Preparation method of purple flower rhizoma bletillae extracellular vesicles

Abstract

The preparation method comprises the following steps: washing fresh rhizoma bletillae, cutting off rhizomes, peeling and slicing tubers to obtain rhizoma bletillae slices, adding a PBS (Phosphate Buffer Solution), juicing, sieving the obtained solution with sterile gauze, carrying out differential centrifugation on the filtrate to obtain supernate, filtering the supernate with a microfiltration membrane, and carrying out freeze drying to obtain the extracellular vesicles of the purple flower rhizoma bletillae extracellular vesicles of the purple flower rhizoma bletillae extracellular vesicles of the purple flower rhizoma bletillae extracellular vesicles of the purple flower rhizoma bletillae. The method comprises the following steps: adding a PBS buffer solution into a rhizoma bletillae water extract rich in extracellular vesicles to obtain the rhizoma bletillae water extract rich in extracellular vesicles, carrying out ultra-high-speed centrifugation on the rhizoma bletillae water extract rich in extracellular vesicles to obtain a supernatant and a precipitate which is a rhizoma bletillae extracellular vesicle crude product, adding the PBS buffer solution into the rhizoma bletillae extracellular vesicle crude product, uniformly mixing, carrying out ultra-high-speed centrifugation, and discarding the supernatant to obtain the purple-flower rhizoma bletillae extracellular vesicles. The method is simple and easy to operate and low in production cost, the prepared rhizoma bletillae extracellular vesicles are high in purity and concentration, the intermediate product rhizoma bletillae water extract can be utilized, the resource utilization degree is high, and important reference value is provided for production of rhizoma bletillae and other traditional Chinese medicine plant extracellular vesicles.

Description

Technical Field

[0001] The invention relates to a method for preparing extracellular vesicles of Bletilla striata, and belongs to the technical field of medicine. Background Art

[0002] Bletilla striata, also known as Lianjicao (from Ben Jing), Ruolan, Zhulan (from Hua Jing), Zilan, Zihui, Baili. It is a traditional Chinese medicine in my country and has been used for thousands of years. As a precious traditional Chinese medicine, it is mainly produced in Anhui, Yunnan, Guizhou and other places. It contains a variety of effective chemical components and has the effects of astringent hemostasis, clearing heat and dampness, and reducing swelling and promoting tissue regeneration. Bletilla has a long history of use and high medicinal value. The earliest existing pharmaceutical monograph "Shennong Bencao Jing" has included Bletilla, which first introduced the medicinal effects and growth environment of Bletilla. Later herbal materials were mostly recorded as "Baiji" and are still used today. Bletilla striata has three effects in treating external injuries, internal injuries and whitening. It not only has the effects of astringent hemostasis, swelling and tissue regeneration, and promotes wound healing, but also can whiten and remove freckles, treat facial sores, smooth skin, remove lines and wrinkles. It is mainly used to treat tuberculosis and hemoptysis, gastrointestinal bleeding, traumatic bleeding, sores, swelling and poison, skin cracks, etc. It is also the main ingredient in traditional whitening prescriptions. For example, "Southern Yunnan Materia Medica" records that Bletilla striata "treats tuberculosis and damages lung qi, replenishes lung deficiency, stops coughing, eliminates tuberculosis and hemoptysis, and astringes lung qi." Zhang Zhongjing's "Treatise on Febrile Diseases" in the Han Dynasty describes that the Bletilla striata glue contained in Bletilla striata can firmly adhere to the surface of the digestive tract mucosa to form a protective film, and effectively protect the ulcer surface and promote its repair and healing. "Yao Xing Lun" states: "Treats facial sores and makes the skin smooth." "Compendium of Materia Medica" says: "Washes the face and removes freckles." Modern pharmacological studies have shown that Bletilla striata has antibacterial, antiviral, anti-tumor, antioxidant, hemostatic and wound healing effects.

[0003] At present, the utilization of fresh Bletilla striata tubers is relatively simple. Bletilla striata extracellular vesicles are vesicles derived from Bletilla striata, which have potential antibacterial, anti-inflammatory and anti-tumor effects. Therefore, the preparation of Bletilla striata extracellular vesicles can be used to broaden the utilization of fresh Bletilla striata. In addition, the extracellular vesicles of Bletilla striata also have the potential for nano-drug delivery. However, there has been no report on the preparation of Bletilla striata extracellular vesicles so far. Summary of the invention

[0004] The purpose of the present invention is to overcome the barrier of insufficient utilization of Bletilla striata in the prior art and provide a method for preparing extracellular vesicles of Bletilla striata with purple flower. The method has a simple process and the Bletilla striata aqueous extract produced as a by-product in the preparation process can also be effectively utilized without the problem of waste of resources.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing extracellular vesicles of Bletilla striata, comprising the following steps:

[0007] (1) Wash fresh Bletilla striata, remove the rhizome, peel the tuber and slice it to obtain Bletilla striata slices;

[0008] (2) adding PBS buffer to the Bletilla striata slices, and then squeezing the juice to obtain a mixed solution;

[0009] (3) sieving the mixed solution with sterile gauze, and then performing differential centrifugation on the obtained filtrate to obtain a supernatant, and then filtering the supernatant with a microporous filter membrane to obtain an aqueous extract of Bletilla striata rich in extracellular vesicles;

[0010] (4) subjecting the water extract of Bletilla striata rich in extracellular vesicles to ultrahigh-speed centrifugation to obtain a supernatant and a precipitate, wherein the supernatant is the water extract of Bletilla striata and the precipitate is a crude product of extracellular vesicles of Bletilla striata;

[0011] (5) Add PBS buffer to the crude Bletilla striata extracellular vesicles, mix well, perform ultrahigh-speed centrifugation, discard the supernatant, and obtain purified Bletilla striata extracellular vesicles.

[0012] Furthermore, in step (2) and step (5), the pH of the PBS buffer is 7.2.

[0013] Furthermore, in step (2), the mass ratio of the Bletilla striata slices to the PBS buffer solution is 1:4.

[0014] Furthermore, in step (3), the sieving is through a 100-mesh sieve.

[0015] Furthermore, in step (3), the differential centrifugation method is: first centrifuge at a centrifugal force of 400g for 20 minutes, take the supernatant, then centrifuge at a centrifugal force of 800g for 20 minutes, take the supernatant, and finally centrifuge at a centrifugal force of 15000g for 20 minutes, and the temperature of the differential centrifugation is 4°C.

[0016] Furthermore, in step (3), the pore size of the microporous filter membrane is 0.22 μm.

[0017] Furthermore, in steps (4) and (5), the ultra-high speed centrifugation conditions are: centrifugal force of 150,000 g, centrifugation time of 2 h, and ultra-high speed centrifugation temperature of 4°C.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a method for preparing extracellular vesicles of Bletilla striata, which has a simple process. The prepared extracellular vesicles of Bletilla striata have potential antibacterial, anti-inflammatory and anti-tumor effects, and also have the potential for nano drug delivery. The extracellular vesicles of Bletilla striata itself have the advantages of low immunogenicity, no toxic side effects, and sustainable sources. The water extract of Bletilla striata produced as a byproduct in the preparation process of the present invention also contains multiple active ingredients and has medicinal potential. Therefore, the method of the present invention does not have the problem of waste of resources, has high resource utilization, provides important reference value for the production of extracellular vesicles of Bletilla striata and other traditional Chinese medicinal plants, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an electron microscopic image of the extracellular vesicles of Bletilla striata prepared in Example 1;

[0021] Figure 2 This is a particle size diagram of the extracellular vesicles of Bletilla striata prepared in Example 1 after being diluted 2000 times with ultrapure water;

[0022] Figure 3 This is the zeta potential diagram of the extracellular vesicles of Bletilla striata prepared in Example 1;

[0023] Figure 4 VIP graph of differential metabolites between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0024] Figure 5 KEGG map of differential metabolites between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0025] Figure 6 The differential lipid classification statistics of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0026] Figure 7 Cluster heat map and VIP bar chart of differential lipids of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0027] Figure 8 KEGG pathway enrichment diagram of differential lipids between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0028] Fig. 9 Length distribution diagram of cleanreads of miRNA libraries of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0029] Fig.10The known_miRNA family classification statistics of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0030] Fig.11 A volcano plot showing the difference in miRNA between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0031] Fig.12 Heat map showing the difference in miRNA between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2;

[0032] Fig.13 The chemical classification of metabolites of the by-product Bletilla striata aqueous extract in Example 1;

[0033] Fig.14 The negative ion chromatogram of the base peak of the component of the water extract of Bletilla striata produced as a byproduct in Example 1;

[0034] Fig.15 The base peak positive ion chromatogram of the component of the by-product Bletilla striata water extract in Example 1;

[0035] Fig.16 The Venn diagram of the difference analysis between the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1;

[0036] Fig.17 This is a principal component analysis diagram of the difference analysis between the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1;

[0037] Fig.18 The volcano plot of the difference analysis between the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1;

[0038] Fig.19 VIP diagram of differential metabolic analysis of by-product Bletilla striata water extract FBSC and fresh Bletilla striata tuber FBST in Example 1;

[0039] Fig. 20 The KEGG diagram of differential metabolic analysis of by-product Bletilla striata water extract FBSC and fresh Bletilla striata tuber FBST in Example 1;

[0040] Fig.21 This is the abundance score diagram of the differential metabolic analysis of the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is clearly and completely described below in conjunction with the accompanying drawings and specific embodiments.

[0042] Example 1

[0043] A method for preparing extracellular vesicles of Bletilla striata, comprising the following steps:

[0044] (1) Select two-year-old fresh tubers of Bletilla striata, wash them, remove the rhizomes, peel and slice the tubers, and obtain 100 g of Bletilla striata slices;

[0045] (2) adding 400 mL of PBS buffer with a pH of 7.2 to the Bletilla striata slices, and then using a juicer to extract juice to obtain a mixed solution;

[0046] (3) The mixed solution was passed through a 100-mesh sieve with sterile gauze, and the obtained filtrate was subjected to differential centrifugation, specifically: first centrifuged at a centrifugal force of 400 g for 20 min, the supernatant was taken, then centrifuged at a centrifugal force of 800 g for 20 min, the supernatant was taken, and finally centrifuged at a centrifugal force of 15000 g for 20 min, the temperature of the differential centrifugation was 4° C.; the obtained supernatant was filtered using a microporous filter membrane with a pore size of 0.22 μm to obtain a water extract of Bletilla striata rich in extracellular vesicles;

[0047] (4) subjecting the water extract of Bletilla striata rich in extracellular vesicles to ultrahigh-speed centrifugation (150,000 g, 2 h, 4° C.) to obtain a supernatant and a precipitate, wherein the supernatant is the water extract of Bletilla striata and the precipitate is the crude product of extracellular vesicles of Bletilla striata;

[0048] (5) Add 20 mL of PBS buffer (pH 7.2) to the crude Bletilla striata extracellular vesicles, mix well, and perform ultrahigh-speed centrifugation (150,000 g, 2 h, 4°C). Discard the supernatant and resuspend the precipitate with 200 μL of PBS buffer (pH 7.2) to obtain purified Bletilla striata extracellular vesicles, which are recorded as WS-BsEVs.

[0049] The concentration of the extracellular vesicles of Bletilla striata obtained in Example 1 was tested by NTA (nanoparticle tracking instrument), and protein quantification was performed by Biyuntian BCA kit (protein quantification kit), and the measured concentration was 3.6×10 11 particles / mL, and the protein content was 2.84 mg / mL.

[0050] Example 2

[0051] A method for preparing extracellular vesicles of Bletilla striata, comprising the following steps:

[0052] (1) Select two-year-old fresh tubers of Leshan Bletilla striata, wash them, remove the rhizomes, peel and slice the tubers, and obtain 100 g of Bletilla striata slices;

[0053] (2) adding 400 mL of PBS buffer with a pH of 7.2 to the Bletilla striata slices, and then using a juicer to extract juice to obtain a mixed solution;

[0054] (3) The mixed solution was passed through a 100-mesh sieve with sterile gauze, and the obtained filtrate was subjected to differential centrifugation, specifically: first centrifuged at a centrifugal force of 400 g for 20 min, the supernatant was taken, then centrifuged at a centrifugal force of 800 g for 20 min, the supernatant was taken, and finally centrifuged at a centrifugal force of 15000 g for 20 min, the temperature of the differential centrifugation was 4° C.; the obtained supernatant was filtered using a microporous filter membrane with a pore size of 0.22 μm to obtain a water extract of Bletilla striata rich in extracellular vesicles;

[0055] (4) subjecting the water extract of Bletilla striata rich in extracellular vesicles to ultrahigh-speed centrifugation (150,000 g, 2 h, 4° C.) to obtain a supernatant and a precipitate, wherein the supernatant is the water extract of Bletilla striata and the precipitate is the crude product of extracellular vesicles of Bletilla striata;

[0056] (5) Add 20 mL of PBS buffer with a pH of 7.2 to the crude extracellular vesicles of Bletilla striata, mix well, and perform ultrahigh-speed centrifugation (150,000 g, 2 h, 4°C). Discard the supernatant and resuspend the precipitate with 200 μL of PBS buffer with a pH of 7.2 to obtain the purified extracellular vesicles of Bletilla striata, which are recorded as LS-BsEVs.

[0057] The concentration of the extracellular vesicles of Leshan Bletilla striata prepared in Example 2 was tested by NTA (nanoparticle tracking instrument), and protein quantification was performed by Biyuntian BCA kit (protein quantification kit), and the measured concentration was 2.4×10 11 particles / mL, and the protein content was determined to be 2.81 mg / mL.

[0058] Figure 1 This is an electron micrograph of the extracellular vesicles of Bletilla striata prepared in Example 1. It can be seen that the extracellular vesicles of Bletilla striata contain a double lipid membrane with a tea tray-like structure in the middle.

[0059] Figure 2 The particle size diagram of the extracellular vesicles of Bletilla striata prepared in Example 1 after being diluted 2000 times with ultrapure water. It can be seen that the diagram is a single peak, with few impurities and pure. The original concentration in the diagram is 10 11 The concentration of extracellular vesicles of Bletilla striata reached the standard.

[0060] Figure 3 The zeta potential diagram of the extracellular vesicles of Bletilla striata prepared in Example 1 is used to describe the interaction and dispersion state between particles and evaluate the stability and surface charge of the particle system. The zeta potential was measured to be -14 mV under alkaline conditions of pH = 8 to simulate the small intestine environment. The dispersion system of BsEVs tends to be stable and can be dispersed in the solution for uniform absorption.

[0061] 1. The extracellular vesicles of Bletilla striata (WS-BsEVs) prepared in Example 1 and the extracellular vesicles of Bletilla striata (LS-BsEVs) prepared in Example 2 were subjected to metabolomics and lipidomics identification of BsEVs. The specific method is as follows: Prepare extracellular vesicles of Bletilla striata (WS-BsEVs) and extracellular vesicles of Bletilla striata (LS-BsEVs), accurately pipette 200 μL of sample into an EP tube, add 80 μL of methanol and 400 μL of MTBE, vortex mix for 30 seconds, and then ultrasonically extract for 30 minutes (5°C, 40KHz). The sample was placed at -20°C, and after 30 minutes, high-speed centrifugation (13000g, 4°C) was performed for 15 minutes. 350 μL of supernatant was transferred, dried in a vacuum concentrator, and redissolved in 100 μL of re-solution (isopropanol: acetonitrile = 1:1). After vortex mixing for 30 seconds, ultrasonicate at 40KHz in an ice water bath for 5 minutes. After high-speed centrifugation (13000g, 4℃) for 5min, 80μL of supernatant was transferred to an injection vial with an inner tube for analysis. The samples were analyzed by LC-MS / MS on the Thermo Fisher Scientific ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system. Chromatographic conditions: 2μL of sample was separated by Accucore C30 column (100mm×2.1mm id, 2.6μm; Thermo) and then entered the mass spectrometry detection. Mobile phase A was 10mM ammonium acetate 50% acetonitrile aqueous solution (containing 0.1% formic acid), and mobile phase B was 2mM ammonium acetate acetonitrile / isopropanol / water (10 / 88 / 2) (containing 0.02% formic acid). Separation gradient: 0-4min, mobile phase B increased from 35% to 60%; 4-12min, mobile phase B increased from 60% to 85%; 12-15min, mobile phase B increased from 85% to 100%; 15-17min, mobile phase B maintained at 100%; 17-18min, mobile phase B decreased from 100% to 35%; 18-20min, mobile phase B maintained at 35%. The flow rate was 0.40mL / min, and the column temperature was 40℃. Mass spectrometry conditions: The sample mass spectrometry signal was collected in positive and negative ion scanning mode, with a mass scanning range of m / z: 200-2000. The positive mode ion spray voltage was 3000V, the negative mode ion spray voltage was -3000V, the sheath gas was 60psi, the auxiliary heating gas was 20psi, the ion source heating temperature was 370℃, the 20-40-60V cycle collision energy, and the data was collected in DDA mode.

[0062] Figure 4VIP diagram of differential metabolites of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2, in which the horizontal axis represents the importance of the metabolite. In this non-targeted metabolomics study, the top 15 differential metabolites were selected from all 403 differential metabolites of WS and LS with VIP ≥ 1 and P ≤ 0.05 for analysis. It was found that the proportion of glycoside metabolites was the largest, and the main effects of glycoside metabolites were anti-inflammatory and antioxidant, while the remaining metabolites had unique antimicrobial and anti-tumor effects.

[0063] All differential metabolites with P_value≤0.05 were selected and annotated with metabolic pathways using the KEGG database (https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways in which the differential metabolites participated. Figure 5 KEGG diagram of differential metabolites of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2. Each bubble in the figure represents a metabolic pathway, and its abscissa and bubble size together indicate the size of the influencing factor of the pathway. The larger the bubble size, the greater the influencing factor. The bubble color represents the p-value of the enrichment analysis, and the redder the color, the higher the degree of enrichment. The analysis found that there are four anti-inflammatory and antioxidant pathways, namely, the biosynthesis of isoquinoline alkaloids, the biosynthesis of isoflavones, the biosynthesis of stilbenes, diarylheptane compounds and gingerol, and the biosynthesis of phenylpropanoids.

[0064] LIPID MAPS was used to perform differential lipid classification statistics. The results are shown in Figure 6 In the figure, the horizontal axis is the number of lipids identified in each subclass, and the vertical axis is the name of the subclass. Different colors are used to mark the name of the major category to which each column belongs, fatty acyl (FA), glyceride (GL), glycerophospholipid (GP), sphingolipid (SP), and sterol lipid (ST). Hex1Cer (monosaccharide sphingosine) and Cer (sphingosine) have the highest content in the SP subclass, which are involved in the formation of cell membranes and cell signal transduction.

[0065] Figure 7The cluster heat map and VIP bar graph of differential lipids of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2 show that there are 6 replicates in each of the WS group and the LS group. The expression patterns in each sample and the P values ​​of metabolites in the VIP and unidimensional statistics of multivariate statistical analysis can intuitively show the importance of differential metabolites and the trend changes in expression levels. The cluster heat map shows that the overall lipid level of the LS group is higher than that of the WS group, and the DG (diglyceride) of WS is significantly higher than that of LS. DG plays a role in cell membranes and energy storage. The VIP diagram shows the contribution of lipid subclass metabolites to the differences between the two groups. The most significant metabolite is dMePE (dimethylphosphatidylethanolamine), which affects the fluidity and stability of the membrane, facilitating the free entry and exit of BsEVs into and out of cells.

[0066] Figure 8 The KEGG pathway enrichment map of the differential lipids of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2 shows that the differential metabolites of WS and LS are enriched in five pathways, namely glycerophospholipid metabolism, glycerophospholipid metabolism, linoleic acid metabolism, glycerolipid metabolism, and α-linolenic acid metabolism. Among them, the most significantly enriched pathway is glycerophospholipid metabolism, which is an important biochemical process in cells, involving the synthesis, decomposition and conversion of glycerophospholipids. Glycerophospholipids are the main components of cell membranes and are involved in cell signal transduction, energy storage and membrane structural stability.

[0067] 2. Non-coding miRNA sequencing was performed on WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2. The specific method is as follows:

[0068] 1) RNA extraction

[0069] Total RNA was extracted from tissues using TRIzol reagent according to the manufacturer's instructions. RNA quality was then determined by 5300 Bioanalyzer (Agilent) and quantified using ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD 260 / 280 = 1.8-2.2, OD 260 / 230 ≥ 2.0, RIN ≥ 6.5, 28S: 18S ≥ 1.0, > 1 μg) were used to construct sequencing libraries.

[0070] 2) Library preparation and BsEVs miRNA sequencing

[0071] The total RNA amount of each sample was 1 μg, which was used as the input material for the small RNA library. The small RNA library was constructed using the QIAseq miRNA Library Kit (Qiagen) (Illumina, San Diego, CA). The single-end RNA-seq sequencing library was sequenced using an Illumina NovaSeqXplus sequencer (Illumina, San Diego, CA, USA).

[0072] The raw data in fastq format were first processed by fastq using default parameters. After this step, clean data (clean reads) were obtained by removing the 3' end adapter, the reads of low-quality bases at the 3' end containing poly-N and the sequencing adapter from the raw data using the fastx toolkit software. All identical sequences in the range of 18 to 32 nt were counted and eliminated from the initial data set.

[0073] 3) Alignment of known miRNAs and prediction of new miRNAs

[0074] The filtered reads were used as references to identify known miRNAs (known-miRNA) using the miRBase database and NCBI blast. The remaining tags were then compared with the Rfam database and the Repbase database to remove ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), other non-coding RNA (ncRNA) and repetitive sequences. Unannotated tags were predicted and identified as novel miRNAs (novel-miRNA) based on the location of the tag in the genome and its hairpin structure using the miRDeep2 software. The expression level of each miRNA was calculated using the transcript reads per million (TPM) method.

[0075] Small RNA sequencing was performed on both WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2. Six miRNA libraries were prepared (two origins, three biological replicates each), and filtered reads with a length of 18–30 nt were mapped. Fig. 9 The length distribution diagram of the clean reads of the miRNA library of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2 shows that in both BsEVs, the most abundant miRNA lengths are distributed in the range of 20-21 lengths (nucleotides, nt).

[0076] Fig.10The known_miRNA family classification statistics of WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2. As shown in the figure, the Bst-ev-miR166 family and the Bst-ev-miR319 family account for the largest proportion of the known-miRNAs in BsEVs.

[0077] Fig.11 The volcano map of the miRNA differences between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2, after preliminary screening, the miRNAs with a read count greater than 10 in any sample among the 2079 miRNAs were retained, and 663 miRNAs were obtained. After the miRNAs were uniformly named, the expression values ​​of the repeated miRNAs were averaged, and 359 miRNAs were finally retained. The volcano map was made by differential analysis to show that there were 178 differentially expressed miRNAs in the miRNAs of BSEVs, including 109 up-regulated and 69 down-regulated miRNAs. Taking the miRNA of LS_BsEVs as the control group, it can be seen that Bst-ev-novel-35 and Bst-ev-novel-8 in the miRNAs are significantly highly expressed in the extracellular vesicles of Bletilla striata, while Bst-ev-novel-61 and Bst-ev-novel-28 are significantly under-expressed.

[0078] Fig.12 The heat map shows the difference in miRNA between WS-BsEVs prepared in Example 1 and LS-BsEVs prepared in Example 2. The columns of the heat map represent BsEVs from two different origins, and the rows represent the types of miRNAs. The colors from blue to red represent the changes in the expression levels of miRNAs from two different origins, where blue represents low expression and red represents high expression. As shown in the heat map, the upregulated miRNAs in WS-BsEVs are BSEVs-specific miRNAs and Bst_ev_miR6300 and Bst_ev__157c. Most of the upregulated miRNAs in WS-BsEVs are from the miR-166 family known to BSEVs, and the rest are BsEVs-specific miRNAs.

[0079] 3. The non-target metabolic identification of the water extract of Bletilla striata produced as a byproduct in the process of preparing WS-BsEVs in Example 1 was performed as follows:

[0080] The differential metabolism of the water extract of Bletilla striata (FBSC) produced as a byproduct during the extraction process of WS-BsEVs was compared with the fresh tuber of Bletilla striata from Wenshan without any treatment (FBST) to explore the pharmacological effects of the water extract of Bletilla striata. 100 μL of the water extract of Bletilla striata was added to 800 μL of a solution (acetonitrile: methanol = 1: 1 (v: v)) containing an internal standard (0.02 mg / mL L-2-chlorophenylalanine) in a 1.5 mL centrifuge tube, the sample was mixed by vortexing for 30 seconds, and low-temperature ultrasonic treatment was performed for 30 minutes (5°C, 40 KHz). The sample was placed at -20°C for 30 minutes to precipitate the protein. The sample was then centrifuged for 15 minutes (4°C, 13000 g). The supernatant was removed and blown dry under nitrogen. The sample was then re-dissolved with 100 μL of a solution (acetonitrile: water = 1: 1) and extracted by low-temperature ultrasonic treatment for 5 minutes (5°C, 40 KHz), followed by centrifugation at 13000 g and 4°C for 10 minutes. The supernatant was transferred to a sample vial for LC-MS / MS analysis. The analysis was performed on an ACQUITY BEH C18 column (100 mm × 2.1 mm id, 1.7 μm; Watts, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Aqueous formic acid: acetonitrile (2:98, v / v) (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The flow rate was 0.40 mL / min and the column temperature was 40°C. The injection volume was 3 μL. The analysis was performed on an ACQUITY BEH Amide column (100 mm × 2.1 mm id, 1.7 μm; Watts, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The flow rate was 0.40 mL / min and the column temperature was 40°C. The injection volume was 3 μL. MS conditions: The ultra-high performance liquid chromatography system was coupled to a Thermo UHPLC-QExactive mass spectrometer equipped with an electrospray ion source (ESI). The optimal conditions were: source temperature 400°C, sheath gas flow 40arb, auxiliary gas flow 10arb, column temperature 100°C. The ion spray voltage floating (ISVF) was -2800V (negative mode) and 3500V (positive mode), respectively; normalized collision energy, 20-40-60V rolling was used for MS / MS. The full MS resolution was 70000 and the MS / MS resolution was 17500. Data acquisition was performed in data-dependent acquisition (DDA) mode. Detection was performed in the mass range of 70-1050 m / z.

[0081] Fig.13The metabolites of the by-product Bletilla striata water extract in Example 1 were chemically classified. The results showed that terpenes (terpenoid compounds have anti-tumor, anti-inflammatory, antibacterial, antiviral, antimalarial effects, promote transdermal absorption, prevent and treat cardiovascular diseases, and have blood sugar lowering effects) accounted for 28.27% of the Bletilla striata water extract, followed by flavonoids (flavonoid compounds have antioxidant, free radical scavenging, anti-tumor, anti-cancer, antibacterial, antiviral, anti-inflammatory immunity, and anti-aging effects), accounting for 17.02%. Both have anti-tumor, anti-inflammatory and antibacterial effects.

[0082] Fig.14 The negative ion chromatogram of the base peak of the water extract of Bletilla striata produced as a byproduct in Example 1, in which the peak number is Fig.13 The compounds shown in . Fig.15 The base peak positive ion chromatogram of the component of the by-product Bletilla striata water extract in Example 1, in which the peak number is Fig.13 The compounds shown in .

[0083] Fig.16 The Venn diagram of the difference analysis between the by-product water extract of Bletilla striata FBSC and the fresh tuber of Bletilla striata FBST in Example 1 is used to reveal the common and unique metabolites between the fresh Bletilla striata extract FBSC and the fresh tuber of Bletilla striata FBST. Fig.16 It can be seen that 1572 identical metabolites were obtained. According to the Venn diagram analysis, most of the metabolites were the same, with only a few being different.

[0084] Fig.17 The principal component analysis diagram of the difference analysis between the by-product water extract of Bletilla striata FBSC and the fresh tuber of Bletilla striata FBST in Example 1 shows the biological variation between FBSC and FBST samples by performing PCA analysis on the samples. The overall metabolite difference and the degree of variation between different groups of samples can be determined. As shown in the figure, the grouped ellipses of FBSC and FBST metabolites are separated, indicating that there are obvious differences between FBSC and FBST metabolites. The total explanation rate is 88.6% (PC1+PC2).

[0085] Fig.18 The volcano plot of the differential analysis of the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1 shows the number of metabolites with differential changes. In this non-targeted metabolomics study, all 703 differential metabolites of FBSC and FBST with VIP ≥ 1 and P ≤ 0.05 were selected for differential metabolite volcano plot analysis, and it was found that 481 were up-regulated and 222 were down-regulated.

[0086] Fig.19This is the VIP diagram of the differential metabolic analysis of the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1. The horizontal axis represents the importance of the metabolite. In the non-targeted metabolomics study, the top 30 differential metabolites were selected from all 703 differential metabolites of FBSC and FBST with VIP ≥ 1 and P ≤ 0.05 for analysis. It was found that most of them were antibacterial metabolites, and the remaining metabolites had antioxidant and anti-inflammatory effects.

[0087] Fig. 20 KEGG diagram of differential metabolic analysis of by-product Bletilla striata water extract FBSC and fresh Bletilla striata tuber FBST in Example 1, each bubble in the figure represents a metabolic pathway, and its abscissa and bubble size together indicate the size of the influencing factor of the pathway. The larger the bubble size, the larger the influencing factor. The bubble color represents the p-value of the enrichment analysis, and the redder the color, the higher the enrichment degree, the smaller the p-value of the differential metabolic pathway, and the more significant the pathway enrichment. Flavone and flavonolbiosynthesis (biosynthesis of flavonoids and flavonols), Isoflavonoid biosynthesis (isoflavonoids biosynthesis), Linoleic acid metabolism (linoleic acid metabolism), and Arachidonic acid metabolism (arachidonic acid) metabolic synthesis pathways are four pathways corresponding to the differential metabolites of the FBSC group and the FBST group, including 4, 8, 11 and 19 metabolites, respectively.

[0088] Fig.21 The abundance score diagram of the differential metabolic analysis of the by-product Bletilla striata water extract FBSC and the fresh Bletilla striata tuber FBST in Example 1, the difference in the score can reflect the overall changes in all pathways. A DA score of 1 indicates that the expression of all identified pathways is on an upward trend, and a DA score of -1 indicates a downward trend. The absolute value of the DA score is represented by the length and size of the line. The abundance difference (DA) of the metabolic pathways between different control groups corresponds to the number of metabolites expressed differently in the pathway. The points are distributed on the left side of the axis, and the longer line indicates the trend of downregulation of general pathway expression. If the points are distributed on the right side of the axis, the longer line indicates that the expression of the entire pathway tends to increase. The more metabolites there are, the more obvious these points are. The size of the p value is shown by the color of the line segment and the point. The more it tends to red, the smaller the p value, and the more it tends to purple, the larger the p value. In the present invention, compared with FBST, the four identified pathways in FBSC, including the biosynthesis of flavonoids and flavonols, isoflavone biology, linoleic acid metabolism, and arachidonic acid, are all upregulated.

Claims

1. A method for preparing extracellular vesicles of Bletilla striata, characterized in that: The steps include: (1) Wash fresh Bletilla striata, remove the rhizome, peel the tuber and slice it to obtain Bletilla striata slices; (2) adding PBS buffer to the Bletilla striata slices, and then squeezing the juice to obtain a mixed solution; (3) sieving the mixed solution with sterile gauze, and then performing differential centrifugation on the obtained filtrate to obtain a supernatant, and then filtering the supernatant with a microporous filter membrane to obtain an aqueous extract of Bletilla striata rich in extracellular vesicles; (4) subjecting the water extract of Bletilla striata rich in extracellular vesicles to ultrahigh-speed centrifugation to obtain a supernatant and a precipitate, wherein the supernatant is the water extract of Bletilla striata and the precipitate is a crude product of extracellular vesicles of Bletilla striata; (5) Add PBS buffer to the crude Bletilla striata extracellular vesicles, mix well, perform ultrahigh-speed centrifugation, discard the supernatant, and obtain purified Bletilla striata extracellular vesicles.

2. The method for preparing the extracellular vesicles of Bletilla striata as claimed in claim 1, characterized in that: In step (2) and step (5), the pH of the PBS buffer is 7.

2.

3. The method for preparing the extracellular vesicles of Bletilla striata as claimed in claim 1, characterized in that: In step (2), the mass ratio of the Bletilla striata slices to the PBS buffer solution is 1:

4.

4. The method for preparing the extracellular vesicles of Bletilla striata as claimed in claim 1, characterized in that: In step (3), the sieving is through a 100-mesh sieve.

5. The method for preparing the extracellular vesicles of Bletilla striata as claimed in claim 1, characterized in that: In step (3), the differential centrifugation method is: first centrifuge at a centrifugal force of 400g for 20 minutes, take the supernatant, then centrifuge at a centrifugal force of 800g for 20 minutes, take the supernatant, and finally centrifuge at a centrifugal force of 15000g for 20 minutes. The temperature of the differential centrifugation is 4°C.

6. The method for preparing the extracellular vesicles of Bletilla striata as claimed in claim 1, characterized in that: In step (3), the pore size of the microporous filter membrane is 0.22 μm.

7. The method for preparing the extracellular vesicles of Bletilla striata as claimed in any one of claims 1 to 6, characterized in that: In steps (4) and (5), the ultra-high speed centrifugation conditions are: centrifugal force of 150000g, centrifugation time of 2h, and ultra-high speed centrifugation temperature of 4°C.

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