A method for preparing a plant-derived vesicle-like nanoparticle, a product thereof, and applications thereof

Abstract

The application belongs to the field of plant extraction, and particularly relates to a preparation method of plant-derived vesicle-like nanoparticles, products and application thereof. The preparation method of the plant-derived vesicle-like nanoparticles provided by the application comprises the following steps: (1) after sample juice is extracted, the sample juice is subjected to preliminary filtration, sterilization filtration and tangential flow filtration, and filtrate is collected; (2) the filtrate is subjected to concentration and washing filtration, and then vesicle-like nanoparticles are obtained; wherein the sterilization filtration is selected from membrane filters with pore sizes of 5 microns, 0.85 microns, 0.45 microns and 0.2 microns. The preparation method has the advantages that the extracted vesicle-like nanoparticles have relatively high concentration and purity, and the prepared cosmetics have good anti-wrinkle firming and repairing effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant extraction, and particularly relates to a preparation method of plant-derived vesicle-like nanoparticles, products and applications thereof. BACKGROUND

[0002] Vesicle-like nanoparticles are nanoscale microvesicles secreted by various cells, containing proteins, lipids, mRNA and miRNA, and are important mediators of intercellular communication by acting on the endocrine system. 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 has been reported that vesicle-like nanoparticles isolated from mammalian cells contain about 20% cholesterol, but plant-derived vesicle-like nanoparticles (PDVLNs) do not contain cholesterol, and the raw plant has 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 a kind of cell-released, lipid bilayer-encapsulated (similar to cell membrane structure) nanoparticles. In order to isolate plant-derived vesicle-like nanoparticles (PDVLNs), a method for selecting the appropriate size and only isolating exosomes is needed. The existing technology for extracting plant-derived exosomes mainly extracts through plant tissue homogenization crushing, ultrahigh-speed centrifugation, etc. For example, when extracting ginger exosomes, the filtered ginger juice is centrifuged at 10,000g at 4℃ for 1h, the supernatant is repeatedly taken for 2-3 times to remove cell debris, and then 100,000g centrifugation is used for 80min to precipitate, and ginger exosomes are obtained. In addition, there is another method of obtaining materials smaller than the pore size of the filter by filtering the isolated extracellular vesicles, that is, when isolating exosomes from a large amount of plant juice, the filtration efficiency is rapidly reduced due to the adsorption and accumulation of impurities or exosomes in the pores of the filter, and the purity and concentration of the obtained exosomes are not high. SUMMARY

[0004] The present application aims to provide a preparation method of plant-derived vesicle-like nanoparticles, products and applications thereof to solve the problems in the prior art.

[0005] To achieve the above-mentioned application purposes, the technical solutions of the present application are as follows:

[0006] On the one hand, the present application provides a preparation method of plant-derived vesicle-like nanoparticles, which comprises the following steps:

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

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

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

[0010] In the present application, the "plant-derived vesicle-like nanoparticles (PDVLNs)" and "exosomes" belong to one meaning.

[0011] Specifically, the sample is added with 1×PBS buffer before being squeezed in step (1).

[0012] Specifically, the primary filtration in step (1) uses a 100-mesh nylon screen.

[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] Further 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 performed at 4-20 times to obtain a concentrated solution.

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

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

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

[0021] In another aspect, the present application provides the use of the 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 a cosmetic or a pharmaceutical product.

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

[0024] Specifically, the product further comprises an excipient, and the excipient comprises an antioxidant.

[0025] In another aspect, the present application provides a lyophilized 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) of the vesicle-like nanoparticle solution, 2.5% (w / w) of trehalose and 7.5% (w / w) of mannitol.

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

[0028] The present application discloses a preparation method of plant-derived vesicle-like nanoparticles, a product and application thereof. BRIEF DESCRIPTION OF DRAWINGS

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

[0030] Figure 2 The results of the change rate of the skin's transepidermal water loss (TEWL) value after 30 minutes, and "**" indicates that there is a very significant difference compared with the negative control area group, P<0.010. DETAILED DESCRIPTION

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

[0032] Experimental consumables:

[0033] Table 1 Experimental consumables

[0034] Name Model requirement Membrane filter 5 μm Membrane filter 0.85 μm Membrane filter 0.45 μm Membrane filter 0.22 μm Membrane filter 0.2 μm Tangential flow filter 800 Kda

[0035] Experimental instruments:

[0036] Table 2 Sources of experimental instruments

[0037] Name Source Model Peristaltic pump Longer Pump BT100-3J Nano Kurt particle size analyzer Ruichizhi R&D RESUN-S09 Microplate reader Thermo Multiskan FC Skin moisture loss test probe Huachetongzheng Group Co., Ltd. Tewameter TM Hex

[0038] Preparation of the lyophilized powder for the basic experiment:

[0039] 1. Preparation of the lyophilized powder:

[0040] (1) Formulation of the freeze-dried solution according to 90% (w / w) of cucumber vesicle-like nanoparticles solution, 2.5% (w / w) of trehalose and 7.5% (w / w) of mannitol;

[0041] (2) The prepared freeze-dried solution was divided into 1.2 mL / bottle in a Schlenk bottle;

[0042] (3) Then, the freeze-drying process was carried out to obtain the freeze-dried powder, and 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 the serum: 0.1 g of freeze-dried powder was added to 1.2 mL of water and mixed to obtain the serum.

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

[0047] S1, about 1 kg of cucumber was washed twice with tap water and pure water, cut into pieces, and squeezed into juice in a juicer, 500 mL of 1 × PBS was added, and the juice was squeezed for 2 min 50 s in juice mode;

[0048] S2, primary filtration was performed with a 100-mesh nylon screen, and the filter liquid was collected by gently squeezing the gauze during filtration;

[0049] S3, sterilization filtration: 5 μm, 0.85 μm, 0.45 μm, and 0.2 μm filters and an 800 KDa tangential flow filter were sequentially connected with a rubber tube, and the sampling tube was installed in the clamping groove of the peristaltic pump. 500 mL of pure water was used to rinse the pipeline, and about 100 mL of 1 × PBS was used to rinse the pipeline. The peristaltic pump speed was 30 rpm, the treated fruit juice was poured into the peristaltic pump, and the peristaltic pump speed was 15 rpm;

[0050] S4, concentration and washing:

[0051] S4.1 washing: washing with 0.5 M NaOH at a shear rate of 6000 m / s. The inlet pressure was 0.5 bar, and the backflow end stop valve was adjusted to ensure that the flow rates of the permeate and the backflow were basically 1:1. Circulation was performed for 60 min, CIP was completed, and the system was emptied of the alkali. The system and the filter column were rinsed with water at a shear rate of 6000 m / s, the backflow end pressure was adjusted to 5 Psi, and circulation rinsing was performed for 5 min. The amount of water used was 2 times the volume of the system. After emptying, the step was repeated twice. The backflow end and the permeation end were continuously washed with clean water. The pH test paper was used to detect the permeation end and the backflow end, and if they were neutral, the rinsing was completed, and the system was emptied.

[0052] S4.2 conditioning: the system was rinsed with 3 system volumes of process buffer, and the solution in the system was emptied.

[0053] S4.3 Sample concentration: Pour the feed into the collection bottle. First close the permeate end with hemostatic forceps, adjust the pump speed to make the sample circulate in the system for 10 min. Then open the permeate end, adjust the transmembrane pressure to 0.2 MPa, start timing and record the weight of the liquid in the permeate end. The TMP may gradually increase during the process, and the backflow end stop valve should be adjusted in time to maintain a constant TMP. According to the weight of the permeate end, calculate the concentration multiple.

[0054]

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

[0056] S5 Obtain enriched vesicle-like nanoparticles.

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

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

[0059] Example 3 A method for preparing a cucumber vesicle-like nanoparticle

[0060] The difference from Example 1 is that “the shear rate in step S4.1 is 8000 m / s, the transmembrane pressure in S4.3 is 0.3 MPa, and the concentration is to 10 times”, and the other steps are the same as those in Example 1, to prepare the vesicle-like nanoparticles.

[0061] Comparative Example 1 A method for preparing a cucumber vesicle-like nanoparticle

[0062] The difference from Example 1 is that “the pore size of the sterilization filter in step S3 is 5 μm, 0.85 μm and 0.2 μm, and the concentration and washing processes in S4.3 and S4.4 are deleted”, and the other steps are the same as those in Example 1, to prepare the exosomes.

[0063] Comparative Example 2 A method for preparing a cucumber vesicle-like nanoparticle

[0064] The difference from Example 1 is that “the pore size of the sterilization filter in step S3 is 5 μm, 0.85 μm, 0.45 μm, and 0.22 μm, and the concentration and washing processes in S4.3 and S4.4 are deleted”, and the other steps are the same as those in Example 1, to prepare the exosomes.

[0065] Comparative Example 3 A method for preparing a cucumber vesicle-like nanoparticle

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

[0067] Effect experiment:

[0068] The enriched vesicle-like nanoparticles were determined for concentration and purity using the Malvern Zetasizer nanoparticle size analyzer.

[0069] 1. Concentration determination: The sample was diluted to an appropriate concentration for determination of the concentration of the vesicle-like nanoparticles;

[0070] 2. Purity determination: Membrane disruption reaction, detection of particle size concentration after 1% Triton x-100 treatment for 15 min,

[0071]

[0072] 3. TEM detection of vesicle-like nanoparticles: 10 μL of the vesicle-like nanoparticle sample was dropped on a copper grid, adsorbed at room temperature for about 10 min, and the excess liquid was absorbed with a small piece of filter paper. Then 10 μL of 2% uranyl acetate was dropped on the copper grid, and the staining was performed at room temperature for 1-3 min. The excess staining liquid was carefully absorbed with filter paper. After drying for 10 min, the copper grid was naturally air-dried, and electron microscopy imaging was performed at 1.5 100 kv. The results are shown in Figure 1 , and the 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 by the preparation method of Example 1 of the present application are relatively high, and the other examples and comparative examples do not achieve the effect of the technical solution of the present application.

[0074] Table 4: Yield, concentration and purity determination results of vesicle-like nanoparticles

[0075]

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

[0077] Elastase inhibition rate experiment is used to evaluate the ability of a cosmetic product or ingredient to inhibit elastase activity. This result can be used as a reference index for measuring whether the cosmetic product has anti-wrinkle firming efficacy. In this experiment, the absorbance value reflects the inhibitory effect of exosomes on elastase activity.

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

[0079] Preparation of samples: 3 mL of liquid was added to the 0.1 g of lyophilized powder bottle, mixed thoroughly, and the sample concentration was prepared to 100% (v / v), 50% (v / v) and 20% (v / v) for testing.

[0080] Experimental steps: 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 enzyme-labeled plate, and add samples in sequence according to the steps in Table 5.

[0081] Table 5 Experimental steps

[0082]

[0083] The enzyme-labeled plate was moved into an enzyme-labeled instrument and incubated at 25°C for 15 min, and the absorbance value was measured at 410 nm. The average absorbance value and average inhibition rate of the sample and positive control are shown in Table 6:

[0084] Table 6 Average absorbance value and average inhibition rate of sample and positive control

[0085] Group Absorbance value 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] Wherein, C represents the enzyme reaction group; T represents the sample group of different concentrations; P represents the positive control group; * represents that the sample group has a statistically significant difference compared with the enzyme reaction group, 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. The CV values of all parallel tubes in the experiment are ≤10%, and the parallelism of the experiment is effective. When the sample concentration is 100%, it has a significant inhibitory effect on elastase, with an inhibition rate of 7.10%, and the sample at this concentration has an anti-wrinkle tightening effect, which also reveals that the vesicle-like nanoparticles of the application have an anti-wrinkle tightening effect.

[0088] Test Example 2 Detection of repair efficacy of cucumber vesicle-like nanoparticles

[0089] This test verifies the repair efficacy of the sample by measuring the trans-epidermal water loss (TEWL) value of the skin in the sample area and the negative control area of 33 subjects before and after use.

[0090] Experimental sample: 0.1 g of lyophilized powder + 3 mL of liquid 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; normal skin; regular work and rest during the test period; able to voluntarily sign the informed consent form; do not use products with equivalent efficacy as the test sample during the test period; do not perform any medical aesthetic procedures during the test period.

[0093] Experimental instruments: skin moisture loss test probe (Huaxi Testing and Certification Group Co., Ltd.);

[0094] Experimental steps:

[0095] (1) The subjects uniformly cleaned the inner side of the forearm, the cleaning method was to wipe clean with dry facial tissues, the test area was the inner side of the forearm, and 1 sample area and one negative control area (each with an area of 3x3 cm 2 ) were randomly selected, with a spacing of at least 1 cm or more, and 3M tape was used for peeling, with a peeling frequency of 10-40 times per block area, and the TWEL value reached 1.25 times before peeling;

[0096] (2) The test sample (essence and lyophilized powder fully mixed) was used in the sample area at a dosage of (2.0±0.1) mg / cm 2 for single coating until the sample was absorbed, and the negative control area was not used with the sample;

[0097] (3) Sit in a laboratory with a temperature of 21±1℃ and a humidity of 50±10% RH for 30 min, and detect the TWEL value with the instrument;

[0098] The skin trans-epidermal water loss (TEWL) value results are shown in Table 7 and Figure 2 The results show that after 30 minutes of use, the skin trans-epidermal water loss (TEWL) value of the sample area of 33 subjects decreased by 36.72%, and the skin trans-epidermal water loss (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 after 30 minutes of use.

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

[0100] Table 7 Skin trans-epidermal water loss (TEWL) value

[0101]

[0102]

Claims

1. A method for preparing a plant-derived vesicle-like nanoparticle, characterized by, The method comprises the following steps: (1) collecting filtrate after initial filtration, sterilization filtration and tangential flow filtration after sample juice extraction; (2) obtaining vesicle-like nanoparticles after concentration and washing of the filtrate; The sterilization filtration uses membrane filters with 5 μm, 0.85 μm, 0.45 μm and 0.2 μm; The tangential flow filtration in step (1) comprises three parameters: shear rate, transmembrane pressure and molecular weight cut-off, the shear rate is 6000 m / s, the transmembrane pressure is 0.2 MPa, and the molecular weight cut-off is 800 KDa; The concentration in step (2) obtains concentrated solution with 15 times; The sample is cucumber.

2. Application of the vesicle-like nanoparticles prepared by the preparation method in claim 1 in the preparation of products with anti-wrinkle tightening and repair efficacy.

3. Use according to claim 2, characterized in that, The products comprise cosmetics or drugs.

4. Use according to claim 3, characterized in that, The products are cosmetics, and the dosage forms comprise aqueous agent, powder or emulsion.

5. Use according to claim 4, characterized in that, The products further comprise auxiliary materials, and the auxiliary materials comprise antioxidants.

6. A lyophilized powder characterized in that, The vesicle-like nanoparticles, trehalose and mannitol are prepared by the preparation method in claim 1.

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