Preparation method of plant cell vesicles from black truffle and application thereof

Abstract

The application discloses a preparation method and application of plant cell vesicles from black truffle, and belongs to the technical field of biology; the raw material of black truffle is pretreated by freeze-drying and ultrafine grinding technology, is subjected to enzymolysis by using a composite enzymolysis system, and is subjected to ultrasonic crushing to release the vesicle structure. Large molecular impurities are intercepted by using an ultrafiltration membrane, and then are separated and purified by sucrose density gradient centrifugation, so that the plant cell vesicles with an average particle size of 127 nm, a Zeta potential of-32 mV and an encapsulation efficiency of 91.7% can be obtained. The vesicles can be used as a drug carrier for anti-inflammatory component delivery, can promote the migration of HaCat cells, have better ability of promoting skin repair, and can be used for preparing medical and cosmetic products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for preparing plant cell vesicles derived from black truffles and their application. Background Technology

[0002] With the rapid development of precision medicine and natural product development, plant-derived extracellular vesicles (PDEVs) have become a research hotspot for novel drug delivery systems due to their unique lipid bilayer structure, low immunogenicity, and natural targeting properties. Black truffles (Tuber spp.), recognized worldwide as a top-tier food ingredient, contain abundant sphingolipids and bioactive peptides in their fruiting bodies. However, the dense polysaccharide-lignin complex structure of their cell walls makes it difficult to efficiently obtain endogenous vesicles using conventional physical disruption methods.

[0003] In existing technologies, the extraction of active ingredients from black truffles mainly relies on supercritical CO2 extraction (pressure 15-25 MPa, temperature 35-45℃) and organic solvent fractionation precipitation. While some existing technologies improve polysaccharide yield through 20 kHz ultrasonic treatment, they do not address the complete separation of vesicle structures. Existing technologies employing high-pressure homogenization combined with membrane filtration can obtain nanoparticles with narrow particle size distributions, but suffer from drawbacks such as high equipment investment and high energy consumption.

[0004] Studies have shown that while traditional supercritical fluid extraction (SCFE) technology offers selectivity in extracting lipid components, it significantly damages biomembrane structures such as extracellular vesicles. When the extraction pressure exceeds 20 MPa, the fluidity of the vesicular lipid bilayer decreases sharply, leading to membrane protein denaturation. Furthermore, commercial supercritical fluid extraction equipment is typically equipped with entrainer systems, which can easily leave residual organic solvents such as ethanol, affecting the compliance of subsequent biopharmaceutical applications.

[0005] To address the shortcomings of existing technologies, this invention innovatively combines enzymatic hydrolysis and physical separation techniques to achieve efficient preparation of black truffle cell vesicles under mild conditions. By optimizing the ratio of the compound enzyme and the enzymatic hydrolysis kinetic parameters, cell wall components are specifically degraded without destroying the structural integrity of the vesicles. Summary of the Invention

[0006] This invention first provides a method for preparing plant cell vesicles derived from black truffles, comprising the following steps:

[0007] (1) After freeze-drying and ultra-fine grinding, black truffle raw materials are mixed with phosphate buffer for enzymatic hydrolysis;

[0008] (2) The enzymatic hydrolysis products were broken down by ultrasound to obtain fractions rich in vesicles;

[0009] (3) Plant cell vesicles with a particle size of 30-200 nm were obtained by ultrafiltration membrane separation and sucrose density gradient centrifugation purification.

[0010] (4) Freeze-dry to obtain the finished product.

[0011] In some embodiments, the enzymatic hydrolysis in step (1) uses a complex enzyme system, including cellulase, β-glucanase and papain.

[0012] In some embodiments, the enzymatic hydrolysis temperature in step (1) is 50-60°C and the time is 1-3 hours.

[0013] In some embodiments, the molecular weight cutoff of the ultrafiltration membrane in step (3) is 10-50 kDa, and the density gradient range of the sucrose density gradient centrifugation is 1.063-1.21 g / mL.

[0014] In some embodiments, the plant cell vesicles have a zeta potential of -20 to -40 mV and an encapsulation rate of ≥85%.

[0015] In some embodiments, the black truffle is Tuber melanosporum or Tuberaestivum.

[0016] The present invention also provides the application of the black truffle-derived plant cell vesicles prepared by the above method as drug carriers for delivering active ingredients.

[0017] The present invention also provides the application of the black truffle-derived plant cell vesicles prepared by the above method, which can be used in cosmetic compositions to improve the transdermal absorption rate of active ingredients.

[0018] This invention also provides the application of the black truffle-derived plant cell vesicles prepared by the above method, which are used to encapsulate antioxidants for the preparation of functional foods.

[0019] Finally, the present invention also provides a cosmetic composition comprising black truffle-derived plant cell vesicles prepared by the above method, as well as polypeptides, polysaccharides and moisturizers.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) An innovative composite enzymatic hydrolysis system is used to achieve selective degradation of the cell wall, with a vesicle integrity rate of >92%;

[0022] (2) Vesicles can be used as drug carriers for the delivery of anti-inflammatory components, can promote HaCat cell migration, have a better ability to promote skin repair, and can be used to prepare medical aesthetic products. Attached Figure Description

[0023] Figure 1 Flowchart of black truffle cell vesicle preparation.

[0024] Figure 2 Transmission electron micrograph of black truffle cell vesicles.

[0025] Figure 3 1. Black truffle cell vesicle size distribution curve.

[0026] Figure 4 1. Black truffle vesicles promote HaCat cell migration (image); control group is the control group. HNV represents the black truffle cell vesicle group. Detailed Implementation

[0027] The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art will be able to make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention.

[0028] Example 1: Preparation of black truffle cell vesicles

[0029] (1) Raw material pretreatment: Take fresh Tuber melanosporum fruiting bodies, freeze dry at -50℃ for 48 hours, then use a planetary ball mill for ultrafine grinding (300 rpm, ball-to-material ratio 10:1), and pass through a 200-mesh sieve for later use.

[0030] (2) Compound enzymatic hydrolysis: Calculate the weight of the solid and add 10 times its weight of pH 5.8 phosphate buffer to obtain a mixture. Calculate the mass of the mixture and add a compound enzyme preparation (composed of enzymes with the following enzyme activity parameters: cellulase 20 U / mg, β-glucanase 10 U / mg, papain 5 U / mg, with a mass ratio of 1:1:1) at a ratio of 2% to 1% in a constant temperature shaker. Place the mixture in a constant temperature shaker at 55°C for 2 hours. During this period, take samples every 30 minutes to determine the reducing sugar content. Terminate the reaction when the increase is <5% for two consecutive times.

[0031] (3) Ultrasonic disruption: The enzymatic hydrolysate was transferred to an ice bath and processed using an ultrasonic cell disruptor with a frequency of 40kHz and a power of 400W. The cycle mode was set with a duty cycle of 30%, a working time of 5s and an interval of 10s, and the total processing time was 15 minutes. During the process, the temperature was maintained at ≤10℃ by circulating water cooling.

[0032] (4) Preliminary separation: Centrifuge the lysate at 8000×g for 20 minutes to remove unbroken cells and large particles of impurities, and collect the supernatant.

[0033] (5) Ultrafiltration concentration: Using a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 30 kDa, cross-flow filtration is performed at a pressure of 0.12 MPa. The permeate is collected and concentrated to 1 / 5 of the original filtrate volume.

[0034] (6) Density gradient centrifugation: Prepare a continuous sucrose density gradient solution (1.063, 1.098, 1.154, 1.21 g / mL), slowly inject the concentrate into the top of the gradient solution, and ultracentrifuge at 120,000 × g for 18 hours at 4°C. Collect different density zones in segments, and observe them with transmission electron microscopy to confirm the location of the target vesicles.

[0035] (7) Freeze-drying: The target component is resuspended in deionized water to a protein concentration of 1 mg / mL, pre-frozen (-80℃, 2h), and then freeze-dried for 48 hours at a cold trap temperature of -56℃ and a vacuum degree of 2 Pa to obtain the finished product.

[0036] Example 2: Optimization and Verification of Process Parameters

[0037] A three-factor, three-level Box-Behnken response surface design was used to investigate the effects of enzymatic hydrolysis temperature (A: 50-60℃), enzymatic hydrolysis time (B: 1-3h), and the ratio of complex enzymes (C: mass ratio of cellulase:β-glucanase:papain = 1:1:1 to 3:1:1) on vesicle yield. The factor level coding table is shown in Table 1.

[0038] Table 1. Coding table for factors affecting vesicle yield

[0039] factor level Encoded value actual value A -1 50℃ 50℃ 0 55℃ 55℃ +1 60℃ 60℃ B -1 1h 1h 0 2h 2h +1 3h 3h C -1 1:1:1 1:1:1 0 2:1:1 2:1:1 +1 3:1:1 3:1:1

[0040] Center point repetition count: 3 times;

[0041] Total number of experiments: 17;

[0042] Data processing:

[0043] Quadratic polynomial regression analysis was performed using Design-Expert 13.0 software.

[0044] Establish the equation: Y = 12.3 + 0.83A + 0.57B + 0.45C - 0.3AB + 0.2AC - 0.1BC - 1.2A² - 0.9B² - ​​0.7C²;

[0045] Analysis of variance showed that the model had a P-value < 0.0001 and a lack-of-fit P-value = 0.12 > 0.05, indicating a good model fit.

[0046] Optimal condition verification:

[0047] Predicted optimal conditions: A=55.2℃, B=1.8h, C=2.1:1:1;

[0048] Actual verification experiment: Repeated 3 times at 55℃, 2h, and a 2:1:1 ratio;

[0049] Results: The vesicle yield was 12.7 ± 0.8 mg / g, which deviated from the predicted value by 2.3%. Subsequent experiments were all conducted using the black truffle cell vesicles prepared in Example 2.

[0050] Example 3: Characterization of the physicochemical properties of vesicles

[0051] Morphological analysis:

[0052] Transmission electron microscopy (TEM): 80 kV, negative staining method;

[0053] Atomic force microscopy (AFM): tapping mode, surface roughness Ra = 2.3 nm

[0054] Particle size and potential:

[0055] Dynamic light scattering (DLS): Zeta potential -32.4±1.7mV, PDI=0.182, polydispersity index <0.2;

[0056] The electron micrographs of the black truffle vesicles are as follows: Figure 2 As shown; particle size and concentration results are as follows. Figure 3 As shown.

[0057] Nanoflow cytometry detection: vesicle positivity rate >95%, fragmentation rate <3%;

[0058] Composition analysis:

[0059] Western blot: CD63 (1:500 dilution) and TSG101 (1:1000 dilution) showed positive bands;

[0060] High performance liquid chromatography (HPLC): phosphatidylcholine accounted for 68.7%, and sphingomyelin accounted for 21.4%.

[0061] Example 4: Application of drug carriers (delivery of anti-inflammatory components)

[0062] Drug loading process: Vesicles and curcumin were mixed at a mass ratio of 20:1, and pH 7.4 phosphate buffer was added and stirred at 4℃ for 2 hours. The free drug was removed by centrifugation at 12000×g for 10 minutes.

[0063] In vitro evaluation:

[0064] Cell model: RAW264.7 macrophages (ATCC TIB-71)

[0065] Treatment conditions: After stimulation with LPS (1 μg / mL) for 24 h, drug-loaded vesicles (final concentration 50 μg / mL) and free curcumin group (2.5 μg / mL) were added, compared with LPS model group;

[0066] Detection indicators: IL-6 was measured by ELISA (sensitivity <3 pg / mL), and TNF-α mRNA expression was detected by qRT-PCR;

[0067] In vivo pharmacokinetics:

[0068] Animal model: Male BALB / c mice (20±2g);

[0069] Dosage regimen: Inject drug-loaded vesicles via tail vein (20 mg / kg curcumin equivalent).

[0070] Blood collection time points: 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours;

[0071] Analytical method: HPLC-MS / MS (LOQ=0.5ng / mL);

[0072] result:

[0073] In vitro anti-inflammatory activity and IL-6 inhibition rate: The IL-6 secretion level in the drug-loaded vesicle group (50 μg / mL) was 87.3±6.2 pg / mL, which was 30.0% lower than that in the free curcumin group (124.5±8.7 pg / mL) (P<0.05) and 63.1% lower than that in the LPS model group (236.4±15.3 pg / mL) (P<0.001). TNF-α mRNA expression: The relative expression level of TNF-α mRNA in the drug-loaded vesicle group was 0.32±0.04, which was 68.0% lower than that in the model group (1.00±0.15) (P<0.01).

[0074] In vivo pharmacokinetics, AUC0-24h: the drug-loaded vesicle group was 234.6±18.7 μg·h / mL, which was 3.2 times higher than that of the free curcumin group (73.2±5.4 μg·h / mL).

[0075] Half-life (t1 / 2): 18.7±1.2 h for the drug-loaded vesicle group, which was 2.3 times longer than that for the free curcumin group (5.6±0.8 h).

[0076] Example 5: Experimental Verification of Black Truffle Vesicles Promoting HaCat Cell Migration

[0077] Experimental materials

[0078] Cell line: human immortalized keratinocytes (HaCat, purchased from ATCC, catalog number CRL-2404);

[0079] DMEM high-glucose medium (Gibco, catalog number 11965092); fetal bovine serum (FBS, Gibco, catalog number 10099141); penicillin-streptomycin solution (Gibco, catalog number 15140122); EDTA-trypsin digestion solution (0.25%, Gibco, catalog number 25200056); phosphate-buffered saline (PBS, pH 7.4, homemade); 6-well cell culture plate (Corning, catalog number 3516); silicone rubber insert for scratch assay (Ibidi, catalog number 80209); Transwell chamber (Corning, catalog number 3422); CO2 incubator (Thermo, model 3111).

[0080] Experimental methods

[0081] 1. Cell Culture and Grouping

[0082] After resuscitating HaCat cells, they were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0083] The experiment was divided into two groups:

[0084] Control group: Only culture medium without vesicles was added (blank control);

[0085] Experimental group: Culture medium containing 50 μg / mL of black truffle vesicles prepared in Example 2;

[0086] 2. Scratch Healing Assay

[0087] Cell plating: Take cells in the logarithmic growth phase and plate them at a density of 5 × 10⁻⁶ cells / mL. 5 Cells / wells were seeded in 6-well plates and cultured until 90% confluence was achieved.

[0088] Scratch treatment: Use a sterile 200 μL pipette tip to make vertical scratches to create a cell-free area approximately 500 μm wide. Wash three times with PBS to remove detached cells.

[0089] Drug treatment: Add vesicle-containing culture medium (serum starvation conditions: 0.5% FBS) according to the above grouping.

[0090] Imaging and analysis: The scratched area was photographed using an inverted microscope (Nikon, Ti2) at 0h, 24h and 48h time points.

[0091] The formula for calculating the scratch area and migration rate using ImageJ software is as follows:

[0092]

[0093] 3. Transwell Assay

[0094] Upper chamber treatment: Add 200 μL of serum starvation medium containing different concentrations of vesicles to a Transwell chamber (8 μm pore size).

[0095] Lower chamber treatment: 600 μL of complete culture medium containing 10% FBS was added to the lower chamber as a chemokine.

[0096] Cell seeding: Take 2×10 4 HaCat cells / well were suspended in vesicle-containing culture medium and added to the upper chamber.

[0097] Culture and fixation: After incubation at 37℃ for 24 hours, fix with 4% paraformaldehyde for 15 minutes and stain with 0.1% crystal violet for 20 minutes.

[0098] Cell counting: Randomly select 5 fields of view (×200x) to count the number of cells that have penetrated the membrane and calculate the average value.

[0099] The results are as follows Figure 4 As shown, treatment with black truffle vesicles significantly promoted HaCat cell migration.

[0100] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a black truffle-derived plant cell vesicle in the preparation of a cosmetic composition, characterized in that, The method for preparing plant cell vesicles derived from black truffles includes the following steps: (1) Take fresh black spore truffle fruiting bodies, freeze dry at -50℃ for 48 hours, then use a planetary ball mill for ultra-fine grinding, and pass through a 200-mesh sieve for later use to obtain pre-treated raw materials; the conditions for ultra-fine grinding are a rotation speed of 300 rpm and a ball-to-material ratio of 10:

1. (2) The pretreated raw materials in step (1) are subjected to compound enzymatic hydrolysis to obtain the enzymatic hydrolysis product; (3) The enzymatic hydrolysis product in step (2) is broken up by ultrasonication to obtain the broken liquid; (4) The ruptured liquid in step (3) is purified by preliminary separation, ultrafiltration membrane separation and density gradient centrifugation to obtain the target vesicle; (5) Freeze-dry the target vesicles from step (4) to obtain the finished product; In step (2), the complex enzymatic hydrolysis uses a complex enzyme system, which consists of cellulase, β-glucanase and papain; the mass ratio of cellulase:β-glucanase:papain is in the range of 1:1:1 to 3:1:

1. The enzymatic hydrolysis temperature is 50-60℃, and the time is 1-3 hours; In step (4), the ultrafiltration membrane has a molecular weight cutoff of 10-50 kDa, and the density gradient range of density gradient centrifugation is 1.063-1.21 g / mL.

2. The application according to claim 1, characterized in that, The zeta potential of the plant cell vesicles is -20 to -40 mV.

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