LOTUS LEAF-DERIVED EXOSOMES HAVING DISPERSION STABILITY AND THEIR USE FOR ATTENUATING INFLAMMATORY REACTION OR FOR WOUND HEALING

Abstract

The present invention provides a lotus-derived extracellular vesicle (LDEV), which is extracted from lotus leaves by a group selected from the following extraction methods: a polymer precipitation method, an ultracentrifugation method, an ultrafiltration method, a density gradient centrifugation method, and size exclusion chromatography. The present invention also provides an anti-inflammatory composition containing the LDEV and the use of the LDEV. The LDEV extracted by different separation methods in the present invention has similar particle sizes and stable zeta potentials. In addition, the present invention has experimentally confirmed that the LDEV can be used to alleviate an inflammatory reaction or heal a wound, and can further be used to prepare anti-inflammatory or wound healing-promoting drugs, compositions, or nutritional supplements.

Description

Title of the invention: LOTUS LEAF-DERIVED EXOSOMES HAVING DISPERSION STABILITY AND THEIR USE FOR ATTENUATING INFLAMMATORY REACTION OR FOR HEALING WOUNDS CONTEXT OF THE INVENTION

[0001] 1. Technical field

[0002] The present invention relates to exosomes of plant origin and more particularly to exosomes derived from lotus leaves.

[0003] 2. Description of the prior art

[0004] Extracellular vesicles (EVs) are vesicles produced by cells and having a lipid bilayer structure. Widely distributed in living organisms, including animals, plants and microorganisms, EVs were originally considered as cellular fragments responsible for removing waste from cells. However, because these vesicles carry a large amount of proteins, lipids, RNA and DNA, their size varies from 50 to 1000 nm and they can freely cross cell membranes, they are now considered not only as a carrier of intracellular waste, but also as an important element of intercellular communication.

[0005] Depending on their mode of formation, EVs can be divided into two major types: exosomes and microvesicles. Exosomes are 30–150 nm in size and are essentially intraluminal vesicles (ILVs) in multivesicular bodies (MVBs). ILVs are released from cells as exosomes when MVBs fuse with cell membranes. Microvesicles, on the other hand, are 50–1000 nm in size and are released from cells through a budding process. With the increase in studies on EVs in the past decade, scientists have gradually discovered that EVs have diverse bioactivity and participate in various physiological and pathological processes in living organisms, such as blood coagulation, angiogenesis, immunoregulation, and inflammation.Studies have also shown that different cell types regulate EV biosynthetic processes according to their respective physiological state to release specific lipids, proteins, and nucleic acids, and therefore, EVs can be used as biomarkers for cancer treatment. Furthermore, due to their ability to cross cells, EVs can be used to treat diseases by serving as nanocarriers for drugs.

[0006] Plant-derived exosome-like nanoparticles (PDENs) are nanoparticles found in plants, either in the cytoplasm or in the extracellular matrix, and are similar to exosomes. Recently, with the progress of research, more and more studies have proven that PDENs have various bioactive functions such as anti-oxidation, anti-inflammation, regulation of enteric bacterial flora, and prevention / treatment of cancer. PDENs can also be used as drug carriers. Many researchers have successfully extracted PDENs from ginger, grape, grapefruit, lemon, apple, cherry, strawberry, tomato, wheat, carrot, Chinese cabbage, and cabbage.

[0007] Although PDENs have enormous potential for application in clinical medicine, it is very difficult to extract them from living organisms efficiently and accurately because, like other exosomes, PDENs are nano-sized particles and, for the most part, heterogeneous. Currently, due to the lack of a standard purification method, the purification of PDENs is carried out by referring to the purification methods of exosomes in mammalian cells. However, if the purification method of exosomes changes, the level of membrane proteins and the content of the obtained exosomes also change. In addition, studies have shown that the lipid composition of PDENs is a key factor in determining the target cells of exosomes.For example, grape-derived exosome-like nanoparticles can only be delivered to cells in the intestinal tract, but lipid-modified grape-derived exosome-like nanoparticles can be delivered to brain cells through the nasal cavity. All the above-mentioned studies indicate that establishing a standard purification process capable of stabilizing the quality of PDENs is a problem in clinical applications that requires a solution.

[0008] Lotus (Nelumbo nucifera Gaertn.), also known as Indian lotus, is a perennial aquatic plant belonging to the genus Nelumbo of the family Nelumbonaceae and widely planted in East Asia and India. It is a decorative plant that can also be used to prepare food and beverages. In traditional Chinese medicine, lotus has long been used to treat diseases such as bloody vomiting, nosebleeds, and high blood lipid levels. Studies have also shown that lotus has a variety of pharmacological and physiological activities, for example, in liver protection, anti-oxidation, treatment of diarrhea, treatment of viral infections, immunoregulation, and reduction of body fat.

[0009] The whole lotus plant, including the leaf, flower, rhizome and pod, is useful, making lotus a crop of high economic value. The pharmacological functions of lotus stem from the various bioactive substances it contains in abundance, including polysaccharides, essential oils, flavonoids, alkaloids, and triterpenoids, among which flavonoids and alkaloids have been the most studied. In particular, the separation, purification, and activity analysis of flavonoid compounds in lotus leaves have been major objectives of lotus leaf research. Summary of the invention

[0010] The content of the section is intended to provide a simplified version of the contents of the present invention so that the reader has a basic understanding of the invention. The summary of the invention does not constitute a complete description of the invention and is not intended to highlight the important / key elements of an embodiment of the invention or to define the scope of the invention.

[0011] Recent studies on PDENs have shed increasing light on the functions and applications of PDENs. Although several kinds of fruits and vegetables have been reported, no research has been conducted on lotus leaf-derived exosome-like nanoparticles. Considering the many bioactive substances present in lotus leaves, it is of interest to evaluate whether or not lotus leaves contain PDENs with bioactivity. It is known that PDENs are capable of transporting mRNAs, miRNAs, bioactive substances, and proteins from various plants into animal cells and thus improving their viability. Previous studies have also shown that lotus leaves contain various active substances and can be used as health foods or nutritional supplements.Therefore, the inventor of the present invention conducted a series of experiments to determine whether lotus leaves also contain exosome-like nanoparticles or not, to evaluate the effects of different plant-derived EV extraction methods on lotus leaf-derived exosome particles, to establish a purification method and a qualitative standard for lotus leaf-derived exosomes, and to analyze whether or not the lotus leaf-derived exosome nanoparticles contain effective bioactive substances.

[0012] In one embodiment of the present invention, lotus leaf-derived exosomes are extracted from lotus leaves by an extraction method selected from the group consisting of: polymer precipitation, ultracentrifugation, ultrafiltration, density gradient centrifugation, and size exclusion chromatography.

[0013] In one embodiment of the present invention, the extraction method is ultrafiltration.

[0014] In one embodiment of the present invention, the lotus leaf-derived exosomes have particle sizes ranging from 50 to 300nm.

[0015] In one embodiment of the present invention, the lotus leaf-derived exosomes have a zeta potential of less than -20 mV.

[0016] In one embodiment of the present invention, lotus leaf-derived exosomes have a zeta potential of less than -30 mV.

[0017] In one embodiment of the present invention, lotus leaf-derived exosomes are used to alleviate an inflammatory response or heal a wound.

[0018] In one embodiment of the present invention, the inflammatory reaction is caused by a lipopolysaccharide (LPS).

[0019] In another aspect, the present invention also provides an anti-inflammatory composition comprising the lotus leaf-derived exosomes described above.

[0020] In one embodiment of the present invention, the anti-inflammatory composition further comprises a carrier.

[0021] In yet another aspect, the present invention also provides a use of the lotus leaf-derived exosomes in the preparation of an anti-inflammatory or wound healing medicament, composition or nutritional supplement.

[0022] The present invention has the following advantages: the present invention discloses the extraction of lotus leaf-derived exosomes by different separation methods and verifies the existence of lotus leaf-derived exosomes. In addition, the characteristics of the obtained lotus leaf-derived exosomes have been identified, and purification methods and quality standards have been established for the lotus leaf-derived exosomes. The lotus leaf-derived exosomes obtained by the above-mentioned methods not only have similar particle sizes, but also zeta potentials that indicate stability. It has also been proven through experimentation that the lotus leaf-derived exosomes can be used to alleviate an inflammatory reaction or heal wounds and can be used to prepare anti-inflammatory or wound healing drugs, compositions, or nutritional supplements. BRIEF DESCRIPTION OF THE FIGURES

[0023] The foregoing and other objects, features and advantages of the present invention may be better understood by referring to the following detailed description of certain embodiments of the invention in conjunction with the accompanying drawings, in which:

[0024] [Fig. 1] shows a process flow of a sample pretreatment of lotus leaf-derived exosomes according to the invention;

[0025] [Fig.2A] [Fig.2B] [Fig.2C] [Fig.2D] [Fig.2E] [Fig.2F] show particle size distributions of lotus leaf-derived exosomes extracted using different methods, among them, [Fig.2A] shows the distribution corresponding to a polymer precipitation method, [Fig.2B] shows the distribution corresponding to an ultrafiltration method, [Fig.2C] shows the distribution corresponding to size exclusion chromatography, [Fig.2D] shows the distribution corresponding to an ultracentrifugation method, [Fig.2E] shows the distribution corresponding to a density gradient centrifugation method and a 30-45% sucrose solution layer, and [Fig.2F] shows the distribution corresponding to the density gradient centrifugation method and the 45-60% sucrose solution layer;

[0026] [Fig.3A] [Fig.3B] [Fig.3C] [Fig.3D] [Fig.3E] [Fig.3F] show observation results of the appearances of lotus leaf-derived exosomes extracted using different methods, among them, [Fig.3A] shows the result corresponding to the polymer precipitation method, [Fig.3B] shows the result corresponding to the ultrafiltration method, [Fig.3C] shows the result corresponding to size exclusion chromatography, [Fig.3D] shows the result corresponding to the ultracentrifugation method, [Fig.3E] shows the result corresponding to the density gradient centrifugation method and the 30-45% sucrose solution layer, and [Fig.3F] shows the result corresponding to the density gradient centrifugation method and the 45-60% sucrose solution layer;

[0027] [Fig.4] is a graph showing the zeta potentials of leaf-derived exosomes lotus extracted using different processes;

[0028] [Fig.5A] [Fig.5B] [Fig.5C] [Fig.5D] show experimental results of samples taken from lotus leaf-derived exosomes extracted using a combined method, among them, [Fig.5A] shows the result of the final density gradient centrifugation, [Fig.5B] shows the particle size distributions corresponding to the combined method, [Fig.5C] shows the yields of lotus leaf-derived exosomes produced by the combined method, and [Fig.5D] shows the zeta potentials corresponding to the combined method;

[0029] [Fig.6A] [Fig.6B] show experimental results of the anti-inflammatory effects of lotus leaf-derived exosomes extracted using different methods, among them, [Fig.6A] shows the cell viability of RAW264.7 macrophages after the intervention of lipopolysaccharide (LPS), and [Fig.6B] shows the result of a nitrite test on the anti-inflammatory effects of the intervention of lotus leaf-derived exosomes in RAW264.7 macrophages having a LPS-induced inflammatory response, in which statistical analysis was performed by one-way analysis of variance (one-way ANOVA) and Tukey post-hoc analysis, and the data of TFF, UC, and SEC groups show significant differences (p < 0.05) compared with the data of LPS group; and

[0030] [Fig.7A] [Fig.7B] show results of an in vitro test on the wound healing capacity of lotus leaf-derived exosomes of the invention, [Fig.7A] shows time-lapse microscopic images showing the migration distances of HaCaT cells after intervention of lotus leaf-derived extracellular vesicles (LDEV) at different concentrations, and [Fig.7B] shows the changes in the wound area, where statistical analysis was performed by one-way ANOVA and Tukey post-hoc analysis, and the data of the LDEV group show significant differences (p < 0.05) compared with the data of the control group (PBS group). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The technical content of the present invention is detailed below with reference to the accompanying drawings. It should be emphasized that, for ease of description, the drawings are not necessarily drawn to scale and that the drawings and the proportions shown therein are not intended to limit the scope of the invention.

[0032] Unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as would generally be attributed to them by a person of ordinary skill in the art. In this application, the following terms have their respective meanings defined as follows.

[0033] In this document, the term "lotus leaf" means a lotus leaf.

[0034] The terms “lotus leaf-derived exosome”, “extracellular vesicle lotus leaf-derived”, “lotus leaf-derived exosome-like nanoparticle” and “lotus leaf-derived exosome nanoparticle” are interchangeable and refer to plant-derived exosome-like nanoparticles extracted from the lotus leaf.

[0035] One aspect of the present invention provides lotus leaf-derived exosomes that are extracted from lotus leaves by an extraction method selected from the group consisting of: a polymer precipitation method, an ultracentrifugation method, an ultrafiltration method, a density gradient centrifugation method, and a size exclusion chromatography method. In a preferred embodiment, the method for extracting the lotus leaf-derived exosomes is an ultrafiltration method.

[0036] Due to the lack of previous studies on lotus leaf-derived exosomes, the inventor of the present invention attempted to use different methods, or a combination thereof, to extract and purify lotus leaf-derived exosomes, before comparing the characteristics of lotus leaf-derived exosomes extracted using the different methods, and thereby finding the differences between these characteristics. The experiments showed that the lotus leaf-derived exosomes obtained by the different extraction methods did not differ much in terms of particle size.In one embodiment of the invention, the lotus leaf-derived exosomes have particle sizes ranging from 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.

[0037] After determining the particle size of the lotus leaf-derived exosomes extracted according to the different methods, the inventor of the present invention analyzed the zeta potentials of the lotus leaf-derived exosomes to determine the stability of these particles. In general, with respect to colloidal particles, the higher the zeta potential, the greater the thickness of the electrical double layer (i.e., the distance between the sliding plane corresponding to a particle and the surface of the particle), and the greater the thickness of the electrical double layer, the less likely adjacent particles are to be attracted to each other and coagulate, i.e., the more likely colloidal particles are to remain stably suspended in a colloidal solution.Conversely, in a solution of particles with a low zeta potential, the weak repulsive forces of the electric charges on the particles tend to cause coagulation (i.e., the formation of large granules) and thus precipitation. Currently, it is generally considered that particles tend to coagulate when their zeta potential is 0+5 mV, that particles are slightly stable when their zeta potential is ±10 to 30 mV, that particles have moderate stability when their zeta potential is ±30 to 40 mV, that particles have good stability when their zeta potential is ±40 to 60 mV, and that particles have excellent stability when their zeta potential is greater than 60 mV or less than -60 mV.In one embodiment of the invention, the lotus leaf-derived exosomes have a zeta potential of 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-derived exosomes have a zeta potential of less than -30 mV.

[0038] Furthermore, the inventor of the present invention used a combination of two or more of the aforementioned extraction and purification methods to extract EVs. The results show that lotus leaf-derived exosomes extracted by a combined method had particle sizes similar to those of exosomes extracted by a single method, had zeta potentials indicating good stability, and had higher yields than those extracted by a single method (see [Fig. 5A] to [Fig. 5D]). Thus, the present invention established methods for extracting lotus leaf-derived exosomes and identified the different characteristics of lotus leaf-derived exosomes extracted with the different methods.

[0039] In one embodiment of the present invention, lotus leaf-derived exosomes are used to alleviate an inflammatory reaction or heal a wound. In a preferred embodiment, the inflammatory reaction is caused by a lipopolysaccharide (LPS). Since lotus leaves are known for their multiple bioactive substances with antioxidant and anti-inflammatory effects, the inventor of the invention conducted an anti-inflammatory experiment to determine whether lotus leaf-derived exosomes also have an anti-inflammatory effect. The anti-inflammatory experiment used LPS to induce an inflammation mode of RAW264.7 mouse macrophages, and lotus leaf-derived exosomes were administered to the macrophages to test the anti-inflammatory ability of the exosomes.The test results show that all lotus leaf-derived exosomes extracted with the different methods were anti-inflammatory and able to attenuate the LPS-induced inflammatory response (see [Fig.6A] and [Fig.6B]).

[0040] Furthermore, wound healing is a complex physiological process that includes a series of inflammatory responses, cell regeneration, cell migration, etc. The healing process of a skin wound usually begins with an inflammatory reaction that occurs immediately after the skin is injured. Although inflammation helps eliminate bacteria and injured tissue around the wound, an excessive inflammatory reaction can negatively impact healing and delay the repair process. Therefore, appropriate anti-inflammatory treatment aimed at reducing this excessive inflammatory reaction can accelerate wound healing while alleviating the pain and discomfort caused by inflammation.Now that the inventor of the present invention has found that lotus leaf-derived exosomes have an anti-inflammatory effect, the wound healing effect of lotus leaf-derived exosomes was then tested. The test results show that lotus leaf-derived exosomes are indeed effective in wound healing and that the higher the concentration of lotus leaf-derived exosomes. The higher the lotus leaf content, the stronger the wound healing ability (see [Fig.7A] and [Fig.7B]). It can therefore be inferred that lotus leaf-derived exosomes have the ability to enhance wound repair and shorten the time required for healing.

[0041] In the preceding experiments, fresh lotus leaves were used as the raw material for extraction. The present invention, however, is not limited to the use of fresh lotus leaves. Withered or dried lotus leaves can also be used as the raw material for extraction.

[0042] Another aspect of the present invention provides an anti-inflammatory composition that comprises the aforementioned lotus leaf-derived exosomes. In one embodiment of the invention, the anti-inflammatory composition further comprises a carrier. In other words, the composition is mixed with a pharmaceutically acceptable carrier according to a conventional drug combination technique in order to meet the requirements of a drug preparation process, to facilitate the preparation of a dose of a drug, or to satisfy the requirements of a dosage form. Suitable pharmaceutically acceptable carriers are well known to a person of ordinary skill in the art. Furthermore, the anti-inflammatory composition may comprise a pharmaceutically acceptable excipient, buffer, or stabilizer, or other ingredients well known in the art.These well-known ingredients must be non-toxic and not interfere with the performance of the active ingredients. The excipient may include one or more surfactants, organic or inorganic salts, diluents, solutes, thickeners, reducing agents, antioxidants, chelating agents, preservatives, etc.

[0043] In one embodiment of the present invention, the anti-inflammatory composition is applied to vertebrates. In a preferred embodiment, the anti-inflammatory composition is applied to mice. In a preferred embodiment, the anti-inflammatory composition is applied to humans. The anti-inflammatory composition of the invention can be administered orally or by injection, for example by intravenous therapy or subcutaneous injection, as a medicament for alleviating the inflammatory reaction.

[0044] Another aspect of the present invention relates to a use of lotus leaf-derived exosomes in the preparation of an anti-inflammatory or healing medicament, composition or nutritional supplement. METHODS OF IMPLEMENTATION

[0045] It is understood that the examples and embodiments described herein serve only to explain the present invention and to suggest to those skilled in the art various modifications or changes which fall within the scope of the present application and the appended claims and which do not depart therefrom.

[0046] To determine whether or not lotus leaves contain exosome-like nanoparticles and whether these nanoparticles can be successfully separated, the inventor of the present invention used a polymer precipitation method, an ultracentrifugation method, an ultrafiltration method, a density gradient centrifugation method, a size exclusion chromatography method and a combined method to extract and separate the exosome-like nanoparticles, a size exclusion chromatography method and a combined method to extract and purify the lotus leaf-derived exosome-like nanoparticles, analyzed the size and concentration of the nanoparticles using a nanoparticle tracking analyzer and then observed the appearance of the nanoparticles under an electron microscope.Furthermore, once the exosome nanoparticles were separated, a comparison was performed to identify the differences between the lotus leaf-derived exosome nanoparticles extracted with the different methods; specifically, the particle size, yields, and appearance of the exosome-like nanoparticles were analyzed.

[0047] Experimental methods

[0048] 1. Extraction and purification of leaf-derived exosome-like nanoparticles lotus

[0049] The lotus leaves used in the experiments were samples provided by BO HUI BIOTECH CO., LTD. To successfully extract and separate the lotus leaf-derived exosome-like nanoparticles, the lotus leaf samples underwent pretreatment (as shown in [Fig.l]) before extracting the lotus leaf-derived exosome-like nanoparticles using the different separation methods.

[0050] 1-1. Precipitation of polymers (PEG)

[0051] The lotus leaves were added to an appropriate amount of double-distilled water, crushed in a juicer, and then filtered through a sieve filter having a pore diameter of 300 μm. Then, the filtrate was subjected to a sequential centrifugation process (2,000xg for 10 minutes; 6,000xg for 30 minutes; and 15,000xg for 60 minutes) to remove large pieces / sections of lotus leaf tissue and cell fragments, and a polyvinylidene difluoride (PVDF) membrane having a pore diameter of 0.8 μm was then used for filtration (see [Fig.l]), before the pH of the filtrate was adjusted to 5.0 with hydrogen chloride. Then, PEG6000 (with a final concentration of 10%) was added, and the mixed solution was left to stand overnight at 4°C, and then centrifuged at 8,000xg for 30 minutes. The precipitate obtained by centrifugation was exosome-like nanoparticles. The exosome-like nanoparticles were suspended in an appropriate amount of double-distilled water, then dialyzed for 24 hours through a 10 kDa dialysis membrane, and then filtered through a membrane filter with a pore diameter of 0.45 μm, so that clean exosomes were obtained.

[0052] 1-2. Ultracentrifugation (UV)

[0053] Large pieces / sections of lotus leaf tissue and cell fragments were removed by the same steps as the initial steps of the polymer precipitation process. A PVDF membrane with a pore diameter of 0.8 μm was then used for filtration (see [Fig.l]), and the filtrate was centrifuged at 120,000xg for 2 hours to obtain exosome-like nanoparticles. The exosome-like nanoparticles were washed twice with double-distilled water (centrifuged at 120,000xg for 2 hours), then suspended in an appropriate amount of double-distilled water, and finally filtered through a membrane filter with a pore diameter of 0.45 μm, so that clean exosomes were obtained.

[0054] 1-3. Ultrafiltration (UF)

[0055] Ultrafiltration was carried out using the tangential flow filtration (TFF) method. Large pieces / sections of lotus leaf tissue and cell fragments were removed by the same steps as the initial steps of the polymer precipitation method. A PVDF membrane with a pore diameter of 0.8 μm was then used for filtration (see [Fig.l]), and the filtrate was introduced into a TFF system and filtered through hollow fiber membranes. Initially, filtration was carried out using a hollow fiber membrane with a pore diameter of 0.65 μm (D02-E65U-07-S). The filtrate was collected and then concentrated with a 750 kDa hollow fiber membrane. Finally, solution displacement was performed with double-distilled water (the displacement volume being 1 liter), and clean exosomes were obtained after filtration through a filter membrane with a pore diameter of 0.45 μm.

[0056] 1-4. Density gradient (DG) centrifugation

[0057] Exosome-like nanoparticles were obtained by the ultracentrifugation method, and the obtained solution was placed above a sucrose solution having a concentration gradient (composed of 8%, 30%, 45% and 60%). After centrifugation at 120,000xg for 2 hours, the solution in the band between the 8% and 30% layers and the solution in the band between the 30% and 45% layers were extracted and washed twice with double-distilled water (centrifugation at 120,000xg for 2 hours). The obtained exosome-like nanoparticles were then suspended in an appropriate amount of double-distilled water, and Clean exosomes were obtained after filtration through a membrane filter with a pore diameter of 0.45 μm.

[0058] 1-5. Size exclusion chromatography (SEC)

[0059] The exosome-like nanoparticles were obtained by the ultracentrifugation method and were reconstituted in an appropriate amount of double-distilled water. The resulting suspension was introduced into a qEV column from the top, and the separation took place without applying pressure. Since each mL defined a unit fraction, the solutions of the different fractions were collected. Then, the solutions in the fractions where the sample was present were mixed and filtered through a membrane filter having a pore diameter of 0.45 μm for further analysis.

[0060] 1-6. Combined method

[0061] When purifying exosomes, the purified exosomes tend to be contaminated with lipoproteins due to the similarity in size and density between exosomes and lipoproteins. Since density gradient centrifugation is based on density differences and size exclusion chromatography performs the separation of exosomes based on molecular weights, the inventor of the present invention evaluated whether higher purity exosomes could be separated by a combination of different methods.

[0062] The combined method was essentially a combination of ultrafiltration, density gradient centrifugation, and size exclusion chromatography. First, lotus leaf-derived exosome-like nanoparticles were obtained by ultrafiltration. Then, the precipitate obtained by centrifugation was reconstituted in an appropriate amount of double-distilled water, before separation took place in a qEV column without applying pressure. The solutions of the fourth to sixth fractions were collected and mixed, and the mixed solution was placed above a sucrose solution having a concentration gradient (composed of 8%, 30%, 45%, and 60%).After centrifugation at 100,000xg for 2 hours, the solutions in the band between the 8% and 30% layers, in the band between the 30% and 45% layers and in the band between the 45% and 60% layers were extracted, washed twice with double-distilled water (centrifugation at 100,000xg for 2 hours), and then filtered through a membrane filter with a pore diameter of 0.45 μm for further analysis.

[0063] 2. Analysis of the characteristics and morphological observation of the nanoparticles of exosome type derived from lotus leaf.

[0064] The analysis of the concentrations and particle size distributions of the obtained lotus leaf-derived exosome-like nanoparticles was entrusted to the Department of Medical Research of National Taiwan University Hospital and was carried out using a nanoparticle tracking analyzer (NTA) (NanoSight NS300). The zeta potentials of lotus leaf-derived exosome-like nanoparticles were measured using a dynamic light scattering particle size analysis and zeta potential analysis instrument (Zetasizer Nano, Malvem). The morphologies of the purified exosome nanoparticles were observed by transmission electron microscopy (TEM).

[0065] 3. Cell culture and induction of inflammation mode

[0066] A cellular inflammation mode was induced in RAW264.7 mouse macrophages primarily by means of lipopolysaccharide (LPS). Specifically, RAW264.7 macrophages were seeded into a 24-well plate at a concentration of 5x105 cells per well and cultured at 37°C for 12 hours. Then, the used Dulbecco's modified Eagle's medium (DMEM), which contained phenol red, was removed, the cells were washed twice with phosphate-buffered saline (PBS), and then DMEM without phenol red was added along with LPS, so that the final concentration of LPS was 100 ng / mL. 10 minutes after LPS addition, samples of lotus leaf-derived exosome-like nanoparticles of different concentrations were added, and the cells were cultured for an additional 24 hours to complete the inflammation induction assay.

[0067] 4. Cell viability test

[0068] Cell viability was analyzed using an MTT assay. 24 hours after the inflammation induction test, the existing cell culture medium was removed, and the cells were rinsed twice with PBS, then added to fresh DMEM plus 2 g / L MTT reagent (the final concentration being 10%). After culturing at 37°C for 90 minutes, the cell culture medium was removed, and the violet crystals were dissolved in dimethyl sulfoxide (DMSO). The absorbance values ​​at the wavelength of 570 nm were then determined.

[0069] 5. Nitrite test

[0070] During cellular inflammation, the affected cells produce NO, which in turn reacts with the culture medium to produce NO2. Therefore, to assess the inflammation degrees of the cultured cells, the NO2 level of the cell culture medium in each well was tested using a Griess reagent. Specifically, after the 24-hour culture in the inflammation mode induction test, 100 qL of cell culture medium was taken from each well and then added with the Griess reagent so that the reactions took place. After the reactions were completed, the absorbance values ​​at the wavelength of 550 nm were determined. The nitrite concentrations were calculated by substituting each absorbance value obtained from the test to a standard calibration curve prepared with a standard nitrite solution.

[0071] 6. In vitro healing test

[0072] The wound healing assay was performed primarily on human keratinocytes (HaCaT cells). First, a two-well culture insert was placed in each well of a 24-well plate, and each well was seeded with 3x104 cells. The cells were cultured overnight in high-concentration DMEM containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 environment. The culture inserts were removed the next day, and LDEVs were added at different concentrations to the culture medium. The migrations of HaCaT cells were then observed, photographed, and analyzed. The cells were observed using a cell heating and culture system (Stage Top Ibidi incubator), and the wounded areas, which changed with cell migration, were analyzed using ImageJ.

[0073] Experimental results

[0074] 1. Effects of different extraction processes on the particle size of exosomes lotus leaf derivatives

[0075] To know the effects of different extraction methods on the separation of lotus leaf-derived exosome-like nanoparticles, the first step was to extract lotus leaf-derived exosomes by the different separation methods, namely polymer precipitation, ultracentrifugation, ultrafiltration, density gradient centrifugation and size exclusion chromatography. The material on which the extraction was carried out was fresh lotus leaves, and before the extraction began, the fresh lotus leaf samples received the pretreatment shown in [Fig.l].

[0076] After sample pretreatment, the different methods were used separately to separate the lotus leaf-derived exosomes. [Fig.2A] to [Fig.2F] show the particle size distributions of the lotus leaf-derived exosomes extracted with the different methods. As shown in the results of [Fig.2A] to [Fig.2F], the lotus leaf-derived exosome particles obtained by the different extraction methods had similar particle sizes that did not differ much among the extraction methods. The particle size was mainly distributed between 50 and 300 nm. The average particle size of the lotus leaf-derived exosomes obtained by the different extraction methods is 152.4+49 nm (for EVs extracted by the polymer precipitation method, hereinafter referred to as PEG-EVs for short; see [Fig.2A]), 159.5+48.5 nm (for EVs extracted by the tangential flow filtration method, hereinafter referred to as TFF-EV for short; see [Fig.2B]), 163.2+70.1 nm (for EVs . extracted by the size exclusion chromatography process, hereinafter referred to as SEC-EV for short; see [Fig.2C]), 166.5+64.3 nm (for EVs extracted by the ultracentrifugation method, hereinafter referred to as UC-EV for short; see [Fig.2D]), about 132.6+39.9 nm (for EV particles obtained from the 30-45% sucrose solution layer of the density gradient centrifugation method, hereinafter referred to as DGU30-45%-EV for short; see [Fig.2E]), and about 136.3+52 nm (for EV particles obtained from the 45-60% sucrose solution layer of the density gradient centrifugation method, hereinafter referred to as DGU45-60%-EV for short; see [Fig.2F]). Of all the lotus leaf-derived exosomes obtained, DGU30-45% and DGU45-60% had the smallest average particle sizes.

[0077] 2. Effects of different extraction processes on the physical properties of exosomes derived from lotus leaves

[0078] In addition to particle size analysis by NTA, the appearance of the obtained EVs was observed under a transmission electron microscope, and the stability of the particles was evaluated by zeta potential analysis. [Fig.3A] to [Fig.3F] show the results of observing the appearance of lotus leaf-derived exosomes extracted with the different methods. It can be seen from [Fig.3A] to [Fig.3F] that there are cup-shaped particle structures (indicated by the white arrows in the images), which are typical structures found when negatively stained EVs are observed under an electron microscope. These special structures were formed during the drying of the samples, during which the surface of a vesicle may sink toward the center of the vesicle due to dryness.The TEM observation results are similar to the NTA analysis results in terms of particle size and size distribution, which proves that the particle sizes obtained by NTA are consistent with the actual particle sizes, and the particles were EVs.

[0079] In the stability analysis of exosomes obtained from lotus leaves, zeta potential was used to determine the characteristics and stability of exosomes, and the results are shown in [Fig.4]. The zeta potentials of all lotus leaf-derived exosomes extracted with the different methods were lower than -20 mV, indicating that the lotus leaf-derived exosomes extracted with the different methods were relatively stable. In particular, the zeta potentials of SEC-EV, UC-EV, DGU30-45%-EV and DGU45-60%-EV were lower than -30 mV.

[0080] 3. Effects of different extraction processes on the yield of derived exosomes of lotus leaves

[0081] To know the effects of different extraction methods on the yield of lotus leaf-derived exosomes, the yields of the obtained lotus leaf-derived exosomes were further analyzed. Referring to Table 1, which shows the result of analyzing the yields of lotus leaf-derived exosomes extracted with the different methods, the tangential flow filtration method produced the highest yield (about 3.69±0.02xl09) of lotus leaf-derived exosomes, followed by the ultracentrifugation method and the PEG precipitation method, the yields of these two methods being 2.20+0.13xl08 and 2.13+0.07xl08, respectively. The size exclusion chromatography method produced the lowest yield (about 4.62±0.06xl07) of lotus leaf-derived exosomes.Furthermore, a comparison of the yields of DGU30-45%-EVs and DGU45-60%-EVs reveals that the yield of DGU30-45%-EVs is approximately twice as high as that of DGU45-60%-EVs. Since the density gradient centrifugation method is equivalent to an even further separation of total EVs, the result of the density gradient centrifugation shows that the lotus leaf-derived exosome particles extracted according to the present invention had densities mostly between 1.13 and 1.2 g / mL. Since this range is considered the density range of animal cell exosomes, it can be inferred that the LDEV particles extracted according to the invention are exosome-like nanoparticles.

[0082] Table 1. Yields of lotus leaf-derived exosomes extracted using different extraction methods [Tables 1] Process Yield (mean±standard deviation; number of particles per gram of fresh leaves) PEG 2.13 + 0.07xl08, b TFF 3.69 + 0.02xl09, a SEC 4.62 + 0.06xl07, d UC 2.20 + 0.13xl08, b DGU30-45% 1.14 + 0.001xl08, c DGU45-60% 5.22 + 0.48xl07, d

[0083] 4. Effects of extraction and purification of lotus leaf-derived exosomes by the combined process

[0084] The literature has shown that using a combination of two or more extraction and purification methods to extract and purify EVs is advantageous for improving the purity of the obtained EV sample. Therefore, to increase the yield of the lotus leaf-derived exosomes of the present invention, the inventor of the invention used a combination of ultrafiltration, density gradient centrifugation, and size exclusion chromatography to separate and purify the lotus leaf-derived exosomes. The purified samples and their particle size distributions are shown in [Fig. 5A] to [Fig. 5D]. Since the combined method includes density gradient centrifugation, there were multiple final samples. The samples taken from different sucrose concentration layers were all analyzed. [Fig.5A] shows the result of the final density gradient centrifugation, and it can be seen from [Fig.5A] that after centrifugation, visible bands appeared in three fractions, namely the fraction between the 8 and 30% layers (hereinafter referred to as the 8-30% fraction), the fraction between the 30 and 45% layers (hereinafter referred to as the 30-45% fraction), and the fraction between the 45 and 60% layers (hereinafter referred to as the 45-60% fraction).

[0085] The particle size of the samples collected from these three fractions was measured using NTA and, according to the measurement results, lotus leaf-derived exosomes corresponding to the 30-45% fraction had the largest average size, while LDEVs corresponding to the 8-30% fraction had the smallest average size. More specifically, the average particle sizes of EVs obtained by combined density gradient centrifugation (CDGU) and corresponding to the 8-30%, 30-45%, and 45-60% fractions (abbreviated as CDGU8-30%-EV, CDGU30-45%-EV, and CDGU45-60%-EV respectively) are 141.8+44.3 nm, 189.6+75.4 nm, and 170+64.6 nm respectively (see [Fig.5B]).Regarding yield, CDGU30-45%-EV had the highest yield (7.93+0.46xl08 particles per gram of fresh leaves), followed by CDGU45-60%-EV (approximately 4.87+0.19xl08 particles per gram of fresh leaves), and CDGU8-30%-EV had the lowest yield (approximately 3.38+0.16xl08 particles per gram of fresh leaves) (see [Fig.5C]). A comparison of the zeta potentials further shows that CDGU8-30%-EV had the highest potential (-60 mV), the potentials of the other two samples were approximately -40 mV, and consequently all samples had good particle stability (see [Fig.5D]).

[0086] 5. Attenuation of LPS-induced mouse macrophage inflammation by to the LDEV

[0087] To find out whether LDEVs have an anti-inflammatory effect, an anti-inflammatory test was performed by inducing an inflammation mode of RAW264.7 mouse macrophages with LPS, and the experimental results are shown in [Fig.6A] and [Fig.6B] When LPS was added during cell culture, the cell viability of RAW264.7 macrophages was significantly reduced by the intervention of LPS. However, when LDEVs obtained by the TFF or SEC purification method were also added, the cell viability increased significantly and increased with the concentration of LDEV (see [Fig.6A]). In addition, the concentrations of nitrite in the cells were tested with Griess reagent, and it was found that the intervention of LDEVs in RAW264 cells.7 under LPS induction caused a significant reduction in the LPS-induced inflammatory response, and that nitrite concentrations were reduced as the LDEV concentration increased (see [Fig.6B]). The experimental results also show that the reduction in nitrite concentration is indeed associated with the intervention of LDEVs. The LDEVs obtained by the UC method failed to reduce the decrease in cell viability caused by LPS but still had an anti-inflammatory effect. Overall, the previous results demonstrate that, regardless of the extraction and purification method used, the resulting LDEVs have an anti-inflammatory effect, with the LDEVs obtained by TFF having the highest anti-inflammatory capacity.

[0088] 6. In vitro healing capacity of LDEVs

[0089] To investigate whether LDEVs have the ability to repair cells and promote wound healing, an in vitro cell migration test was performed on human keratinocytes (HaCaT cells), and the test results are shown in [Fig.7A] and [Fig.7B]. The intervention of LDEV samples at different concentrations accelerated the reduction of wound area significantly, and the cell migration speed increased with the sample concentration (see [Fig.7A]). The wound treated with the concentration of IxlO11 particles / mL healed completely at the end of the third day (see [Fig.7B]) while the wound treated with PBS only showed a 40% reduction in area at the end of the third day. From the experimental results, it can be concluded that LDEVs have the ability to improve wound repair and shorten the time required for healing.

[0090] Thus, it has been proven that lotus leaf-derived exosomes can be extracted using different methods, and that the tangential flow filtration system of the ultrafiltration method is particularly effective in extracting and purifying lotus leaf-derived exosomes, with a yield ten times higher than that of other methods.

[0091] In view of the above, the present invention discloses the extraction of lotus leaf-derived exosomes by different separation methods and verifies the existence of lotus leaf-derived exosomes. Furthermore, the characteristics of the obtained lotus leaf-derived exosomes were identified, and purification methods and quality standards were established for the lotus leaf-derived exosomes. The lotus leaf-derived exosomes obtained by the above-mentioned methods not only have similar particle sizes, but also zeta potentials that indicate stability. It has also been proven through experimentation that the lotus leaf-derived exosomes can be used to alleviate an inflammatory reaction or heal wounds and can be used to prepare anti-inflammatory or wound healing drugs, compositions, or nutritional supplements.

[0092] Although the present invention has been described in detail above, the embodiments described herein are only preferred embodiments of the invention and should not be construed as limiting the scope of the invention. Any equivalent changes or modifications based on the appended claims are within the scope of the invention.

Claims

Claims

1. Exosomes extracted from lotus leaves by an extraction method selected from the group consisting of: polymer precipitation, ultracentrifugation, ultrafiltration, density gradient centrifugation, and size exclusion chromatography.

2. Exosomes extracted from lotus leaves according to claim 1, wherein the extraction method is ultrafiltration.

3. Lotus leaf-extracted exosomes according to claim 1, which lotus leaf-extracted exosomes have particle sizes in a range of 50 to 300 nm.

4. Lotus leaf-extracted exosomes according to claim 1, which lotus leaf-extracted exosomes have a zeta potential of less than -20 mV.

5. Lotus leaf-extracted exosomes according to claim 4, which lotus leaf-extracted exosomes have a zeta potential of less than -30 mV.

6. Exosomes extracted from lotus leaves as defined according to any one of claims 1 to 5, for their use in alleviating an inflammatory reaction or healing a wound.

7. Exosomes extracted from lotus leaves for their use according to claim 6, said inflammatory reaction being caused by a lipopolysaccharide (LPS).

8. An anti-inflammatory composition comprising exosomes extracted from lotus leaves as defined in any one of claims 1 to 5.

9. A composition according to claim 8, further comprising a vehicle.

10. Use of exosomes extracted from lotus leaves as defined according to any one of claims 1 to 5, in the preparation of an anti-inflammatory or healing medicament, a composition or a nutritional supplement.

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