exosomes.

[0065] Comparative Example 3 Preparation method of cucumber vesicle-like nanoparticles

[0066] The difference from Example 1 is that: "S4.3 concentration and S4.4 diafiltration process are deleted", and the other steps are the same as those in Example 1 to prepare exosomes.

[0067] Effect experiment:

[0068] The concentration and purity of the enriched vesicle-like nanoparticles were determined using a Rockchip Coulter nanoparticle sizer.

[0069] 1. Concentration determination: dilute the sample to an appropriate concentration for vesicle-like nanoparticle concentration determination;

[0070] 2. Purity determination: membrane permeabilization reaction, 1% Tritonx-100 treatment for 15 minutes and then the particle size concentration is detected.

[0071]

[0072] 3. TEM detection of vesicle-like nanoparticles: Take 10μL of vesicle-like nanoparticle sample and drop it on the copper mesh. Let it adsorb at room temperature for about 10 minutes. Use a small piece of filter paper to absorb the excess liquid. Then add 10μL of 2% uranyl acetate and drop it on the copper mesh. Let it stain at room temperature for 1-3 minutes. Carefully use filter paper to absorb the excess staining liquid. Dry for 10 minutes, let the copper mesh dry naturally, and use 1.5 100kv for electron microscopy imaging. The results are as follows: Figure 1 As shown, vesicle-like nanoparticles can be clearly seen.

[0073] The experimental results are shown in Table 4 below. It can be seen that the concentration and purity of the vesicle-like nanoparticles obtained in Example 1 of the preparation method of the present invention are relatively high, and the other examples and comparative examples cannot achieve the effect of the technical solution of the present invention.

[0074] Table 4 Results of yield, concentration and purity determination of vesicle-like nanoparticles

[0075]

[0076] Test Example 1: Anti-wrinkle and firming efficacy test of cucumber vesicle-like nanoparticles

[0077] The elastase inhibition rate test is used to evaluate the ability of cosmetic products or ingredients to inhibit the activity of elastase. This result can be used as a reference indicator to measure whether the cosmetic product has anti-wrinkle and firming effects. This experiment reflects the inhibitory effect of exosomes on elastase activity through absorbance value.

[0078] Experimental sample: 0.1 g lyophilized powder + 3 mL liquid, 2 mg / mL epigallocatechin gallate solution (EGCG) (positive control) (from Merck);

[0079] Sample preparation: Add 3 mL of liquid into a 0.1 g freeze-dried powder bottle and mix thoroughly to prepare samples with concentrations of 100% (v / v), 50% (v / v) and 20% (v / v) for testing.

[0080] Experimental steps: Set 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 ELISA plate, and add samples in sequence according to the steps in Table 5.

[0081] Table 5 Experimental procedures

[0082]

[0083] The ELISA plate was transferred to the ELISA reader, incubated at 25°C for 15 minutes, and 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:

[0084] Table 6 Average absorbance and average inhibition rate of samples and positive controls

[0085] Group Absorbance Inhibition rate (%) C 0.3641±0.0014 0.00±0.38 T-100 0.3383±0.0036 7.10±0.99* T-50 0.3620±0.0033 0.59±0.90 T-20 0.3723±0.0020 -2.23±0.55 P-2 0.1144±0.0008 68.59±0.21

[0086] Among them, C represents the enzyme reaction group; T represents the sample groups with different concentrations; P represents the positive control group; * represents that the difference between the sample group and the enzyme reaction group is statistically significant, P<0.05.

[0087] The results in Table 6 show that under the experimental conditions, the inhibition rate of the positive control EGCG is 68.59% (≥50%), and the experimental system is effective. In addition, the CV values ​​of the OD values ​​of all parallel tubes in this experiment are ≤10%, and the parallelism of this experiment is effective. When the sample concentration is 100%, it has a significant inhibitory function on elastase, and the inhibition rate is 7.10%. The sample at this concentration has an anti-wrinkle and firming effect, which also reveals that the vesicle-like nanoparticles of the present invention have anti-wrinkle and firming effects.

[0088] Test Example 2: Detection of the Repairing Efficacy of Cucumber Vesicle-like Nanoparticles

[0089] This test verifies the repair efficacy of the samples by measuring the transepidermal water loss (TEWL) values ​​of the sample area and negative control area of ​​33 subjects before and after use.

[0090] Experimental sample: 0.1g lyophilized powder + 3mL liquid as in Test Example 1;

[0091] Subject information: subject gender (male: female = 1:32); subject age (20-60 years old);

[0092] Subject screening criteria: Age 18-60 years old; normal skin quality; regular work and rest during the test period; able to voluntarily sign the informed consent form; no products with the same efficacy as the test sample may be used during the test period; no medical beauty projects may be performed during the test period.

[0093] Experimental instruments: Skin moisture loss test probe (HuaCe Testing & Certification Group Co., Ltd.);

[0094] Experimental steps:

[0095] (1) The subjects cleaned the inner forearms of both hands uniformly by wiping them with dry tissue paper. The test area was the inner forearm. One sample area and one negative control area (area 3×3 cm) were randomly selected. 2 ), with an interval of at least 1 cm, use 3M tape for peeling, the number of peeling times is 10-40 times per area, and the TWEL value reaches 1.25 times before peeling;

[0096] (2) Use the test sample (essence and freeze-dried powder are fully mixed) in the sample area at (2.0±0.1) mg / cm 2 Apply a single application of the amount until the sample is absorbed, and do not use the sample in the negative control area;

[0097] (3) Sit still in a laboratory at a temperature of 21±1°C and a humidity of 50±10% RH for 30 min, and measure the TWEL value with an instrument;

[0098] The results of TEWL values ​​of skin are shown in Table 7 and Figure 2 As shown, the results show that after 30 minutes of use, the TEWL value of the sample area of ​​33 subjects decreased by 36.72%, and the TEWL value of the negative control area decreased by 16.02%. There is a very significant difference between the two groups of data, indicating that the test sample has a repair effect 30 minutes after use.

[0099] The above examples not only illustrate that the vesicle-like nanoparticles have anti-wrinkle and firming effects, but also have repair effects.

[0100] Table 7 Transepidermal Water Loss TEWL Values

[0101]

[0102]

Claims

1. A method for preparing plant-derived vesicle-like nanoparticles, characterized in that: The method comprises the following steps: (1) After the sample is squeezed, it is filtered, sterilized and filtered, and the filtrate is collected; (2) The filtrate is concentrated and filtered to obtain vesicle-like nanoparticles; Wherein, the sterilizing filtration is selected from 5μm, 0.85μm, 0.45μm and 0.2μm membrane filters.

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 cut-off, the shear rate is 4000-8000m / s, the transmembrane pressure is <0.5MPa, and the molecular weight cut-off is 100-1000KDa.

3. The preparation method according to claim 2, characterized in that: The shear rate is 6000-8000 m / s, the transmembrane pressure is less than 0.3 MPa, and the molecular weight cut-off is 500-1000 KDa.

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

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

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

7. The use according to claim 6, characterized in that: The product includes a cosmetic or a medicine.

8. The use according to claim 7, characterized in that: The product is a cosmetic, and its dosage form includes water, powder or emulsion.

9. The use according to claim 6, characterized in that: The product also includes auxiliary materials, and the auxiliary materials include antioxidants.

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

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