Lotus leaf exosome
Lotus leaf exosomes were extracted from lotus leaves through different extraction methods, which solved the problems of purification and stability of plant-source exosome nanoparticles, and achieved effective application of lotus leaf exosomes in inflammation response and wound healing.
Patent Information
- Application Number
- CN202311639316.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-30
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively purify and stabilize plant-derived exosome nanoparticles, especially lotus leaf exosome nanoparticles, which lead to challenges in clinical applications.
Through different extraction methods, such as polymer precipitation method, ultra-high-speed centrifugation method, over-filtration method, density gradient centrifugation method and column chromatography method, lotus leaf exosomes were successfully extracted from lotus leaves, and their purification methods and qualitative standards were established.
The stability and similar particle size of lotus leaf exosomes are achieved, the application potential of them in alleviating inflammation responses and wound healing, and the basis for the preparation of anti-inflammatory or wound healing drugs, compositions or nutritional supplements.
Smart Images
Figure CN120060116A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a plant exosome, particularly a lotus leaf exosome.
Background Art
[0002] Extracellular vesicles (EVs) are lipid bilayer vesicles produced by cells, widely present in organisms, including animals, plants, and microorganisms. Initially, these extracellular vesicles were considered cell debris responsible for carrying intracellular waste out of the cell. However, since these vesicles carry a large amount of proteins, lipids, RNA, and DNA and their size ranges from 50 to 1000 nm, allowing them to freely penetrate cell membranes, extracellular vesicles are no longer regarded as mere intracellular waste carriers but as important players in cell - to - cell communication.
[0003] According to the formation method of extracellular vesicles, extracellular vesicles can be mainly divided into two types, namely exosomes and microvesicles. Exosomes are approximately 30 - 150 nm in size and are essentially intraluminal vesicles (ILVs) within multi - vesicular bodies (MVBs). Subsequently, they are released into the extracellular space when the multi - vesicular body fuses with the cell membrane; microvesicles are 50 - 1000 nm in size and are released from the cell by budding. In the past decade, with the increasing number of studies on extracellular vesicles, scientists have gradually discovered that these extracellular vesicles have various biological activities and are involved in various physiological and pathological processes of organisms, such as blood coagulation, angiogenesis, immune regulation, and inflammation. More studies have shown that various types of cells regulate the generation method of extracellular vesicles according to their physiological state to release extracellular vesicles with specific lipid, protein, and nucleic acid components. Therefore, these extracellular vesicles can be used as biomarkers for cancer treatment. In addition, due to their ability to shuttle between cells, extracellular vesicles can also be used as nano - drug carriers in the treatment of diseases.
[0004] Plant-derived exosome-like nanoparticles (PDENs) are nano-sized particles similar to exosomes in plants and exist in the plant cytoplasm or extracellular matrix. In recent years, with the progress of research, many studies have confirmed that these plant-derived exosome-like nanoparticles have a variety of bioactive functions, such as antioxidant, anti-inflammatory, modulation of gut microbiota, anti-cancer, etc. In addition, plant-derived exosome-like nanoparticles can also be used as drug carriers. Currently, many studies have successfully extracted plant-derived exosome-like nanoparticles from various plants, including ginger, grapes, grapefruit, lemon, apple, cherry, strawberry, tomato, wheat, carrot, Chinese cabbage, and cabbage.
[0005] Although plant-derived exosome-like nanoparticles have great application potential in clinical medicine, due to the fact that plant-derived exosome-like nanoparticles are similar to other exosomes, being nano-sized particles and most of them being heterogeneous particles, it is a great challenge to extract them efficiently and precisely from organisms. Currently, the purification methods of plant-derived exosome-like nanoparticles all refer to the exosome purification methods of mammalian cells, there is no standard purification method, and purifying exosomes by different methods will cause changes in the membrane protein content on exosomes and the contents will also be different. Existing studies have shown that the lipid composition of plant-derived exosome-like nanoparticles is considered to be the key factor determining the target cells of exosomes. The exosome-like particles obtained from grapes will only be delivered to intestinal cells; the grapefruit exosome-like particles modified with lipids can be delivered to brain cells via the nasal cavity. These studies all show that establishing a standard purification method to stabilize the quality of plant-derived exosome-like nanoparticles is an urgent problem to be solved in clinical applications.
[0006] Lotus (Nelumbo nucifera Gaerm), belonging to the genus Nelumbo in the family Nelumbonaceae, also known as the lotus flower, is a perennial aquatic plant widely cultivated in East Asia and India and can be used for ornamental and dietary purposes. In traditional Chinese medicine, the lotus has a long history of use and can be used to treat various diseases, such as hematemesis, epistaxis, hyperlipidemia, etc. In addition, there are also studies showing that the lotus has a variety of pharmacological and physiological activities, including liver protection, antioxidant, antidiarrheal, antiviral, immune regulation, anti-obesity and other effects.
[0007] Since the whole lotus plant, including leaves, flowers, rhizomes, and seed pods, can be utilized, it is a crop with high economic value. The pharmacological effects of the lotus mainly come from its rich variety of bioactive substances, including polysaccharides, essential oils, flavonoids, alkaloids, and triterpenoids. Among them, the flavonoids and alkaloids in lotus leaves have been the most studied. Due to their high biological activity and low cost, the isolation, purification, and activity analysis of flavonoid compounds in lotus leaves have become the main objectives in the research of lotus leaves. SUMMARY OF THE INVENTION
[0008] The summary of the present invention aims to provide a simplified summary of the present invention to enable readers to have a basic understanding of the present invention. The summary of the present invention is not a complete review of the present invention, and it does not intend to point out the important / critical elements of the embodiments of the present invention or define the scope of the present invention.
[0009] In recent years, with the emergence of research on various plant-derived exosome-like nanoparticles, the functions and applications of plant-derived exosome-like nanoparticles have become increasingly obvious. However, although several fruits and vegetables have been reported, there has been no relevant research on lotus leaf exosome-like nanoparticles in past studies. In view of the fact that lotus leaves contain a variety of bioactive substances, it is a worthy research topic to evaluate whether lotus leaves contain plant-derived exosome-like nanoparticles and have bioactive characteristics; it is known that plant-derived exosome-like nanoparticles can carry mRNA, miRNA, bioactive substances, and proteins of various plants into animal cells and improve their cell activity. Past studies have also shown that lotus leaves contain various active substances and can be used as health foods. Therefore, in order to understand whether lotus leaves also contain exosome-like nanoparticles, the inventors of this case conducted a series of experiments to evaluate the effects of various methods for extracting plant-derived extracellular vesicles on lotus leaf exosome particles, to establish its purification method and qualitative criteria, and to analyze whether the lotus leaf exosome-like nanoparticles contain effective bioactive substances.
[0010] Accordingly, on the one hand, the present invention provides a lotus leaf exosome, which is extracted from lotus leaves by a group consisting of the following extraction methods: polymer precipitation method, ultra-high speed centrifugation method, ultrafiltration method, density gradient centrifugation method, and column chromatography method.
[0011] According to an embodiment of the present invention, the extraction method is the ultrafiltration method.
[0012] According to an embodiment of the present invention, the lotus leaf exosome has a particle size of 50 to 300 nm.
[0013] According to an embodiment of the present invention, the lotus leaf exosomes have a zeta potential (electrokinetic potential) of less than -20 mV.
[0014] According to an embodiment of the present invention, the lotus leaf exosomes have a zeta potential of less than -30 mV.
[0015] According to an embodiment of the present invention, the lotus leaf exosomes are used for alleviating inflammatory responses or wound healing.
[0016] According to an embodiment of the present invention, the inflammatory response is caused by LPS.
[0017] Another aspect of the present invention provides an anti-inflammatory composition comprising the lotus leaf exosomes as described above.
[0018] According to an embodiment of the present invention, the anti-inflammatory composition further comprises a carrier.
[0019] Another aspect of the present invention provides a use of lotus leaf exosomes, which is for preparing a drug, composition or nutritional supplement for anti-inflammation or wound healing.
[0020] The present invention has the following advantages: The present invention extracts lotus leaf exosomes from lotus leaves by different separation methods, thereby establishing the existence of lotus leaf exosomes; in addition, the present invention further determines the characteristics of the lotus leaf exosomes, establishes its purification method and qualitative standard, and the lotus leaf exosomes obtained by the above methods have similar particle size and stable zeta potential. In addition, the present invention is experimentally confirmed that the lotus leaf exosomes can be used for alleviating inflammatory responses or wound healing, and can be further used for preparing drugs, compositions or nutritional supplements for anti-inflammation or wound healing.
Description of the Drawings
[0021] In order to make the above-mentioned present invention and other objectives, features, advantages and embodiments more obvious and understandable, the drawings are described as follows:
[0022] Figure 1 It is a schematic diagram of the pretreatment process of the lotus leaf exosome sample of the present invention.
[0023] Figure 2 A to F of are the particle size distribution diagrams of lotus leaf exosomes extracted by different methods of the present invention; wherein, A is the result of using the polymer precipitation method; B is the result of using the ultrafiltration method; C is the result of using the column chromatography method; D is the result of using the ultra-high speed centrifugation method; E is the result of using the density gradient centrifugation method and collecting the 30%-45% sucrose solution layer; F is the result of using the density gradient centrifugation method and collecting the 45%-60% sucrose solution layer.
[0024] Figure 3Figures A to F are the observation results of the appearance of lotus leaf exosomes extracted by different methods in the present invention; among them, A is the result of using the polymer precipitation method; B is the result of using the ultrafiltration method; C is the result of using the column chromatography method; D is the result of using the ultra-high speed centrifugation method; E is the result of using the density gradient centrifugation method and collecting the 30%-45% sucrose solution layer; F is the result of using the density gradient centrifugation method and collecting the 45%-60% sucrose solution layer.
[0025] Figure 4 is the zeta potential result diagram of lotus leaf exosomes extracted by different methods in the present invention.
[0026] Figure 5 Figures A to D are the sample and particle size distribution diagrams of lotus leaf exosomes extracted by the combination method in the present invention; among them, A is the result diagram after the final density gradient centrifugation; B is the particle size result diagram using the combination method; C is the lotus leaf exosome yield result diagram using the combination method; D is the zeta potential result diagram using the combination method.
[0027] Figure 6 Figures A to B are the result diagrams of the anti-inflammatory effect of lotus leaf exosomes extracted by different methods in the present invention; among them, A is the result diagram of the cell viability of RAW264.7 macrophages after intervention with LPS; B is the anti-inflammatory effect of RAW264.7 macrophages after intervention with lotus leaf exosomes under LPS induction detected by nitrite. Statistical analysis was performed using one-way analysis of variance and Tukey's post hoc analysis, and the data marked with letters indicate significant differences compared with the LPS group (p < 0.05).
[0028] Figure 7 Figures A to B are used to test the wound healing ability of the lotus leaf exosomes of the present invention in vitro; among them, A is the migration distance of the HaCaT cell line at different concentrations of LDEVs shown by time-lapse micrographs; B is the wound area change diagram. Statistical analysis was performed using one-way analysis of variance and Tukey's post hoc analysis, and the data marked with letters indicate significant differences compared with the control group (p < 0.05).
Detailed Description of the Invention
[0029] The detailed description and technical content of the present invention will be described below in conjunction with the drawings. Furthermore, for the convenience of illustration, the proportions of the drawings in the present invention are not necessarily drawn according to the actual proportions, and these drawings and their proportions are not intended to limit the scope of the present invention, which is hereby stated in advance.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. The following terms used throughout this application shall have the following meanings.
[0031] Unless otherwise specified, "or" means "and / or". "Comprising" means not excluding the presence or addition of one or more other components, steps, operations or components on the described components, steps, operations or components respectively. The terms "include", "comprise", "contain", "encompass" and "have" described herein are interchangeable and non-limiting. The singular forms "a" and "the" used in this text and the appended patent claims include plural referents unless the context otherwise indicates. For example, the terms "a", "the", "one or more" and "at least one" are interchangeable in this text.
[0032] As used herein, "lotus leaf" refers to the leaf of a lotus flower (or water lily).
[0033] As used herein, "lotus leaf exosomes", "lotus leaf extracellular vesicles", "lotus leaf exosome-like particles" and "lotus leaf exosome-like nanoparticles" are used interchangeably and refer to plant-derived exosome-like nanoparticles extracted from lotus leaves.
[0034] In one aspect, the present invention provides a lotus leaf exosome, which is extracted from lotus leaves by a method selected from the group consisting of: polymer precipitation method, ultra-high speed centrifugation method, ultrafiltration method, density gradient centrifugation method and column chromatography method. In a preferred embodiment, the method for extracting lotus leaf exosomes is the ultrafiltration method.
[0035] Since there is currently no research on lotus leaf exosomes, the inventors of this case have tried to extract and purify lotus leaf exosomes using different methods or a combination of the above methods, and further compare the differences in the characteristics of lotus leaf exosomes obtained by different extraction methods. It has been found through experiments that there is not much difference in the particle size of lotus leaf exosomes obtained by these different extraction methods; according to an embodiment of the present invention, these lotus leaf exosomes have a particle size of 50 to 300 nm, such as but not limited to: 50 nm, 63 nm, 70 nm, 84 nm, 90 nm, 100 nm, 115 nm, 126 nm, 132 nm, 136 nm, 152 nm, 159 nm, 163 nm, 166 nm, 200 nm, 217 nm, 223 nm, 235 nm, 248 nm, 251 nm, 266 nm, 273 nm, 286 nm, 298 nm or 300 nm.
[0036] After confirming the particle size of the lotus leaf exosomes, the present invention further analyzes the zeta potential of the lotus leaf exosomes extracted by these methods to detect the stability of these particles. Generally speaking, the larger the zeta potential of the colloidal particles, the greater the distance between the sliding surface of the particles and the surface of the particles (i.e., the thickness of the electric double layer), and it is not easy for the particles to attract and agglomerate, which is beneficial to maintaining a stable suspension state of the colloidal solution. On the contrary, in the particle solution with a low zeta potential, due to the small charge repulsion force between the particles, it is easy to aggregate into large particles and cause precipitation. It is generally believed that when the zeta potential of the particles is between 0±5, the particles are in an easily agglomerated state; when it is between ±10-30 mV, the particles are slightly stable; when it is between ±30-40 mV, it is moderately stable, when it is between ±40-60 mV, it is well stable, and when it is greater than ±60 mV, it is excellently stable. According to an embodiment of the present invention, the lotus leaf exosomes have a zeta potential less than -20 mV, such as but not limited to: -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, -50 mV, -51 mV, -52 mV, -53 mV, -54 mV, -55 mV, -56 mV, -57 mV, -58 mV, -59 mV or -60 mV. In a preferred embodiment, the lotus leaf exosomes have a zeta potential less than -30 mV.
[0037] In addition, the present invention also combines the above methods to extract extracellular vesicles by two or more extraction and purification methods. The results show that the lotus leaf exosomes extracted by the combined method can also obtain a similar particle size and a well-stable zeta potential, and the exosomes extracted by the combined method have a higher yield ( Figure 5 ). Thus, it is obvious that the present invention has established a method for extracting lotus leaf exosomes and analyzed the different characteristics of lotus leaf exosomes extracted by different methods.
[0038] According to an embodiment of the present invention, the lotus leaf exosomes are used for alleviating inflammatory responses or wound healing; in a preferred embodiment, the inflammatory response is caused by LPS. Since it is known that lotus leaves have a variety of bioactive substances and have antioxidant and anti-inflammatory effects, in order to understand whether lotus leaf exosomes also have anti-inflammatory effects, the present invention conducted anti-inflammatory experiments. The anti-inflammatory experiments used lipopolysaccharide (LPS) to induce an inflammatory model in mouse macrophages RAW264.7, and lotus leaf exosomes were administered to detect their anti-inflammatory ability. The results showed that lotus leaf exosomes extracted by different methods all had anti-inflammatory effects and could reduce the inflammatory response induced by LPS( Figure 6 ).
[0039] Wound healing is a complex physiological process, including a series of inflammatory responses, cell regeneration, and cell migration, etc. The healing process of skin wounds usually begins with an inflammatory response, which is an immediate response after skin damage. Although inflammation helps to clear germs and damaged tissues around the wound, however, excessive inflammatory responses may have a negative impact on wound healing and delay the repair process. Therefore, appropriate intervention in anti-inflammatory treatment can help reduce this excessive inflammatory response, accelerate wound healing, and also reduce the pain and discomfort caused by inflammation. Since the present invention found that the lotus leaf exosomes have anti-inflammatory effects, the influence of the lotus leaf exosomes on wound healing was further tested. The results showed that the lotus leaf exosomes indeed had the effect of promoting wound healing, and the higher the concentration of the lotus leaf exosomes, the better the wound healing ability( Figure 7 ). It can be seen that the described lotus leaf exosomes have the ability to improve wound repair and can shorten the time course of wound healing.
[0040] In addition, in the above experiments, the present invention used fresh lotus leaves as the extraction raw material; however, the present invention is not limited to fresh lotus leaves, and other materials such as withered lotus leaves or dried lotus leaves can also be used as the extraction raw material.
[0041] On the other hand, the present invention provides an anti-inflammatory composition, which comprises the lotus leaf exosomes as described above; according to an embodiment of the present invention, the anti-inflammatory composition further comprises a carrier. The carrier refers to mixing the composition with a pharmaceutically acceptable carrier according to conventional pharmaceutical combination techniques for the needs of the drug manufacturing process, facilitating the preparation of drug dosages, or different dosage forms, etc. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. In addition, the anti-inflammatory composition may comprise pharmaceutically acceptable excipients, buffers, stabilizers, or other materials well known to those skilled in the art. These materials should be non-toxic and should not interfere with the efficacy of the active ingredients. Among them, these excipients include one or more surfactants, inorganic or organic salts, diluents, solubilizers, thickeners, reducing agents, antioxidants, chelating agents, preservatives, etc.
[0042] According to an embodiment of the present invention, the anti-inflammatory composition is for vertebrates; in a preferred embodiment, the anti-inflammatory composition is for mice; in a more preferred embodiment, the anti-inflammatory composition is for humans. The administration route of the anti-inflammatory composition of the present invention is oral or injection administration, such as intravenous or subcutaneous, as a drug for alleviating inflammatory symptoms.
[0043] Another aspect of the present invention is the use of lotus leaf exosomes, which is for preparing drugs, compositions or nutritional supplements for anti-inflammatory or wound healing. Examples
[0044] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art, and such modifications or changes will be included within the spirit and scope of this application and the scope of the appended patent applications. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[0045] In order to understand whether lotus leaves also contain exosome-like nanoparticles and successfully isolate them, the present invention first uses polymer precipitation, ultra-high speed centrifugation, ultrafiltration, density gradient centrifugation, column chromatography and combined methods for the extraction and purification of lotus leaf exosome-like nanoparticles, analyzes their particle size and concentration with a nanoparticle tracking analyzer, and then observes their appearance with an electron microscope. Then, after isolating the exosome nanoparticles, the differences in lotus leaf exosome-like nanoparticles extracted by different methods are further compared, and their size, yield and appearance of the exosome-like nanoparticles are analyzed.
[0046] Experimental methods
[0047] 1. Extraction and purification of lotus leaf exosome-like nanoparticles
[0048] The lotus leaves used in the present invention are samples provided by Bohui Biotechnology Co., Ltd. In order to successfully extract and isolate lotus leaf exosome-like nanoparticles, first perform sample pretreatment (as Figure 1 shown), and then use different separation methods to extract lotus leaf exosome-like nanoparticles.
[0049] 1-1. Polymer precipitation method (PEG)
[0050] Add an appropriate amount of secondary distilled water to the lotus leaves and break them with a juicer, and then filter with a 300 μm pore size filter. Then, centrifuge the filtrate sequentially (2,000×g for 10 minutes, 6,000×g for 30 minutes, 15,000×g for 60 minutes) to remove large fragments of lotus leaf tissue and cell debris, and filter with a 0.8 μm pore size PVDF membrane ( Figure 1) Then, the pH of the filtrate was adjusted to 5.0 using hydrogen chloride. PEG6000 (final concentration of 10%) was added, and the mixture was left overnight at 4°C. It was then centrifuged at 8,000×g for 30 minutes. The precipitate obtained from centrifugation was the exosome-like nanoparticles. The exosome-like nanoparticles were suspended in an appropriate amount of double-distilled water and dialyzed through a 10 kDa dialysis membrane for 24 hours. Finally, it was filtered through a 0.45 μm filter membrane to obtain clean exosomes.
[0051] 1-2. Ultracentrifugation, UV
[0052] Remove large fragments of lotus leaf tissue and cell debris through the same pre-treatment steps as in the polymer precipitation method, and filter through a PVDF membrane with a pore size of 0.8 μm ( Figure 1 ) Then, the filtrate was centrifuged at 120,000×g for 2 hours to obtain exosome-like nanoparticles. Wash twice with double-distilled water (centrifuged at 120,000×g for 2 hours). Finally, the exosome-like nanoparticles were suspended in an appropriate amount of double-distilled water and filtered through a 0.45 μm filter membrane to obtain clean exosomes.
[0053] 1-3. Ultrafiltration, UF
[0054] The ultrafiltration method uses tangential flow filtration (TFF). Remove large fragments of lotus leaf tissue and cell debris through the same pre-treatment steps as in the polymer precipitation method, and filter through a PVDF membrane with a pore size of 0.8 μm ( Figure 1 ) Then, the filtrate was introduced into a tangential flow filtration system and filtered through a hollow fiber membrane. First, filter through a hollow fiber membrane with a pore size of 0.65 μm (D02-E65U-07-S), collect the filtrate, and then concentrate it through a 750 kDa hollow fiber membrane. Finally, replace the solution with double-distilled water (replacement volume of 1 liter) and filter through a 0.45 μm filter membrane to obtain clean exosomes.
[0055] 1-4. Density gradient centrifugation, DG
[0056] After obtaining exosome-like nanoparticles by ultra-high speed centrifugation, the solution was placed on the upper layer of a sucrose concentration gradient solution (composed of 8%, 30%, 45% and 60%), and centrifuged at 120,000×g for 2 hours. After centrifugation, the band solutions between 8%-30% and 30%-45% were aspirated, and washed twice with double distilled water (centrifuged at 120,000×g for 2 hours). The exosome-like nanoparticles were suspended in an appropriate amount of double distilled water and filtered through a 0.45μm filter membrane to obtain clean exosomes.
[0057] 1-5. Size-exclusion chromatography (SEC)
[0058] After obtaining exosome-like nanoparticles by ultra-high speed centrifugation, they were redissolved in an appropriate amount of double distilled water. Subsequently, the suspension was placed on a qEV column and separated without pressure. Each ml was taken as a unit interval (fraction), and different intervals were collected separately. Finally, the intervals where the samples were located were mixed, filtered through a 0.45μm filter membrane, and then subjected to subsequent analysis.
[0059] 1-6. Combining method
[0060] In exosome purification, exosomes are often contaminated by lipoproteins because the size and density of exosomes are similar to those of lipoproteins. Considering that density gradient centrifugation is based on density differences and column chromatography uses molecular weight to separate exosomes, the present invention simultaneously evaluates whether it is possible to isolate exosomes with higher purity by combining different methods.
[0061] In the part of the combining method, it is mainly carried out by combining ultrafiltration, density gradient centrifugation and column chromatography. After obtaining lotus leaf exosome-like nanoparticles by ultrafiltration, the centrifuged precipitate was redissolved in an appropriate amount of double distilled water, then separated without pressure using a qEV column, and the 4th to 6th intervals were collected. After mixing, the mixture was centrifuged with a sucrose concentration gradient solution (composed of 8%, 30%, 45% and 60%) (centrifuged at 100,000×g for 2 hours). After centrifugation, the band solutions between 8%-30%, 30%-45% and 45%-60% were aspirated, washed twice with double distilled water (centrifuged at 100,000×g for 2 hours), and filtered through a 0.45μm filter membrane for subsequent analysis.
[0062] 2. Analysis of the nano-characteristics and morphological observation of lotus leaf exosome-like
[0063] The concentration and particle size distribution of lotus leaf-derived exosome nanoparticles will be analyzed by the Medical Research Department of National Taiwan University using a nanoparticle tracking analyzer (NTA, NanoWSight NS300). The zeta potential of lotus leaf-derived exosome nanoparticles will be measured using a dynamic light scattering particle size and zeta potential analyzer (Malvern, Zetasizer Nano). The morphology of the purified exosome particles will be observed using transmission electron microscopy (TEM).
[0064] 3. Cell culture and induction of the inflammatory model
[0065] The cell inflammatory model is mainly induced by lipopolysaccharide (LPS) in the mouse macrophage RAW264.7 cell line. Specifically, the RAW264.7 cell line is seeded into a 24-well plate at a cell concentration of 5×10 5 cells per well and cultured at 37°C for 12 hours; subsequently, the DMEM medium containing phenol red is removed, the cells are washed twice with PBS, and then fresh phenol red-free DMEM medium is added. At the same time, LPS is added to a final concentration of 100 ng / mL. After 10 minutes of adding LPS, samples with different concentrations are added and the cells are cultured for another 24 hours to complete the inflammation induction experiment.
[0066] 4. Detection of cell viability
[0067] Cell viability is analyzed by MTT assay. After 24 hours of the inflammation induction experiment, the old cell culture medium is removed, and the cells are rinsed twice with PBS. Subsequently, fresh DMEM medium is added, and 2 g / L of MTT reagent (final concentration 10%) is added. After culturing at 37°C for 90 minutes, the cell culture medium is removed, the purple crystals are dissolved with DMSO, and the absorbance is measured at a wavelength of 570 nm.
[0068] 5. Detection of nitrite
[0069] Since NO is produced during cell inflammation - and reacts with the culture medium to form NO 2 - , the present invention uses Griess reagent to detect NO in the cell culture medium 2 -The degree of inflammation is evaluated by its content. After culturing the cells in the inflammation induction mode test for 24 hours, 100 μL of cell culture medium was collected, and then Griess reagent was added for reaction respectively. After the reaction was completed, the absorbance was measured at a wavelength of 550 nm. The concentration of nitrite was calculated by substituting the absorbance value obtained from the test into the standard calibration curve (standard curve) prepared from the nitrite standard solution.
[0070] 6. In vitro wound healing assay
[0071] The wound healing assay was mainly performed with human keratinocytes (HaCaT cells). First, two-well culture inserts were placed in a 24-well plate, and 3×10 4 cells were seeded in each well and cultured overnight at 37 °C and 5% CO 2 in high-concentration DMEM culture medium containing 10% fetal bovine serum (FBS). The next day, the culture inserts were removed, and then different concentrations of lotus leaf extracellular vesicles were added to the culture medium, and the migration of HaCaT cells was photographed and observed and analyzed. The cells were observed using an Ibidi Stage Top Incubator cell heating and culture system, and the cell migration area was analyzed using ImageJ.
[0072] Experimental results
[0073] 1. Effects of different extraction methods on the particle size of lotus leaf exosomes
[0074] To understand the effects of different extraction methods on the separation of lotus leaf-derived exosome nanoparticles, lotus leaf exosomes were first extracted by different separation methods, including polymer precipitation method, ultra-high speed centrifugation method, ultrafiltration method, density gradient centrifugation method, and column chromatography method, and fresh lotus leaves were used as extraction materials. The sample pretreatment process is as Figure 1 shown.
[0075] After sample pretreatment, lotus leaf exosomes were separated by different methods respectively, Figure 2 is the particle size distribution diagram of lotus leaf exosomes extracted by different methods. The results showed that the particle sizes of lotus leaf exosomes obtained by different extraction methods were similar and there was not much difference. The particle sizes were mainly distributed between 50 - 300 nm. The average particle sizes of lotus leaf exosomes extracted by different methods were 152.4 ± 49 (extracellular vesicles extracted by the polymer precipitation method, hereinafter referred to as PEGEVs) ( Figure 2A), 159.5 ± 48.5 (extracellular vesicles extracted by tangential flow filtration method, hereinafter referred to as TFF-EVs)( Figure 2 B), 163.2 ± 70.1 (extracellular vesicles extracted by column chromatography method, hereinafter referred to as SEC-EVs)( Figure 2 C) and 166.5 ± 64.3 (extracellular vesicles extracted by ultra-high speed centrifugation method, hereinafter referred to as UC-EVs)( Figure 2 D); Among them, the extracellular vesicle particles obtained from the 30%-45% and 45%-60% sucrose solution layers in the density gradient centrifugation method (hereinafter referred to as DGU30-45% and DGU45-60%) have a smaller particle size, about 132.6 ± 39.9( Figure 2 E) and 136.3 ± 52 nm( Figure 2 F).
[0076] 2. Effects of different extraction methods on the physical properties of lotus leaf exosomes
[0077] In addition to analyzing the particle size by NTA, the present invention also observes the appearance of extracellular vesicles by transmission electron microscopy and detects their particle stability through the analysis of zeta potential. Figure 3 For the appearance observation results of lotus leaf exosomes extracted by different methods, from Figure 3 A to 3F, it can be clearly seen that there is an obvious cup-shaped particle structure (at the white arrow in the figure). This is a typical structure of extracellular vesicles observed by electron microscopy with negative staining. The main reason for this is that when the sample dries, the middle of the vesicles will form this special structure due to drying depression. Under the observation of TEM, the particle size distribution is similar to that measured by NTA analysis, proving that the particle size measured by NTA is consistent with the actual particle size, and these particles are extracellular vesicles.
[0078] In the analysis of the stability of lotus leaf exosomes, the present invention uses zeta potential to judge its characteristics and stability. The results are as Figure 4 shown. The zeta potentials of lotus leaf exosomes extracted by different methods are all less than -20 mV, indicating that the lotus leaf exosomes extracted by the present invention by different methods are all relatively stable. Among them, the potentials of SEC-EVs, UC-EVs, DGU30-45%-EVs and DGU-45-60%-EVs are even lower than -30 mV.
[0079] 3. Effects of different extraction methods on the yield of lotus leaf exosomes
[0080] To understand the effects of different extraction methods on the yield of lotus leaf exosomes, the present invention further analyzes the yield of lotus leaf exosomes. Table 1 shows the analysis results of the yield of lotus leaf exosomes extracted by different methods; the results show that when extracting and separating lotus leaf exosomes by tangential flow filtration, the maximum yield can be obtained, approximately 3.69±0.02×10 9 , followed by ultra-high speed centrifugation and PEG precipitation, with yields of 2.20±0.13×10 8 and 2.13±0.07×10 8 , while the yield of lotus leaf exosomes purified by column chromatography is the lowest, approximately 4.62±0.06×10 7 . In addition, comparing the yields of DGU30-45%-EVs and DGU45-60%-EVs, it is found that the yield of DGU30-45%-EVs is approximately twice that of DGU45-60%-EVs; since the density gradient centrifugation method is equivalent to the further separation of total extracellular vesicles, it indicates that the density of the lotus leaf exosome particles extracted in the present invention is mostly 1.13-1.2 g / mL; since this range is considered to be the density range of animal cell exosomes, it is inferred that the lotus leaf extracellular vesicle particles extracted in the present invention are exosome-like nanoparticles.
[0081] Table 1. Yield of lotus leaf exosomes extracted by different extraction methods
[0082] 4. Influence of extracting and purifying lotus leaf exosomes by the combination method
[0083] Previous literature has shown that using a combination of two or more extraction and purification methods for the extraction and purification of extracellular vesicles is a way to improve the purity of extracellular vesicle samples. Therefore, to increase the yield of lotus leaf exosomes in the present invention, the present invention combines ultrafiltration, density gradient centrifugation and column chromatography to separate and purify lotus leaf exosomes. Figure 5 For the purified sample and particle size distribution diagram, since the combination method includes density gradient centrifugation, the final number of samples is plural, so the present invention also analyzes the samples in different sucrose concentration layers. Figure 5 A shows the result after final density gradient centrifugation. After centrifugation, obvious bands can be observed in three intervals, namely 8%-30%, 30%-45% and 45%-60%.
[0084] After collecting samples from these three intervals and measuring their particle sizes by NTA, the results showed that the lotus leaf extracellular vesicles in the 30%-45% interval had the largest average particle size, while the average particle size of the lotus leaf extracellular vesicles isolated in the 8%-30% interval was the smallest. The average particle sizes of the combined density gradient ultracentrifugation (CDGU) 8-30% EVs (CDGU8-30%-EVs), 30-45% EVs (CDGU30-45%-EVs), and 45-60% EVs (CDGU45-60%-EVs) were 141.8±44.3 nm, 189.6±75.4 nm, and 170±64.6( Figure 5 B). In terms of yield, CDGU30-45%-EVs had the highest yield, which was 7.93±0.46×10 8 particles per gram of fresh leaf weight. Followed by CDGU45-60%-EVs, which was approximately 4.87±0.19×10 8 particles per gram of dry leaf weight. The yield of CDGU8-30%-EVs was the lowest, approximately 3.38±0.16×10 8 particles per gram of dry leaf weight( Figure 5 C). Further comparison of their zeta potentials revealed that CDGU8-30%-EVs had the largest potential of approximately -60 mV, while the other two samples were approximately -40 mV, and the particle stability was good( Figure 5 D).
[0085] 5. Lotus leaf extracellular vesicles alleviate the inflammatory symptoms induced by LPS in mouse macrophages
[0086] To understand whether lotus leaf extracellular vesicles (LDEVs) have anti-inflammatory effects, the present invention conducted an anti-inflammatory test using the LPS-induced inflammation model of mouse macrophages RAW264.7. The experimental results are as Figure 6 shown. When LPS was added during cell culture, the cell viability of the RAW264.7 cell line decreased significantly due to the intervention of LPS. However, when LDEVs obtained by the TFF or SEC purification method were added, it was found that the cell viability increased significantly and increased with the increase in the concentration of LDEVs( Figure 6 A). On the other hand, by detecting the nitrite concentration of the cell line with Griess reagent, it was found that when the RAW264.7 cell line was intervened with LDEVs under LPS induction, the inflammatory response induced by LPS could be significantly reduced, and its nitrite concentration would also decrease with the increase in the concentration of LDEVs( Figure 6B). The experimental results also showed that the reduction of nitrite concentration was indeed related to the intervention of LDEVs. Although LDEVs obtained by the UC method could not effectively alleviate the decrease in cell viability caused by LPS, they still had an anti-inflammatory effect. Based on the above results, the present invention confirmed that LDEVs extracted and purified by any method had an anti-inflammatory effect, and among them, TFF had the best anti-inflammatory ability.
[0087] 6. Lotus leaf extracellular vesicles have the ability to heal wounds in vitro
[0088] To understand whether lotus leaf extracellular vesicles have the ability to repair cells and promote wound healing, the present invention used human skin keratinocytes, the HaCaT cell line, and explored it through cell migration assays in in vitro experiments. The results are as Figure 7 shown. When different concentrations of lotus leaf extracellular vesicle samples were intervened, the reduction of the wound area was significantly accelerated, and the cell migration speed increased with the increase of the sample concentration ( Figure 7 A), and at a concentration of 1×10 11 particles / ml, the wound had completely healed after the third day ( Figure 7 B). In contrast, in the group treated with PBS, the wound area only shrank by 40% after three days. This experimental result shows that lotus leaf extracellular vesicles have the ability to improve wound repair and can shorten the wound healing time course.
[0089] The present invention confirmed that lotus leaf exosomes can be extracted by different methods, and among them, the tangential flow filtration system in the ultrafiltration method can effectively extract and purify lotus leaf exosome particles, and its yield is ten times that of other methods.
[0090] In summary, the present invention extracted lotus leaf exosomes from lotus leaves by different separation methods, and established the existence of lotus leaf exosomes; in addition, the present invention further determined the characteristics of the lotus leaf exosomes, established its purification method and qualitative standard, and the lotus leaf exosomes obtained by the above methods had similar particle sizes and stable zeta potentials. In addition, the present invention was experimentally confirmed that the lotus leaf exosomes can be used to alleviate inflammatory reactions or wound healing, and can be further used to prepare anti-inflammatory or wound healing drugs, compositions or nutritional supplements.
[0091] The present invention has been described in detail above. However, the above are only the preferred embodiments of the present invention, and the scope of implementation of the present invention cannot be limited thereby. That is, all equivalent changes and modifications made according to the scope of the patent application of the present invention should still fall within the scope of the patent of the present invention.
Claims
1. A lotus leaf exosome, which is extracted from lotus leaves by a method selected from the group consisting of: polymer precipitation method, ultra-high speed centrifugation method, ultrafiltration method, density gradient centrifugation method, and column chromatography method.
2. The lotus leaf exosome according to claim 1, wherein the extraction method is the ultrafiltration method.
3. The lotus leaf exosome according to claim 1, which has a particle size of 50 to 300 nm.
4. The lotus leaf exosome according to claim 1, which has a zeta potential of less than -20 mV.
5. The lotus leaf exosome according to claim 4, which has a zeta potential of less than -30 mV.
6. The lotus leaf exosome according to any one of claims 1 to 5, which is used for relieving inflammatory reactions or wound healing.
7. The lotus leaf exosome according to claim 6, wherein the inflammatory reaction is caused by LPS.
8. An anti-inflammatory composition, which comprises the lotus leaf exosome according to any one of claims 1 to 7.
9. The composition according to claim 8, which further comprises a carrier.
10. A use of a lotus leaf exosome, which is used for preparing a drug, composition or nutritional supplement for anti-inflammation or wound healing.