CN117264871A - Artemisia annua outer vesicle extract and application thereof - Google Patents

Abstract

The invention belongs to the technical field of extraction of plant-derived outer vesicles, and particularly discloses an artemisia annua outer vesicle extract and application thereof, wherein the artemisia annua outer vesicle extract is an extract extracted from natural plants such as artemisia annua and containing natural spherical outer vesicle-like nanoparticles. The application of the artemisia annua exovesicle in medicines for regulating immune microenvironment is characterized in that the artemisia annua exovesicle is used as an immunomodulator and is co-cultured with macrophages to polarize macrophages in vitro and in vivo from an M2-like phenotype to an M1 phenotype.

Description

A kind of Artemisia annua exovesicle extract and its application

Technical field

The invention belongs to the technical field of plant-derived exovesicle extraction, and specifically relates to an Artemisia annua exovesicle extract and its application.

Background technique

External vesicles are biological nanostructures of 40 to 150 nm that are secreted by most types of cells and transmit information between cells and organisms in all areas of life. External vesicles mainly contain components such as proteins, lipids, and RNA. Multiple studies have shown that external vesicles have various biological activities such as anti-inflammatory, anti-tumor, and anti-aging. Plant external vesicles were observed about 15 years before mammalian exosomes, but little is known about them other than old microscopy images suggesting their presence. In recent years, with the in-depth research on external vesicles, it has been found that plant external vesicles also have certain biological activities, and medicinal plant external vesicles have been found to have more prominent biological activities than common plant-derived external vesicles. As a therapeutic agent and drug carrier, it shows unique advantages and has very broad application prospects.

my country has a long history of using medicinal plants to treat diseases. The medicinal substances in medicinal plants exert their therapeutic effects through complex mechanisms and play an important role in ensuring human health. As an important medium for intercellular communication, the extravesicles of medicinal plants play an important role in the therapeutic effects of medicinal plants. There are existing literatures on exosomes derived from common medicinal materials or basic plant sources such as ginger, ginseng, turmeric, rhodiola mistletoe, astragalus, and asparagus. A large number of studies have shown that medicinal plant extravesicles have considerable potential in the prevention and treatment of diseases and deserve more extensive research. At present, there are no reports on the extraction method of external vesicles derived from Artemisia annua and the application of Artemisia annua external vesicles. Artemisia annua L. is a plant of the genus Artemisia in the Asteraceae family. The Chinese Pharmacopoeia records that Artemisia annua has a bitter and cold nature and flavor. It can be used as medicine after being cut into sections and dried. It has the effects of clearing away heat from deficiency, relieving summer-heat, relieving malaria and reducing jaundice. The fresh product can be taken internally or externally after being squeezed into juice.

Contents of the invention

In order to further develop the medicinal scope and medicinal value of the medicinal plant Artemisia annua, the present invention provides a method for extracting Artemisia annua exovesicles, that is, it provides a method for extracting Artemisia annua-containing exosomes from the natural plant Artemisia annua. A method of producing natural spherical extravesicle-like nanoparticles with protein content. Extracellular vesicles are a kind of nanoparticle structure secreted by mammals or plants. They are generally loaded with proteins, nucleic acids, lipids and other biomolecules. Extracellular vesicles are used as Biological nanocarriers play a vital role in promoting long-distance communication between cells. They are a natural drug delivery nanocarrier. By extracting external vesicles from plants, they have low cost, high delivery efficiency and non-existent Artificial nanocarriers are used to solve the empty-loading problem when preparing drugs. The other components of the outer vesicles extracted by the Artemisia annua outer vesicle extraction method provided by the present invention also have no toxic or side effects.

Technical solutions:

In a first aspect, the present invention provides a method for extracting outer vesicles of Artemisia annua. The extraction method includes the steps of: cleaning the whole fresh Artemisia annua plant with pure water, then adding pure water and grinding to obtain juice; After centrifugation at 200 × g for 10 minutes, 2000 × g for 20 minutes, and 10,000 × g for 30 minutes, the supernatant was taken to remove large particles and fibers. The supernatant was centrifuged at 100,000 × g for 1 hour, and the precipitate was suspended in PBS. ; Use sucrose density gradient centrifugation to transfer the sample to 15%, 30%, 45%, and 60% sucrose solutions and centrifuge at 150,000×g for 1 hour. Collect the 45% sucrose solution layer and add an equal volume of PBS to the 45% sucrose solution layer. , centrifuge at 150,000 × g for 1 hour to wash away the sucrose, collect the precipitate, and resuspend it in sterile PBS. Use it fresh or store it at -80°C. The centrifugal radius of the above centrifugation is 39.5mm.

The Artemisia annua exovesicles extracted using the above extraction method are spherical exovesicle-like nanoparticles with a hydrated particle size of 124.8±0.9nm, and the spherical exovesicle-like nanoparticles have a negative Zeta potential value of -26.7±0.87mV.

In a second aspect, the present invention also provides the application of the Artemisia annua exovesicle extract extracted by the above extraction method in the preparation of drugs for regulating the immune microenvironment, that is, as an immunomodulator. As an immunomodulator, the above-mentioned Artemisia annua exovesicles It can promote the polarization of macrophages from M2-like phenotype to M1 phenotype and exert an immunoregulatory effect;

In a third aspect, the present invention also provides the use of the above-extracted Artemisia annua exovesicle extract in the preparation of anti-tumor drugs. Macrophages stimulated by the Artemisia annua exovesicles can inhibit tumor growth and can be used to produce tumors. Nanomedicines for immunotherapy.

Beneficial effects:

The Artemisia annua exovesicle extract extracted using a method for extracting Artemisia annua exovesicles provided by the present invention contains spherical exovesicle-like nanoparticles. Specifically, through verification, this kind of spherical exovesicle nanoparticles have a particle size of about 124.8 nm. Vesicle-like nanoparticles contain artemisinin.

In terms of medicinal efficacy, the outer vesicles of Artemisia annua extracted using the extraction method of the present invention can change the polarization of M2-like macrophages. In the tumor microenvironment, M2-like macrophages account for the majority of tumor-related macrophages, inhibiting or Changing M2-like cells is considered an effective cancer treatment strategy; real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay ELISA further confirmed that Artemisia annua exovesicles can change the M2 of macrophages. Such polarization. The experiment also verified that treatment with macrophage supernatant stimulated by Artemisia annua exovesicles significantly increased the apoptosis of A549 and LLC lung cancer cells. In vivo experiments in mice verified that treatment with Artemisia annua external vesicles significantly inhibited tumor growth. The weight of tumors in mice treated with Artemisia annua external vesicles was reduced by 23%; HE staining results showed that in the heart, liver, spleen, and In the kidney, lung, and brain tissues, there was no obvious drug toxicity damage to the outer vesicles of Artemisia annua. The above experimental results suggest that Artemisia annua exovesicles can change the polarization of M2-like macrophages and regulate the tumor microenvironment to inhibit the growth of lung cancer cells.

In conclusion, the Artemisia annua exovesicles provided by the present invention can change macrophage polarization in vivo and in vitro and increase the number of M1 phenotype macrophages, which contributes to anti-tumor response. The Artemisia annua exovesicles extracted by the present invention serve as an immune modulator to participate in the immune response of mammals and can be used as a new type of nanomedicine for cancer immunotherapy.

Description of the drawings

Figure 1A is a photo of the sample after sucrose gradient ultracentrifugation described in Example 1;

Figure 1B is a comparison chart of high performance liquid chromatography analysis of different samples in Example 1;

Figure 1C is a transmission electron microscope TEM photo of the Artemisia annua extract in Example 1;

Figure 1D is the Zeta potential analysis of the nanoparticle structure in Example 1;

Figure 1E is the ZetaView nanoparticle tracking analysis results of the nanoparticle structure in Example 1;

Figure 1F is a transmission electron microscope TEM photo of the nanoparticle structure in Comparative Example 1;

Figure 1G shows the results of ZetaView nanoparticle tracking analysis of the nanoparticle structure in Comparative Example 1;

Figure 2A shows the polarization-related surface labeling levels in Experiment 1;

Figure 2B is the ELISA measurement result of changes in M1 markers in macrophages in Experimental Example 1;

Figure 2C shows the RT-PCR measurement results in Experimental Example 1;

Figure 3A shows the apoptosis results of tumor cells in Experiment 2 after incubation of Artemisia annua outer vesicles and macrophages under the action of cell supernatant;

Figure 3B is a picture of mouse tumors isolated from LLC tumor-bearing mice of different administration groups in Experiment 3 after 14 days of treatment;

Figure 3C shows the changes in tumor volume of LLC tumor-bearing mice in different administration groups in Experimental Example 3 after 14 days of treatment. ;

Figure 3D shows the body weight changes of LLC tumor-bearing mice in different administration groups in Experiment 3 after 14 days of treatment;

Figure 3E shows the tumor volume inhibition rate of LLC tumor-bearing mice in different administration groups in Experimental Example 3 after 14 days of treatment;

Figure 3F shows the weight of isolated mouse tumors in LLC tumor-bearing mice of different administration groups in Experimental Example 3 after 14 days of treatment;

Figure 3G is an H&E stained image of sections of various organs of LLC tumor-bearing mice in different administration groups after 14 days of treatment in Experiment 3. The scale bar is 100 μm;

Figure 4A shows the determination of alanine aminotransferase content in the serum of LLC tumor-bearing mice in different administration groups in Experiment 4 after 14 days of treatment;

Figure 4B shows the determination of aspartate aminotransferase content in serum of LLC tumor-bearing mice of different administration groups in Experiment 4 after 14 days of treatment;

Figure 4C shows the measurement of serum urea nitrogen content in LLC tumor-bearing mice of different administration groups in Experiment 4 after 14 days of treatment;

Figure 4D shows the measurement of blood creatinine content in the serum of LLC tumor-bearing mice of different administration groups in Test Example 4 after 14 days of treatment.

Detailed ways

In order for those in the relevant technical field to better understand the content of the patent of the present invention, the embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation modes and specific details are given. Operation process, but the content of the present invention is not limited to the following examples.

The sources of reagents, materials and instruments involved in the examples of the present invention are as follows: fresh Artemisia annua was purchased from Henan Province in November 2022; mouse monocyte macrophage RAW264.7 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; human monocytes Cells THP-1 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; mouse lung cancer cell LLC cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

Example 1 Extraction and characterization of Artemisia annua exovesicle NPs

Take 5kg of fresh Artemisia annua, wash the whole plant with pure water three times, add 100ml of pure water, and grind it with a juicer to obtain the juice. Centrifuge the juice at 200×g for 10 minutes, 2000×g for 20 minutes, and 10,000×g for 30 minutes to remove large particles and fibers. Take the supernatant, centrifuge at 100,000 × g for 1 h, and suspend the pellet in 1 mL PBS. Using sucrose density gradient centrifugation, the sample was transferred to 15%, 30%, 45%, and 60% sucrose solutions and centrifuged at 150,000 × g for 1 h. As shown in Figure 1A, 4 bands were formed after sucrose gradient ultracentrifugation, and most NPs accumulated. At the 45% interface, that is, between band 3 and band 4; collect the 45% sucrose solution layer, add an equal volume of PBS to the 45% sucrose solution, centrifuge at 150,000×g for 1 hour to wash away the sucrose, collect the precipitate, and use 1 mL of sterile The Artemisia annua extract containing Artemisia annua exovesicles can be obtained after being resuspended in PBS. It can be used fresh or stored at -80°C. The centrifugal radius of the above-mentioned centrifugation is 39.5 mm.

Dilute the above Artemisia annua extract with pure water to 1mg/mL, then drop it on the copper grid of the electron microscope and dry it naturally. After washing with water, stain it with 2% phosphotungstic acid for 5 minutes. After drying, pass it through the FEI-TALOS-F200X transmission electron microscope. Shoot. As shown in Figure 1C, which is a transmission electron microscope TEM photo, it can be seen that there is a spherical nanoparticle structure in the Artemisia annua extract;

The above-mentioned Artemisia annua extract was first diluted to 1 mg/mL, and the hydrated particle size of the nanoparticle structure present in the Artemisia annua extract was measured by ZetaView nanoparticle tracking analyzer. As shown in Figure 1E, it was measured by ZetaView nanoparticle tracking analyzer. The hydrated particle size of purified NPs is 124.8nm; the Zetasizer Nano ZS90 potential meter measured the Zeta potential of the nanoparticle structure. The Zeta potential analysis in Figure 1D shows that the nanoparticle NPs have a negative Zeta potential value of -26.7mV; through hydration The measurement of particle size and zeta potential of the nanoparticle structure can determine that the nanoparticle structure in the Artemisia annua extract is Artemisia annua exovesicles, so the Artemisia annua extract in Example 1 is an Artemisia annua exovesicle extract.

An Agilent HP1100 high-performance liquid chromatograph was used to analyze the components in the extract solution at different stages in the above extraction process, the artemisinin standard, and the extract solution with the nanoparticle structure removed. The analysis results are shown in Figure 1B, where a represents Artemisinin standard, b represents the extract solution before sucrose density gradient centrifugation, c represents the extract solution after density gradient centrifugation purification, and d represents the extract solution after centrifugation to remove the nanoparticle structure. Through comparison, it can be found that Qinghaosu The peak time of the characteristic peak of artemisinin is about 10 minutes. The characteristic peak of artemisinin can be observed in both solution b and solution c. It can be determined that artemisinin exists in the nanoparticle structure. This may be due to the nanoparticle structure exerting the drug. effective material basis.

Comparative example 1

The only difference between the extraction steps of the Artemisia annua exovesicle extract in this Comparative Example 1 and Example 1 is that the supernatant to remove large particles and fibers is centrifuged at 14000g for 30 minutes at 4°C.

Figure 1F shows the electron microscopy image of the external vesicles extracted in Comparative Example 1; Figure 1G shows the particle size distribution chart of the external vesicles extracted in Comparative Example 1. From Figure 1G, it can be seen that in Comparative Example 1 The particle size of the extracted external vesicles is distributed at both 131.6nm and 122.1nm, and the particle size distribution is not uniform enough, which will affect the distribution and stability of the external vesicles in the body and thus affect the final therapeutic effect. In addition, uniform outer vesicle particle size is a necessary condition for large-scale production to ensure good repeatability between different extraction batches, thereby ensuring that different batches have stable and good therapeutic effects.

The outer vesicles extracted according to the ultra-high-speed centrifugation method in Example 1, as shown in Figure 1E, have particle sizes mainly distributed at 124.8 nm, indicating that high-concentration and uniform particle sizes can be obtained by using the ultra-high-speed centrifugation in Example 1. The outer vesicle structure is not damaged by long-term ultrahigh-speed centrifugation. Compared with the traditional extraction method through suction filtration and low-speed centrifugation after incubation, no additional reagents are added and time and cost are saved.

Test Example 1 Verification of Artemisia annua exovesicle extract inhibiting M2-like macrophage polarization in vitro

Mouse monocyte macrophage RAW264.7 and human monocyte THP-1 were evenly spread on a 6-well plate at a density of 3×10 ^6. After 24 hours, stimulating factors 20ng/mL IL-4 and 20ng/mL IL were added. -13 induced differentiation into M2-like macrophages. M2-like macrophages were incubated with 20 μg/mL Artemisia annua exovesicle extract in Example 1 for 48 h. The supernatant was collected and used for enzyme-linked immunosorbent assay (ELISA). The kit detects M1-related cytokines, including IL-6 and TNF-α. Then, cells are collected, gene expression is measured and surface markers detected. Prepare macrophage single cell suspension in PBS. Nonspecific labeling was blocked with anti-CD16/32 and macrophage surface markers were detected using the following mouse monoclonal antibodies: anti-CD206 APC; anti-CD80APC; anti-CD86 PE; anti-MHC-II FITC; anti- CD11bAPC; anti-F4/80PE; anti-TLR2FITC; anti-TLR4 PE/Cy7. Incubate for 30 minutes and detect using CytoFLEX S flow cytometer.

M2-like macrophages account for the majority of tumor-associated macrophages. Inhibiting or altering M2-like cells is considered an effective cancer treatment strategy. First, polarization confirmation was performed by flow cytometry analysis to detect the levels of surface markers related to polarization. As shown in Figure 2A, NPs treatment in RAW264.7 cells resulted in a significant decrease in CD206 levels in M2-like macrophages, while M1-like The expression of macrophage-related surface marker proteins CD80, CD86, MHC-II, TLR2 and TLR4 was increased. Next, to further determine whether NPs can alter the M2-like polarization of macrophages. The expression of M1 and M2-related genes was measured using real-time fluorescence quantitative polymerase chain reaction (RT-PCR). Analysis of RT-PCR results in Figure 2C showed that NPs treatment significantly induced an increase in M1-related markers IL-6 and TNF-α, while M2-related markers Arg-1 and IL-10 were down-regulated. The enzyme-linked immunosorbent assay ELISA method was used to further verify the changes in M1 markers in NPs-treated macrophages. As shown in Figure 2B, in mouse monocyte macrophages RAW264.7 and human monocyte THP-1, using The production of IL-6 and TNF-α increased after treatment with the Artemisia annua external vesicle extract extracted in Example 1.

Test Example 2: In vitro test of macrophages treated with Artemisia annua exovesicle extract to inhibit the growth of lung cancer cells

In vitro test: Mouse lung cancer cell LLC cells were evenly spread on a 6-well plate at a density of 3 × 10 ⁵ and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After the cell density reached 70%, M2 macrophages were The cell supernatant was used as the drug in the control group, and the supernatant after incubation of M2 macrophages with 20 μg/ml Artemisia annua exovesicle extract extracted in Example 1 was used as the drug in the experimental group. The drugs were added for 24 hours respectively. After the drug effect ended, Digest the cells in each well with trypsin and place them in an EP tube. Centrifuge at 4°C and 1500 rpm for 5 min. Discard the supernatant. Collect the cells. Add PBS to wash the cell pellet. Centrifuge at 4°C at 1500 rpm for 5 min. Discard the PBS. Add 200 μL Binding Buffer to each tube. Fully suspend the cells, add 5 μL of Annexin V-FITC and 5 μL of 7-AAD in the dark, mix well, and let stand at room temperature for 30 minutes in the dark. After the incubation is complete, detect the cells with the CytoFLEX S flow cytometer.

As shown in Figure 3A, A549 and LLC lung cancer cells were treated with different cell supernatants for 24 h, and stained with Annexin V-PE/7-AAD apoptosis detection kit. found that NPs-stimulated macrophage supernatant treatment significantly increased the apoptosis of A549 and LLC lung cancer cells compared with unstimulated macrophage supernatant.

Test Example 3: In vivo test of inhibiting the growth of lung cancer cells by macrophages treated with Artemisia annua exovesicle extract

In order to verify whether Artemisia annua exovesicle extract treatment has similar effects in vivo, that is, to verify that Artemisia annua exovesicle extract has a positive effect on the interaction between macrophages and tumor cells both in vivo and in vitro, the following in vivo experiments were conducted test:

(1) Construct an LLC tumor-bearing mouse model. The establishment process specifically includes: first, resuspend the collected LLC cells in pre-cooled PBS, and promptly inject them near the right lower limb of BALB/c mice (1.0×10 ⁷ cells/mouse ), and observe tumor growth every other day. When the tumor begins to grow, use vernier calipers to measure the length and width of the tumor, and calculate the tumor volume according to the following formula: tumor volume = length × width ² /2. Drug treatment was given when the tumor size reached ^100mm3 .

(2) When the tumor size reaches about ^100mm3 , the above mouse model is randomly allocated into 3 groups, with 5 mice in each group. After grouping, the tumor site is injected subcutaneously every two days, with a total of 7 injections in each group. Each injection is 100ul. The first group is injected with normal saline Control group in situ. The second group is injected with macrophage scavenger in situ. The second group is injected with 20 μg/ml of Artemisia annua exovesicle extract extracted in Example 1 in situ. and 5 mg/ml macrophage scavenger mixture NPs+ClodronateLiposomes group. The third group was injected in situ with 20 μg/ml Artemisia annua external vesicle extract extracted in Example 1 as the NPs group. Starting from the first day of treatment, By measuring the tumor volume, as shown in Figures 3B and 3C, the Artemisia annua exovesicle extract treatment group significantly inhibited tumor growth; at the same time, the body weight and tumor volume of the mice were measured every two days. After two weeks of drug treatment, The nude mice in each group were euthanized, and the tumors and major organs including the heart, liver, spleen, lungs and kidneys were collected. The tumors were cleaned with physiological saline, photographed and weighed. After the end, two parts of the tumors from each group were taken and the organs were collected. Soak in 4% paraformaldehyde fixative for hematoxylin-eosin staining (H&E) staining for subsequent observation;

Test results: As shown in Figure 3F, the tumor weight of mice in the Artemisia annua external vesicle extract treatment group was reduced by 23%, indicating that macrophages treated with Artemisia annua external vesicle extract can also inhibit the growth of cancer cells in vivo. ; As shown in Figure 3G, no obvious toxicity was found in the H&E staining examination of tissue sections of Brain, Kidney, Lung, Liver, Heart and Spleen; and as shown in Figure 3D, during the administration period, no obvious toxicity was found. There was no significant difference in the body weight changes of the mice in the two groups, indicating that Artemisia annua external vesicle extract has no side effects of drug toxicity.

Test Example 4 In vivo safety test of Artemisia annua exovesicle extract

In order to further verify the in vivo safety of Artemisia annua external vesicle extract, the following test was conducted: mice were randomly divided into 2 groups, 3 mice in each group: PBS group and Artemisia annua external vesicle extract group. The drug was administered intravenously once every two days, for a total of 7 administrations. The first group is the physiological saline Control group, and the second group is 20 μg/ml Artemisia annua external vesicle extract NPs extracted in Example 1. After continuous administration, the mice were weighed, the eyeballs were removed to collect blood, and the blood was stored in heparin-containing EP tubes. Serum was separated by gradient centrifugation for subsequent experiments. Take 5 μl of serum sample and operate according to the instructions of the Soleba test kit, as shown in Figure 4A, Figure 4B, Figure 4C and Figure 4D, which are the LLC tumor-bearing mice in different administration groups of Test Example 4 after 14 days of treatment. After the test, the results of the determination of alanine aminotransferase, aspartate aminotransferase, urea nitrogen and creatinine in the serum showed that they were all within the normal range.

Claims (6)

1. A method for extracting outer vesicles of Artemisia annua, characterized in that the extraction method includes the steps of: cleaning the whole fresh Artemisia annua plant with pure water, then adding pure water and grinding to obtain the juice; passing the juice in sequence Centrifuge at 200 × g for 10 minutes, 2000 × g for 20 minutes, and 10,000 × g for 30 minutes. Take the supernatant to remove large particles and fibers. Centrifuge the supernatant at 100,000 × g for 1 hour, and suspend the precipitate in PBS; Use the sucrose density gradient centrifugation method to transfer the sample to 15%, 30%, 45%, and 60% sucrose solutions and centrifuge at 150,000×g for 1 hour. Collect the 45% sucrose solution layer and add an equal volume of PBS to the 45% sucrose solution layer. Centrifuge at 150,000 × g for 1 hour to wash away the sucrose, collect the precipitate, and resuspend in sterile PBS. Use fresh or store at -80°C. The centrifugal radius of the above centrifugation is 39.5 mm. 2. The Artemisia annua exovesicle extract extracted by the extraction method of claim 1, wherein the Artemisia annua exovesicle extract contains Artemisia annua exovesicles, and the Artemisia annua exovesicles are hydrated Spherical external vesicle-like nanoparticles with a particle diameter of 124.8±0.9 nm, and the spherical external vesicle-like nanoparticles have a negative Zeta potential value of -26.7±0.87mV. 3. Application of the Artemisia annua exovesicle extract according to claim 2 in the preparation of drugs for regulating the immune microenvironment. 4. Application of the Artemisia annua external vesicle extract according to claim 2 in the preparation of anti-tumor drugs. 5. The application of claim 3 or claim 4, wherein the Artemisia annua exovesicle is the only active ingredient. 6. The application according to any one of claim 3 or claim 4, characterized in that the concentration of the Artemisia annua external vesicle extract is 20 μg/mL.