Lutherion and its isolation and cultivation method

Abstract

This invention provides a method for isolating and culturing luteolion, a mitochondrial-like microstructure. [Solution] A method for separating luteion, a mitochondrial-like microparticle, comprising the following steps: (a) A step of filtering a condensate obtained by cooling steam or gas obtained by boiling a plant or food extract using a filter having a void size of 0.8 to 1.2 μm; (b) the step of centrifuging the filtered condensate; and (c) A step in which luteions are separated from the centrifuged supernatant by irradiating with IR light with a wavelength of 200 to 600 μm in order to collect mobile luteion particles.

Description

Technical Field

[0001] The present invention relates to lutelion, which is a mitochondria-like fine substance, a method for separating the same, and a method for culturing the same, and specifically relates to a method for separating lutelion using a filter having pores of a specific size, lutelion having specific characteristics separated by such method, and a culturing method for proliferating the same.

Background Art

[0002] The inventors of the present application developed a method capable of effectively separating luterial, which is a fine substance present in already excreted body fluids of patients or healthy subjects, and filed a patent application on May 9, 2014 disclosing the clarified characteristics of the separated luterial (WO2015 / 108246). Further, they discovered that diseases can be diagnosed and predicted by observing the characteristics of fine substances present in already excreted body fluids of patients, and filed a patent application on January 14, 2014 disclosing the above (WO2015 / 005553).

[0003] Such luterial is (1) a cell or cell analog having intermediate fusion characteristics between prokaryotic cells and eukaryotic cells; (2) it is present in body fluids such as blood, semen, intestinal fluid, saliva, and cell fluid; (3) it shows positive color reaction to Janus green B, Acridine Orange, and Rhodamine 123 in immunofluorescence tests; (4) under optimal conditions (pH 7.2 to 7.4), it exhibits expression characteristics of genes derived from beta-proteobacteria and gamma-proteobacteria, and has a size of 30 to 800 nm; (5) under acidic conditions, it ​​​​​Expression characteristics of not only Rhea and gamma-proteobacteria-derived genes, but also eukaryotic cell-derived genes. Although it exhibits sexual characteristics, it is mainly the Sterptophyta gene and Homologue features that are expressed. (6) Under normal conditions, ATP production occurs. (7) Unlike mitochondria, exosomes are in a completely different cell or It is a cell analog.

[0004] While these luteal compounds are mainly found in the blood of animals, including humans, luteal compounds are Lutherion is a microscopic substance with a structure and function similar to ruthenium, and is mainly found in plants or food. The existence of (Luterion) was confirmed.

[0005] Under these technical circumstances, the inventors of this application can apply luterion clinically. As a result of diligent efforts to effectively separate and cultivate plants or food, After adding the gas and collecting the resulting vaporized gas while shaking, the sample is filtered and centrifuged before processing. It is possible to effectively separate luterion contained in the vaporized gas, and the luterion separated from there We have confirmed that it is possible to culture Therion under specific conditions and in a culture medium, and we have established the present invention. It's finished. [Overview of the project] [Problems that the invention aims to solve]

[0006] The objective of this invention is to provide luterion. Another object of the present invention is to use a filter having voids of a specific size to process luterion. The aim is to provide an effective separation method that can be applied clinically. Another object of the present invention is to provide an efficient method for culturing luteolions. [Means for Solving the Problem]

[0007] To achieve the above object, the present invention provides a lute rion having one or more properties selected from the following: (a) It is circular or elliptical with a size of 50 to 800 nm and has motility; (b) It contains nucleic acid; (c) It exhibits a reaction similar to mitochondria during immunochemical fluorescent staining; (d) It exhibits an ecological mode of fusion and / or fission; (e) In the absence of fusion, it matures to a size of up to 500 nm, matures into a mitochondrion-like structure containing DNA, and exhibits a structure similar to mitochondria in SEM or TEM electron micrographs; ; (f) It exhibits a photoreaction different from that of exosomes; (g) Fission occurs upon IR irradiation or pressurization; (h) It expresses CD332, CD133, CD73 or CD39 as surface antigens; (i) It exhibits autofluorescence; (j) It produces ATP with a size of 200 to 400 nm; (k) It has a double-membrane or multi-membrane structure; (l) It has adhesiveness; (m) It inhibits telomerase activity in cancer cells; (n) It promotes the activation of telomerase in normal cells; (o) It has cell permeability; and (p) It has blood-brain barrier (BBB) permeability.

[0008] The present invention also provides a method for isolating a luterion comprising the following steps: (a) Cooling a steam or gas shaking extract of a plant or food, and subjecting the obtained condensate to 0.8 filtering using a filter having pores of ~1.2 μm; (b) centrifuging the filtered condensate; and (c) step of separating luteonerion from said centrifuged supernatant.

[0009] The present invention also provides a method for separating luteonerion comprising the following steps: (a) luteonerion an antibody or aptamer that specifically binds to a luteonerion surface antigen is immobilized on an extract containing the immobilized particles are added to induce binding between luteonerion and the particles; and (b) to said particles step of recovering bound luteonerion.

[0010] The present invention also provides a method for culturing luteonerion comprising adding water to said luteonerion and culturing at 18 to 3 0°C under irradiation with IR light or under pressure.

[0011] The present invention also provides a method for culturing luteonerion comprising culturing said luteonerion in a sugar-containing medium under conditions of pH 5 to 9 and 18 to 30°C . BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Figure 1] This is a photograph of luteonerion taken with a Confocal Laser Scanning Microscope (Zeiss), and the size thereof is also shown. [Figure 2] This figure shows a photograph obtained by observing whether color development occurred after staining luteonerion with Mito-tracker. [Figure 3] This figure shows a photograph obtained by observing whether color development occurred after staining luteonerion with Rhodamine 123. [Figure 4]This image shows a comparison of fluorescent and non-fluorescent photographs of luteion (A: Janus Green B Positive; B: Rhodamine 123 Positive; C: Mito-tracker Red Positive; D: Unstained). [Figure 5] This shows the presence or absence of color development after luteolion was stained with Rhodamine 123, Mito-tracker Red, and Janus Green B (A: Janus Green B Positive; B: Rhodamine 123 Positive; C: Mito-tracker Red Positive; D: Unstained). [Figure 6] The size of the fluorescently stained lutein (50-800 nm) was measured to indicate the size of the lutein (A: Janus Green B reaction size measurement; B: Rhodamine 123 reaction size measurement; C: Mito-tracker Red reaction size measurement; D: Unstained reaction size measurement). [Figure 7] This shows the life cycling of a normal luteion (A) and the life cycling of a mutated luteion (B). [Figure 8] This is a TEM electron microscope image confirming that luteion has a bilayer structure. [Figure 9] This is an atomic microscope image confirming that luterion contains nucleic acids internally. [Figure 10A] This is the result of a bioanalyzer analysis to determine whether or not RNA is present inside the luteion (L: Control; 1: less than 50 nm, 2: 50-100 nm, 3: 100-200 nm, 4: 200-400 nm). [Figure 10B] This shows the total RNA of luteion in the 200-400 nm range. [Figure 11]This shows the results of luteinization induction under conditions of 5,000 psi and 10-20°C. [Figure 12] This shows the results of luteinization induction under conditions of 15,000 psi and 10-20°C. [Figure 13] This shows the results of luteinization induction under conditions of 35,000 psi and 10-20°C. [Figure 14] This image shows the results of introducing luterins into the nucleus by fluorescently staining luterins with PKH26-1, PKH26-2, and PKH26-3 red fluorescence. [Figure 15-16] This image shows the results of a magnified view confirming that luteion, stained with red fluorescence, is introduced into the intracellular nucleus. [Figure 17] This shows the results of administering fluorescently stained luteion orally via a gastric tube. [Figure 18-19] This shows the results of intraperitoneal injection of fluorescently stained luteolion. [Figure 20] This shows the telomerase activity in normal cells (fibroblasts) and cancer cells (lung cancer, breast cancer, colorectal cancer) after treatment with luterion. [Figure 21] This shows the cell viability of normal cells (fibroblasts) and various cancer cells (lung cancer, breast cancer, colorectal cancer, liver cancer, leukemia) according to the luterion treatment concentration. [Figure 22] This study demonstrates the suppression of cell proliferation in pancreatic cancer cell lines (AsPC-1) treated with luterins of diverse origins. [Figure 23] This study demonstrates the suppression of cell proliferation in pancreatic and lung cancer cell lines (A549) treated with lutein of various origins. [Figure 24] This study demonstrates the suppression of cell proliferation in pancreatic breast cancer cell lines (BT-20) treated with lutein of various origins. [Figure 25] This shows the telomerase activity in normal cells (Fibroblasts) treated with luterion. [Figure 26]This is a schematic diagram of an ATP assay design to confirm whether or not ATP generation is suppressed by luterion treatment. [Figure 27] This image shows the results of imaging luteion with a Carl Zeiss scanning electron microscope (SEM). [Figure 28] This image shows the results of imaging luteolins using a Bruker Fast Scan AFM atomic microscope. [Figure 29] Lutherions were stained using anti-CD39 antibody conjugate, Mito-tracker, and DAPI, and the presence or absence of color development is shown individually or combined. [Figure 30] Lutherions were stained using anti-CD73 antibody conjugation, Mito-tracker, and Hoechst, and the presence or absence of color development is shown individually or combined. [Figure 31] This shows the results of confirming whether or not Lutherion is positive for anti-CD332 antibody via fluorescence emission. [Figure 32] The graph shows the results of confirming whether luterion is positive for anti-CD133 antibody via fluorescence luminescence. The upper panel shows the results of PE (Phycoerythrin) fluorescence staining, and the lower panel shows the results of anti-FITC fluorescence staining. [Figure 33] This diagram shows the step of coating particles, on which antibodies that specifically bind to the luteion surface antigen are immobilized, with carbon and carboxyl groups. [Figure 34] This diagram illustrates the process of separating a complex formed by luteion and magnetic particles, to which an antibody that specifically binds to the luteion surface antigen is bound, using a magnet. [Figure 35]The images show the following photos taken with a fluorescence microscope after lutein separation: (a) shows a photo taken with a fluorescence microscope after lutein separation using a nanofilter; (b) shows a photo taken with a fluorescence microscope after lutein separation using a separation method that utilizes particles immobilized with antibodies that specifically bind to the lutein surface antigen; (c) shows a photo taken with an electron microscope after lutein separation using a nanofilter; and (d) shows a photo taken with an electron microscope after lutein separation using a separation method that utilizes particles immobilized with antibodies that specifically bind to the lutein surface antigen. [Figure 36] The images show the aqueous extracts of luteion, with (a) showing the aqueous extracts of luteion after separation using a nanofilter, and (b) showing the aqueous extracts of luteion after separation using a separation method that utilizes particles immobilized with antibodies that specifically bind to the luteion surface antigen. [Figure 37] This is a schematic diagram illustrating the specific steps in the extraction of luterion. [Figure 38] This shows a decrease in ATP production in cancer cells due to luterion treatment. [Figure 39] This shows ATP production in normal cells (Fibroblasts) treated with luterion. [Modes for carrying out the invention]

[0013] Unless otherwise defined, any technical or scientific use as used herein The terms have the same meaning as they would normally be understood by experts skilled in the art to which this invention pertains. It has. The nomenclature used herein and the experimental methods detailed below are generally considered to be the same as those used herein. It is well-known and widely used in the technical field.

[0014] The terms "luterial" and "luterion" used in this invention "Terion" refers to 50-400 ohms present in all living organisms, including animals and plants. These are nano-organisms the size of a m, ranging from those similar to viruses to those reaching 800-1200 nm when fused. While possessing a certain size, this mutant exhibits genetic abnormalities and pathological phenomena at wavelengths above 1200 nm. The inventors have named the fine materials classified under this category.

[0015] Luterials and luterins contain DNA and RNA and possess motility and adhesiveness. It is distinct from exosomes and microvesicles. Mitochondria are detected by Janus green B and fluorescence The dyes are Rhodamine 123 and Mito Tracker. tracker), Acridine Orange, and D The coloration is confirmed by API, but the aforementioned luteion and luteal are also mitochondria. The same staining agent was used to confirm the color development (Figures 1-6), and it was double, similar to mitochondria. A membrane structure that has a membrane but has not completed its internal cristae structure. It has a similar structure (Figure 7) and is observed in the same laser wavelength range as mitochondria, hence it is "similar to mitochondria." "Mitochondria," "Mitochondrial analog," or "Mitochondrial precursor (prot)" It can also be called "o-mitochondria".

[0016] Luterial refers to nanoorganisms that exist within the host body in an ecosystem, including humans and other animals. In some cases, blood, saliva, lymphatic vessels, semen, vaginal fluid, breast milk (especially colostrum), umbilical cord blood, brain cells, It can be present in the spinal cord and bone marrow. On the other hand, luteon can be found when the host uses it as food, etc. It refers to a type of nanoorganism that can exist primarily in plants and food.

[0017] Unlike blood-derived luteal, luteion does not dissolve or disappear quickly at room temperature. It has the characteristic of not fusing or undergoing mutation even when stored for a long period of time.

[0018] In one view, the present invention relates to a separated lute having one or more characteristics selected below. Regarding Rion: (a) Circular or elliptical in shape, 50-800 nm in size, and mobile; (b) containing nucleic acids; (c) Shows a reaction similar to that of mitochondria during immunochemical fluorescence staining; (d) Exhibiting fusion and / or fission ecological patterns; (e) If fusion does not occur, the mitochondria mature to a size of 500 nm and contain DNA-like mitochondria. In the rear, it matures and develops a structure similar to mitochondria in SEM or TEM electron microscopy images. To show; (f) Exhibits a different photoreaction than exosomes; (g) Fission occurs upon IR irradiation or pressurization; (h) Expresses CD332, CD133, CD73, or CD39 as a surface antigen; (i) Exhibits autofluorescence; (j) Produces ATP at a size of 200-400 nm; (k) Bilayer or multilayer structure; (l) Adherent; (m) Inhibition of telomerase activity in cancer cells; (n) Promoting telomerase activation in normal cells; (o) Possesses cell penetrating ability; and (p) Possesses the ability to permeate the blood-brain barrier (BBB).

[0019] According to one embodiment of the present invention, luterion is a living organism ) ranging from a degree similar to that of a virus to approximately 500 nm (normal division stage 50-500 nm) A name coined by the inventor for a fine material having a size of m (800 nm or more in the anomalous fusion stage). It is a microvesicle (microv) that contains DNA and / or RNA and is motile. It is distinct from esicle. Also, autofluorescence It also possesses the characteristics of photosynthesis. Mitochondria are stained with the fluorescent dye rhodamine 123 (Rh Odamine 123), Mitotracker, Acridine Ole Acridine Orange, DAPI, and Janus Green B The coloration can be confirmed by US Green B, but lutein is the same as mitochondria. Since the color development is confirmed by the aforementioned dye, the luteion is correctly separated by the aforementioned dye. You can check if it has been done (Figures 1-6).

[0020] Furthermore, RNs were detected within the lutein by DAPI and acridine orange (AO) staining. It can be confirmed that it contains not only A but also DNA. Specifically, RNA is A At a level where excitation occurs at 460 nm and emission occurs at 650 nm using a cridine orange stain. Stained orange, the DNA is excited at 502 nm and emitted at 525 nm. It is stained green, and according to the DAPI staining method, it can be confirmed that DNA is present. This can be done. The luteion of the present invention utilizes the aforementioned staining method to allow RNA and DN to be incorporated into the luteion. It can be confirmed that A is included.

[0021] In one embodiment of the present invention, the structure of luteion was confirmed to have a double or multilayer structure. It can be confirmed that the lutein contains nucleic acids, particularly RNA (RUT), inside. This was confirmed (Figures 9 and 10).

[0022] Previously, the fact that luterion exists in plants or food was not known, therefore, It can be said that there is no technology whatsoever for methods of isolating or culturing Lyon. According to the present invention This provides an effective method for isolating and culturing Therion.

[0023] In other words, the present invention relates to a method for separating luteion, comprising the following steps: (a ) A condensate obtained by cooling a steam or gas-shaking extract of a plant or food is 0.8-1 (b) filtering using a filter having voids of 0.2 μm; (b) the filtered condensate (c) the step of centrifuging the liquid; and (c) separating luterion from the centrifuged supernatant. The stage of doing so.

[0024] First, step (a) involves cooling and removing a steam or gas-shaking extract of a plant or food. The obtained condensate is filtered using a filter with voids of 0.8 to 1.2 μm. be.

[0025] In one embodiment, the shaking extraction involves adding a solvent to a plant or food and heating it at 50-90°C until it becomes a gas. This allows the process to proceed by intermittently bubbling and shaking. The luterion used has a density of less than 1, and is denser than fat and geology. Because it has a lower density than protein, it can be separated from plants or food by steam distillation. This is possible, but not limited to this.

[0026] When luterion is shaken while intermittently bubbling with a gas at 50-90°C, It is released along with the gas in the form of water vapor or gas, but when shaken together with the water vapor or gas... This causes the boiling point of plants or foods containing luterion to drop, leading to the decomposition and alteration of luterion. It can prevent quality or damage.

[0027] Depending on the circumstances, the mixture may be shaken for 8-10 hours in step (a) above, and then shaken for 20-30 minutes every 2-3 hours. An additional step is taken to bubble the plant or food luteinization to prevent clumping. This can increase the separation efficiency of luterion.

[0028] The specific details of the extraction-filtration in step (a) above are shown in Figure 37. Plant or After adding a solvent, such as distilled water, to the food and extracting by shaking, approximately 100-250g is obtained. Preferably, the mixture is subjected to primary centrifugation at 190g, then spun down to remove impurities, and then approximately 1000g -5000g, preferably about 3000g, is subjected to secondary centrifugation for several minutes to several tens of minutes (preferably two After stabilization (within or outside the specified time), the supernatant can be collected. In some cases, at this time C After staining D332 and confirming its motility, the presence or absence of luterins is first confirmed. This can be done. Subsequently, to obtain pure luterion, 100,000-150,000g, Preferably, a tertiary centrifugation step can be performed at 120,000 g.

[0029] Subsequently, the lower layer filtered through an approximately 800nm ​​filter is collected, and internal implants such as plasma are used. Removes debris pellets such as material or exosomes. Afterward, repeat 1 When the amount exceeds 40,000g, the supernatant liquid free of pellets can be collected by centrifugation. The supernatant liquid is filtered through a 400nm filter to collect the lower layer, and then the CD332 solution is used again. The motility can be confirmed by staining. Furthermore, it can be filtered with a 500nm filter. The supernatant liquid that settles on the filter can be collected to gather lutein, and pH 1 Alternatively, it can be stored at temperatures below -90°C.

[0030] In one embodiment, the plant can be any medicinal plant selected from Tables 1-4. Since Lyon is present in all plants, it is not limited to the medicinal plants listed in Tables 1-4.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] Furthermore, it is expected that luterion contained in plants will be more abundantly distributed in the stem of the plant, and the stem part It is preferable to isolate the luterion by including [a specific component].

[0036] The condensate is cooled after collecting the water vapor or gas that is vaporized by the shaking. It can be obtained by separating the collected vaporized water vapor or gas from the luteo. It is possible to obtain n, or luteion in a mixed state with it, vaporized water vapor Alternatively, if the gas and luteion are mixed, an additional separation procedure is required to obtain the luteion. It is possible.

[0037] Depending on the circumstances, the process may further include irradiating the condensate obtained in step (a) with IR light. That's fine too. For example, irradiate with IR light for 20-60 minutes, preferably 30-40 minutes to irradiate lutein This prevents deformation of the ON state, and before performing step (c), the mobility is controlled by IR light irradiation. By gathering luteions that possess certain properties, you can obtain high concentrations of luteion. To make it.

[0038] Step (b) is performed using a filter having a gap of 0.8 to 1.2 μm to obtain This is the step of filtering the condensed liquid. The filter has voids of 0.8 to 1.2 μm. This is the optimal size derived by the inventor of this application, taking into account the major axis of the luterion. The condensate obtained in step (a) above is filtered to obtain the desired luterion-containing solution. It is possible.

[0039] Step (c) is the step of centrifuging the filtered condensate. This allows the Furthermore, luteion of high purity can be obtained. The centrifugation is performed at 1200-500°C. This is repeated at 0 rpm for 5-10 minutes, but under any centrifugal conditions for improving purity. It can also be adjusted and applied.

[0040] Step (c) is the step of separating luteion from the centrifuged supernatant. For example, the supernatant liquid obtained by centrifugation is irradiated with IR light with a wavelength of 200-600 μm to analyze the movement. The luteion particles that gather together due to their properties are separated, and the luteion surface antigens CD332 and CD1 33. Check for the presence or absence of binding with a conjugate that recognizes CD73 or CD39 to determine the presence or absence of luteinized grains. Children can be separated, but are not limited to this.

[0041] Even after separating the luteion through step (c), the separated luteion After filtering the particles with a filter having voids of less than 50 nm, the part caught in the filter It is also possible to collect and filter out only the luteions that have passed through the aforementioned process. This allows for the removal of fine particles other than luteion, and removes luteion with a size of 50 nm or larger. You can acquire Therion.

[0042] Depending on the case, additional 200nm, 400nm, 600nm, 800nm ​​and 1000nm may be added. By sequentially using nm filters, we can achieve the following wavelengths: 50-200 nm, 200-400 nm, and 4 nm. Luterios with sizes of 00-600nm, 600-800nm, and 800-1000nm It can be classified into [type]. The luteion according to the present invention is used in dark-field microscopy or confocal microscopy. Observation is possible through a mirror, at 200nm, 400nm, 600nm, and 800nm ​​respectively. By sequentially using m filters, depending on the size, 50-200nm (nascent stage) / 200-40 0nm (mature stage) / 400-600nm (mitotic stage) / 600-800nm ​​(hypermitotic stage) They can be classified (Figures 27 and 28).

[0043] Subsequently, the process may include an additional step of preserving luteion under specific pH and temperature conditions. It can be stored under conditions of pH 7 or lower and 0°C or lower. Preferably, It can be stored at pH 1 to 5 and -90°C to 0°C. In one embodiment of the present invention, particularly p When storing at temperatures below 0°C under conditions H1-3, preserve the motility of luteion. We confirmed that this is possible. Preserving luterion can improve separation efficiency. ru.

[0044] Furthermore, it may include additional growth stages, such as a fission induction stage. Applying a pressure of 5,000 psi (pounds per square inch) or more It can induce fission, but in one embodiment of the present invention, particularly 25,000 to 35,000 We confirmed that splitting is induced when a pressure of psi is applied. At this time, the temperature was 10-20°C. The temperature was maintained at °C, and the pH 1-3 conditions used during storage were also maintained.

[0045] In other words, the present invention relates to a method for separating luteion, comprising the following steps. (a) an extract containing luterion and an antibody that specifically binds to the luterion surface antigen. Alternatively, a step in which particles with immobilized aptamers are added to induce binding between luteion and the particles; and (b) a step of recovering the luteion bound to the particles.

[0046] In this invention, based on the expression of specific antigens on the surface of lutein, a large amount of lutein is effectively utilized. To achieve high-rate separation, particles immobilized with antibodies or aptamers against the specific antigen are used. Then, an attempt was made to separate the luteion. As a result, in one embodiment of the present invention, the time required was shorter than in the conventional method. We confirmed that a large amount of luterion can be separated in between.

[0047] The extract containing luterion is, for example, (i) a hot water extract of a plant or food, ( ii) Solvent extracts of plants or food, or (iii) plants or in the presence of an antibiotic in the solvent It may also be the condensed liquid of gases generated by heating food.

[0048] The aforementioned hot water extract was obtained by extracting physiologically active substances from plants using water at a temperature of 40°C or higher. If it is used, there are no particular restrictions, but using hot water extraction may cause problems due to the toxicity of organic solvents. It does not produce. The solvent extract is, for example, ether, methanol. Solvents such as anol, ethanol, and hexane It may also be extracted using the same method.

[0049] The aforementioned condensate is obtained by adding distilled water to plants or food and using the gas at 50-90°C. The water vapor or gas vaporized by the shaking is captured by intermittently bubbling and shaking. The condensed liquid may be obtained by cooling the collected material.

[0050] The "Luteion surface antigen" according to the present invention refers to the luteion surface antigen located in the lipid layer of the luteion membrane. It is attached to the cell surface membrane via a carboxyl-terminal domain containing a sexual amino acid, These are substances that can exert a physical antigenic effect, such as CD39 (Ectonucleoside triphosphate diphosphohydrolase 1;ENTPD 1), CD73 (Ecto-5'nucleotidase; NT5E), HBsAg, SLC3A2, CD109, LY9, CD332, CD133, or CD53 are preferred. Alternatively, CD332, CD133, CD39, or CD73 may be used.

[0051] An "antibody" is a protein molecule that is directed to and specifically binds to an antigenic site. The present invention involves immobilizing particles on a substance that can specifically bind to lutein surface antigens and using that substance. However, antibodies that specifically bind to the antigen can be used. Antibodies available in the present invention The body may, for example, use anti-CD332 antibodies, anti-CD133 antibodies, anti-CD39 antibodies, or anti- A CD73 antibody may also be used. Commercially available, manufactured antibodies can be used. Alternatively, instead of such antibodies, aptamers that bind to the surface-expressed antigen may be used. good.

[0052] "Particles" are those that possess magnetic properties, luminescence properties, electrostatic properties, and ionic properties depending on their characteristics. This refers to things that can do that, and things that have a micro or nano size depending on their size. There are no particular limitations as long as it can separate luteion via binding to an antibody. However, for example, magnetic particles, silica particles, quantum dot particles, glass particles, polymer particles, fiber particles The particles are selected from a group consisting of and fluorescent particles. Furthermore, the particles possess magnetic properties and fluorescent properties. The particles simultaneously include particles in which quantum dots and gold and silver particles are bonded to magnetic particles. It is possible.

[0053] The aforementioned particles are, for example, magnetic particles. Magnetic particles refer to particles that are magnetic and move in the presence of a magnetic field. The aforementioned magnetism refers to paramagnetism. It may possess. Magnetic particles are, for example, metallic materials. ), magnetic material or magnetic alloy It may include c alloy. Magnetic particles are particles that are magnetic, but in this invention However, magnetic particles are not limited to these; they may not be magnetic themselves but may possess magnetism. They may be metal particles that are pulled by or magnetic force. Furthermore, the metal particles may be, for example, Iron, aluminum, cobalt, nickel, manganese, tin, zinc, cadmium, magnesium, copper , any of the following selected from the group consisting of barium, lithium, or yttrium oxides It can be manufactured using one material. Iron is preferably selected. The particles are micrometers While particles of a certain size exhibit ferromagnetism, tiny particles of nanometer size exhibit supermagnetism. It acquires properties. Such particles are easy to synthesize and their size can be easily controlled. Example For example, having a size of 1 to 1,000 nm, preferably 10 to 1,000 nm, more preferably Particles with a size of 10 to 500 nm, more preferably 10 to 100 nm, are used. It is possible.

[0054] Depending on the circumstances, the magnetic particles may be modified in order to maintain dispersibility and safety. It is possible to modify it, for example, by coating it with carbon.

[0055] Additionally, it may further contain functional groups suitable for antibody or aptamer binding. Active groups include, for example, amide groups and ester groups. oup), amine group, carboxyl group group), thiol group (SH), epoxy group (epoxy group), phenyl Phenyl group, sulfone group, a alkoxy group, aldehyde group p) may be selected from the ketone group. The amine group (NH2) is found in monoamines and diamines. ), triamine, ethylenediamine (ine) or diethylenetriamine That's fine.

[0056] In the step of recovering the luteion bound to the aforementioned particles, if the particles are magnetic particles, (b) Step involves applying magnetism externally to recover the magnetic nanoparticles to which the luterion has been bonded. It is characterized by the following.

[0057] The method for collecting and recovering magnetic nanoparticles to which luteion has been bonded after applying the aforementioned magnetism is as follows: The luteion and the luteion surface antigen-binding antibody or aptamer immobilized on the magnetic field After mixing and reacting the sample containing particles, use a magnet or magnetically activated cell separator (Ma) at room temperature. Using gnetic-activated cell sorting (MACS) After collection, the supernatant is removed and the material is resuspended in a buffer solution, then the flocculation film and capture film are used. By sequentially passing the material through a filtration membrane, the luterion-antibody-magnetic particle complex can be identified. Cut.

[0058] In the present invention, if the particles are fluorescent particles, step (b) is performed using a fluorescence-based cell sorter. This method is characterized by using this to recover particles to which luterions are bound.

[0059] The recovery method using the aforementioned fluorescence-based cell sorter is such that cells can be recovered depending on the type and degree of fluorescence. A method for classifying microscopic substances of various forms, in which fluorescent cells are used for quantitative analysis and classification. A "fluidic system" that allows fluid to flow through a pipe, and This control system is used to observe the flow of fine materials through an optical system. "electronic system" which converts optical signals from the optical system into electrical signals and processes them. It consists of a "ronic system". The fluorescent cell classification device is used to classify various cells according to their characteristics. It is a device for more efficient classification and collection, and is useful in life sciences (zoology, botany, microbiology, agriculture, water science). This device is widely used in industry-academia, forestry, and medical research. The fluorescent particles of this invention contain Any fluorescent substance that can be used for bioimaging is acceptable. The fluorescent substance may be, but is not limited to, rhodamine and its derivatives, fluorescein and Its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, e Litrosine and its derivatives, eosine and its derivatives, and 4-acetamido-4'-iso Selected from the group consisting of thiothianatostilbene-2,2'disulfonic acid. This is also acceptable. More specifically, examples of fluorescent substances are as follows: Rhodamine and its derivatives: 6-carboxy-X-rhodamine (ROX), 6-carb Xylodamine (R6G), Lisamin Rhodamine B Sulfonyl Chloride, Rhodamine (R hod), Rhodamine B, Rhodamine 123, Rhodamine X isothiocyanate, sulfo Rhodamine B, Sulforhodamine 101, Sulfonyl Chloride of Sulforhodamine 101 Derivative (Texas Red), N,N,N',N'-tetramethyl-6-carboxylate TAMRA, Tetramethylrhodamine, Tetramethylrhodamine isothiocyanate Anate (TRITC), riboflavin, rosolic acid, terbium chelate derivative, Al exa derivatives, Alexa-350, Alexa-488, Alexa-547, Ale xa-647; Fluorescein and its derivatives: 5-carboxy-fluorescein (F AM), 5-(4,6-dichlorotriazine-2-yl)aminofluorescein (DTA) F), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluoroseine (JO E) Fluorocaine, Fluorocaine Isothiocyanate, QFITC ​​(XRITC), F Luoresamin, IR144, IR1446, malachite green isothiocyanate, 4 - Methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosa Diphosphorus, phenol red, β-phycoerythrin, o-phthalaldehyde; coumarin and its derivatives: Coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120) ), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 4'-6-Diamidino-2-phenylindole (DAPI), 5',5''-Dibromo Pyrogallol-sulfonphthalein (Bromopyro-gallol Red), 7 -Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarindie Chilentriamine pentaacetate, 4-(4'-diisothiocyanatodehydrosyl) Ben-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'- Disulfonic acid, 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, da nsyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid ( DABCYL) 4-dimethylaminophenylazophenyl-4'-isothiocyanate ( DABITC); Acridine and its derivatives: Acridine, Acridine isothiocyanate 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-Amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfo Nate (LuciferYellow VS), N-(4-anilino-1-naphthyl)ma Reimide, anthranilamide, Brilliant Yellow; pyrene and Derivatives: Pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reac tive Red 4(Cibacron Brilliant Red 3B-A); Erythrosine and its derivatives: Erythrosine B, Erythrosine isothiocyanate, E Thidium; Eosin and its derivatives: Eosin, Eosin isothiocyanate; 4-A Cetoamide-4'-isothiocyanatostilbene-2,2'disulfonic acid

[0060] In the present invention, if the particles are electrostatically charged particles, step (b) is performed to ensure a non-uniform electric charge A field is formed, causing a dipole moment to form in the particles, and the luteions are bound together by electrostatic attraction. This method is characterized by recovering the ionic particles that have been removed.

[0061] The aforementioned non-uniform electric field is formed to create a dipole moment in the particle, and the electrostatic attraction is One method for recovering the ionic particles to which therion is bound is an electrical particle separation device. A particle classifier (Different mobility analyzer; DMA) This is made possible through the use of electrostatic force. The electrostatic particle classifier utilizes the difference in particle mobility to classify particles. A device for classifying particles, also known as a differential-type electrical mobility analyzer or differential-type electrostatic classifier. In essence, an electrical particle classifier utilizes the fact that the movement velocity of charged particles is a function of particle diameter. Using this method, monodisperse particles with a desired diameter are obtained from polydisperse particles. This equipment is designed to sort out (no-disperse) particles.

[0062] In the present invention, the particles are ionic particles, for example, anionic or cationic particles. In this case, step (b) above is an ionic particle to which luteions are bound using electrostatic attraction. The offspring can be retrieved.

[0063] The method for recovering fiber particles to which luteions are bound by the electrostatic attraction is the electrostatic particle Via a differential mobility analyzer (DMA) This becomes possible. The present invention further includes the step of separating only luteion from luteion bound to particles. It is possible.

[0064] Add BSA / PBS buffer solution to the luteion bound to the aforementioned particles and incubate at 25°C. After incubation, BSA is used by utilizing magnetism or ionic properties. By separating only the particles to which the adsorbed substance is located, BSA (Bovine Serum Albumin) is used. Adding PBS again to the particles that have adsorbed ) and incubating By doing this, particles can be detached and separated, allowing only the luteion to be isolated.

[0065] In another aspect, the present invention involves adding water to luterion and irradiating it with IR light or This invention relates to a method for culturing luteolions, which includes a step of growing them under pressure at 18-30°C.

[0066] In one embodiment, the water added during the culture may be saline solution or PBS solution. This is not limited to this.

[0067] Furthermore, in another respect, the present invention provides luteolion in a sugar-containing medium at pH 5-9 (when stored) A method for culturing luteion that includes a step of growing it under conditions of pH 1-3 and 18-30°C. To relate to.

[0068] In one embodiment, the sugars are rhamnose and glucose. e) galactose, fructose or It may be xylose, and preferably glucose. In one embodiment of the invention, a culture medium containing glucose, such as DMEM medium or blood medium, is used. Add the solution at a concentration of approximately 1-10%, preferably 2-8%, and set the pH to 7 and the temperature to approximately 20°C. We confirmed that we could optimally culture luteon to maintain the number of cases. The number of lyons is determined by the luterion-specific surface antigens CD332, CD133, CD73, and This counts the number of luteions containing CD39 and luteions stained with Mitotracker Red. I checked and confirmed it.

[0069] The luteion before culture may be 50-200 nm in size, but in the culture of the present invention The size of luteion cultured by the cultivation method may be 300-500 nm after cultivation. At this time, while observing with a microscope, ensure that the size of the rutheion does not exceed 500 nm. Once the culturing is complete, they can be sorted by size and stored at -80°C, or It can be stored filled with nitrogen or at temperatures above 0°C, and preservatives can be added during storage.

[0070] As described above, the cultured luteion grew to the desired size (50) without any change in its properties for a certain period of time. It can be stored at a size of 0 nm or less and can be effectively used in the treatment of diseases using lutein. can.

[0071] Specifically, the luterion according to the present invention can be introduced into the nucleus of a cell. The results of fluorescently staining the cells with red fluorescence to confirm whether or not luteion is introduced into the intracellular nucleus showed that We confirmed that luterins are introduced into the intracellular nucleus stained with DAPI.

[0072] Furthermore, the luteion according to the present invention is fluorescently stained, and the fluorescently stained luteion is injected intraperitoneally. Results from administration by injection (IP) and oral gastric tube: At least 3 hours after oral administration Furthermore, it can be confirmed that the blood-brain barrier (BBB) ​​is crossed within 5 minutes of intraperitoneal injection. By utilizing its properties, the luteion according to the present invention can be used in the treatment of degenerative brain diseases and the like, by crossing the blood-brain barrier. This overcomes the limitations of drugs that could not pass through the filtration pathway and therefore could not be used as therapeutic agents. It can be used as a treatment for degenerative brain diseases that can cross the blood-brain barrier, or as a conventionally known drug It can also be used as a drug delivery system that allows drugs to cross the blood-brain barrier.

[0073] Furthermore, the luteion according to the present invention inhibits telomerase activity in cancer cells and in normal cells. It has no effect or enhances telomerase activity, effectively suppressing only the proliferation of cancer cells. It has an anti-cancer effect.

[0074] On the other hand, in normal cells, luterion treatment increases telomerase expression and lengthens telomeres. As the amount increases, luteion exhibits anti-aging activity by enhancing telomerase activity in normal cells. We were able to confirm that it was indeed there.

[0075] In this invention, "normal cells" are defined as cells that have increased telomerase activity along with cancer cells, and which have an infinitely increasing telomerase activity. This refers to cells that undergo a normal aging process, rather than cells that exhibit a phenotype of proliferation.

[0076] In this invention, the term "telomerase" refers to the telomere at the end of the telomere. RIC refers to ribonucleic acid proteins that catalyze the addition of repeats. Telomeres are the ends of chromosomes. It is a long extension of the repetitive sequence covering the ends, and is thought to stabilize the chromosome. In this context, telomeres are typically 7-10kb in length and consist of multiple repeats of the sequence -TTAGGG-. It includes. Telomerase is not expressed in many adult cells, and telomere length is continuous. It reduces cell replication. When cell replication exceeds a certain number of times, telomeres gradually shrink. The cells enter a stage of terminal breakdown, which leads to cellular aging. Telomerase is inactive in somatic cells, but is active in 90% of cancer cells, inhibiting telomerase. The drug may be useful in fighting cancer.

[0077] Furthermore, as a result of treating healthy fibroblasts with luterion, the telomeres of the aforementioned normal human cells We were able to confirm that enzyme expression increased and ATP production increased. To confirm that therion can suppress aging by increasing telomerase activity in normal cells. This was achieved. Therefore, the luteion according to the present invention enhances telomerase expression and / Alternatively, it can be used to treat diseases or conditions that are susceptible to telomerase activity, and treatment This includes administering it to patients who require it to increase telomerase activity in their cells or tissues. It is possible.

[0078] "Diseases or illnesses associated with aging" include tumor formation and malignant development of cancer, myocardial infarction (visceral paralysis). ), cerebellar infarction, stroke, Parkinson's disease, heart failure, atherosclerosis, hypertension, white blood Internal organ damage, age-related vision loss, muscle loss, osteoarthritis, osteoporosis, bone marrow loss, multiple sclerosis, Sjögren's syndrome, rheumatoid arthritis, immunodeficiency, diabetes, idiopathic pulmonary fibrosis, and Neurodegenerative diseases, Alzheimer's disease, Huntington's disease, and testosterone, estrogen Disorders caused by reduced energy production, growth hormone, IGF-I, or other factors. It means...

[0079] "Anti-aging effect" refers to increased mitochondrial biodevelopment and function, and reduced R OS level, extended lifespan of somatic cells such as senescent cells and neuronal cells, tumor Table including prevention of age-related phenomena such as ulcer formation, malignant progression of cancer, cerebellar infarction, and myocardial infarction. It means the current form.

[0080] Mitochondria, the powerhouses of cells, are primarily responsible for respiratory function. As a place of active oxygen consumption, it is where all cells carry out metabolic activities, and this activity is carried out by the cells It supplies the energy necessary for all other activities (Boveris A et al.). (Biochem.J.134:707-716, 2973). Mitochondrial oxygen Reactive electrons such as O2·- or OH· are generated during the transport process. Oxygen species (ROS) disrupt the structure and components of mitochondria. This induces changes in respiration rate and oxidative phosphorylation reactions, ultimately determining ATP, which in turn determines cellular metabolism. It begins to affect production and the NADH / NAD+ ratio. The energy of many higher animals ATP synthesized in mitochondria is used for energy metabolism that takes place in mitochondria. Its effects are thought to be closely related to aging (Lee JW et al., J Ko rean Med Assoc.52(10):1007-19, 2009).

[0081] We confirmed that luterion treatment increased ATP production in normal cells. The term "preventing diseases or symptoms associated with aging" means reducing the chances of them occurring. It refers to delaying or reversing age-related diseases.

[0082] As used herein, the terms “senescence” or “senescent cells” refer to telomere dysfunction, D Mitotic fluid may be induced by NA damage or oncogene activation. This shows the cell cycle arrest state in fissured cells. In budding yeast, telomere dysfunction also occurs. The senescent cells that have been subjected to this process are arrested in the G2 / M phase of the cell cycle. In mammalian cells, senescence The cell is arrested in the G0 phase, a non-mitotic phase outside the cell cycle. WI-38 fibroblast Cellular aging is observed under a microscope, and the number of β-galls does not increase for 10 days after the passage. This refers to cells that show positive staining for galactosidase.

[0083] As used herein, the term "post-division cell" refers to a non-dividing cell outside the cell cycle. Cells that are in a dormant G0 phase but still perform their main functions for the survival of the rest of the organism. This refers to a group. After somatic cell division, cells become neuronal cells, cardiac muscle cells, and muscle cells. It contains cells. Like the parenchymal cells of liver and kidney tissue, mature Some types of cells in organisms enter the G0 phase semi-permanently and only divide again in very specific environments. These types of cells can be induced to begin splitting. At some point, cells are considered to be cells after somatic cell division.

[0084] The aforementioned age-related diseases or symptoms include mitochondrial dysfunction, telomere dysfunction, and aging. Cellular degeneration and age-dependent cell loss, or mitochondrial degeneration. It is associated with Dorian degeneration or the arrest of the cell cycle in somatic cells after division.

[0085] In one mode, luteion interacts with the telomerase enzyme to affect the tissues or cells of an individual. Stimulates and / or increases telomerase expression and / or activity in cells. In this state, such activity may be reduced or absent, which can lead to disease in the individual, or This can lead to an increase or development of the etiology or syndrome associated with the symptoms. In particular, by increasing telomerase expression and / or activity in healthy tissues. In other words, to extend the lifespan of the individual and maintain its health, as described in this application. The term "treatment" includes any mode of implementation included in the term, including clinical treatment and / or It can include the field of diagnosis. [Examples]

[0086] The present invention will be described in detail below with reference to examples. These examples merely provide more specific details of the present invention. This is for illustrative purposes only, and the scope of the present invention is not limited to these embodiments. This is self-evident to anyone with ordinary knowledge.

[0087] Example 1: Separation of luterion (1) Lutherion extraction 100g of the medicinal plant Rhus verniciflua stokes (Rhus verniciflua stokes) Cut to 0-30 times its original size, that is, to fit the size of a 2-3 liter container, and place it in the container. And, 500-800g (preferably 6 times the amount of the plant, i.e., 600g) which is 5-8 times the amount of the plant. After adding the distilled water from g) to the container, the entire mixture was heated at a temperature of 80°C or lower. The entire mixture was heated for approximately 8 hours. However, adding a bubble ring with oxygen for 20-30 minutes every 3 hours helps the plant's luteinization to solidify. To prevent this from happening, the vaporized water vapor produced during extraction after bubbling was transferred to the flask. The water vapor was collected. The collected water vapor was cooled and condensed to obtain a condensate, which was then exposed to IR light (wavelength: Irradiate with 3-1000 μm (preferably 200-600 μm) for 1-2 hours to luteo The process was designed so that the cell was not mutated and cell division was induced. This process is schematically shown in Figure 37. .

[0088] (2) Separation by centrifugation and filtration (1) The condensate collected in step (1) is subjected to primary centrifugation with 190g and then spun down to remove impurities. Then, centrifuge the mixture at 3000g and stabilize it for several minutes to several tens of minutes (preferably around two hours). Afterward, the supernatant was collected. At this time, CD332 staining was performed to confirm motility, and then the luteal phase was observed. The presence or absence of ON was initially confirmed. Subsequently, in order to obtain pure luterion, 120, It was subjected to tertiary centrifugation at 000g.

[0089] The lower layer, filtered through an approximately 800nm ​​filter, is collected, and the plasma and other internal plants are also collected. After removing the exosomes and other dead cell debris pellets, it is then re-processed to 140,000 The supernatant liquid was collected by centrifugation of 0g or more to remove the pellet. The supernatant liquid was then heated to 400°C. After filtering with an m filter to collect the lower layer, the motility was confirmed by staining with CD332 again. I did that. I filtered it with a 500nm filter and collected the supernatant liquid that remained on top of the filter. Lutherions were collected and stored at pH 1 or below -90°C.

[0090] The above process can yield rutheions with a major axis of 50-800 nm, which are dark Observation and confirmation were possible via a field microscope or confocal microscope. The acquired luteol The size of the n depends on the stage: 50-200nm (developmental stage) / 200-400nm (maturation stage) / 400 The cells were divided into two phases: ~600nm (mitotic phase) and 600~800nm ​​(hypersequencing phase). Similarly, luteolion was obtained from the medicinal plants listed in Tables 1-4, Angelica sinensis, Porcelain bark, and Kiwifruit using the same method. I obtained it.

[0091] (3) Separation using surface antigen-specific antibodies 3-1) Confirmation of lutein surface antigen Anti-CD39 antibody (sc-18766, Santa C) was used to isolate luteion from plants. ruz Biotechnology), anti-CD73 antibody (sc-25603, San (ta Cruz Biotechnology) or anti-CD332 antibody (BS-06 We attached 75R (Bioss Inc.) and then attached anti-FITC to observe whether or not color development occurred. (Blue fluorescence: CD133 / 1-VIOBRIGHT-FITC manufactured). Red fluorescence: PE (Phycoerythrin: Manufactured by Miltenyi Bitech GmbH) It was stained with Mito-tracker, DAPI, and Hoec. After staining with HST, the presence or absence of color development was observed using a fluorescence microscope.

[0092] As a result, luteria stained by Mitotracker and DAPI or Hoechst Turn on anti-CD39 antibody, anti-CD73 antibody, anti-CD133 antibody, or anti-CD332 antibody. By confirming that antibodies bind to CD39, CD73, CD133, or C We were able to confirm that D332 is a surface antigen of lutein (Figures 29 to 32). ).

[0093] 3-2) Production of antibody-immobilized particles Step 1. Fabrication of carbon-coated iron (Fe) magnetic nanoparticles Iron acetylacetonate hydrate (0.5) mmol in 10 mL of octyl ether After preparing the metal precursor solution by stirring it in the Erlenmeyer flask, the solution is then subjected to ultrasonic irradiation. The metal precursor solution was irradiated with ultrasound at 20 kHz (50%) intensity for 10 minutes using a blasting device. As ultrasound is applied, the solution, which is initially orange, changes to a dark brown color over time. We were able to observe the above changes, but the iron oxide (Fe2O3) magnetic nanoparticles formed This means that it was manufactured in an excess amount. After adding ethanol to precipitate the resulting magnetic nanoparticles, centrifugation was performed to remove the magnetic nanoparticles. The particles and the supernatant were separated, and the supernatant was removed. The washing process described above was repeated at least three times. Afterward, the magnetic nanoparticles were dried at a temperature of 50°C for 12 hours to form an oxide with a size of 300 nm. Iron magnetic nanoparticles were manufactured. The manufactured iron oxide magnetic nanoparticles were subjected to 600°C for 3 hours. Iron-magnetic nanoparticles coated with carbon were obtained by heat treatment under a gon (Ar) atmosphere.

[0094] Step 2. Fabrication of carboxyl-coated magnetic nanoparticles The carbon-coated iron-magnetic nanoparticles (0.5g) obtained in step 1 and succinic 1g of anhydride together with 5ml of silane polyethylene glycol carboxylate After dispersing in 25 ml of ethanol, the mixture was reacted for 24 hours. After the reaction was complete, the mixture was centrifuged. The precipitate obtained was washed with ethanol and then dried in a vacuum oven to separate the carbon and carbon. Iron-magnetic nanoparticles with a double coating of ruboxyl groups were obtained. The construction stages are shown in a schematic diagram (front of Figure 33).

[0095] Stage 3: Production of antibody-immobilized particles Anti-CD39 antibody, anti-CD73 antibody, anti-CD133 antibody, or anti-CD332 antibody The carboxyl was reacted with a thiol-reactive reagent to produce thiolation. The iron magnetic nanoparticles into which the functional group has been introduced are reacted with the antibody having the thiol group. The antibody was attached to magnetic nanoparticles. A schematic diagram of the antibody-immobilized particles is shown (Figure). (Behind 33).

[0096] 3-3) Isolation of luterion from plants or food sources 100-200 µl plant extract and 5 µl CD39 antibody-iron magnetic nanoparticles or CD7 3. Place the antibody-iron magnetic nanoparticles in a beaker and allow to bind for 30 minutes, then transfer to a magnetic separator. After 2 minutes, the luterion-magnetic nanoparticles were collected, the supernatant was discarded, and the mixture was washed. Iron magnetic nanoparticles bonded with 0.033 wt% BSA (Bovine Serum) Add Albumin / PBS buffer solution and incubate at 25°C for 1 hour. After incubation, a magnet is used to remove the iron magnetic nanoparticles to which the BSA has been adsorbed. The ink is separated, and a certain amount of PBS is added again to the iron magnetic nanoparticles on which the BSA has been adsorbed. Detachment was performed via incubation. The adsorbed BSA was F P-640 spectrofluorometer (JASCO) Quantitative analysis was performed using the standard calibration curve method at 280 nm (emission slit: 0.5 nm). (Absorption slit: 0.5 nm). Plants or plant lutea using the iron magnetic nanoparticles. The ON separation stage is shown schematically in Figure 34.

[0097] 3-4) Confirmation of separated luterions The rutherion isolated in 3-3) above and the rutherion isolated by the separation method via (2) filtration were combined with anti-FITC bound to antibodies that bind to CD39, CD73, CD133 or CD332 , and the presence or absence of color development was observed ((a), (b), (c), (d) of Figure 35). Fluorescently activate d cell sorter was used to compare the extraction yield of the two separation methods described above.

[0098] As a result, when separated using the nanofiltration method, the number of rutherion was 1.5× 10 8 cells / ml, while when separated using particles that bind to CD39, CD73, CD133 or CD332 surface antigens , the number of rutherion was 7.18×10 8 cells / ml, and it wa confirmed that the number of rutherion increased by 4 times or more compared to the nanofiltration method (Figure 3 6 (a) and (b)).

[0099]

Table 5

[0100] As shown in Table 5, when the nanofiltration method is used, for rutherion with a size of 50 to 400 nm, the final extraction rate is 40 to 45% and the loss rate is 50 to 60%, whereas in the present invention when using the antibody-iron nanopar ticle separation method that binds to CD39, CD73, CD133 or CD332 surface antigens, the final extraction rate of rutherion with a size of 50 to 400 nm is 90 to 95% or more, and the loss rate is 10% or less, whereby it was confirmed that the extraction rate and efficiency are greatly improved .

[0101] From these results, compared with the conventional rutherion separation method using a nanofilter, the present invention ​Separation using particles immobilized with antibodies or aptamers that bind to the luterion surface antigen. The method can be confirmed to increase the final extraction rate and decrease the loss rate that occurs during the separation process. Ta.

[0102] (4) Confirmation of differences between plant mitochondria and luterion As shown in Table 6, plant mitochondria are CD332, CD39, CD73, CD133 Furthermore, not only is it impossible to express any one of the surface antigens in CD326, but 140,000 At concentrations above g, mitochondria rupture during centrifugation, but lutein remains intact. It was confirmed that this occurs. Furthermore, pressurization to induce fission, for example, 35,000 psi or more, is required. When applied, mitochondria rupture, but luteon remains intact. I confirmed that.

[0103] [Table 6]

[0104] Example 2: Preservation of luterion Motile luterins and CD39, CD73, CD133, or CD332 The lutein contained as a surface antigen was stored and its number was confirmed. Ruterin that accumulates upon irradiation with IR. Check and add anti-CD332, anti-CD39, anti-CD133, or anti-C to your luterion. Luterion and Mito, which were confirmed to develop color when D73 antibody was conjugated and anti-FITC was conjugated. The number of stained rutheions was counted after staining with a tracker. At this time, the pH ranged from 1 to 3. The result of storing rutheons under conditions below ℃, from -90 to 0℃, and counting the number of rutheons is p The following Tables 7 and 8 show the H and temperature conditions (+ indicates the number of luterins counted) (Indicates a number).

[0105]

Table 7

[0106]

Table 8

[0107] According to Tables 7 and 8, when maintained for 1 to 3 months under the conditions of pH 1 to 3 and -90 to 0°C , it can be confirmed that the lute lion can be preserved without affecting the number of lute lions.

[0108] Example 3: Induction of lutein fission using pressure Pressure was applied via a French press to the lute lion isolated in Example 1 as shown in Table 9 . In addition, the effect of temperature on the induction of division of lute lion was confirmed (+ indicates the degree of division).

[0109]

Table 9

[0110] According to Table 9 and Figures 11 to 13, under the conditions of pH 1 to 3, 25000 to 35,000 psi, it can be confirmed that division is induced under the temperature condition of 10 to 20°C, and proliferation of lute lion occurs .

[0111] Example 4: Properties of luterion (1) Structure Among the lute lions obtained in Example 1, lute lions having a size of about 50 to 400 nm were analyzed by Confocal Laser Scanning Mic roscope, Zeiss) and Transmission Elec (Tron Microscope), Scanning Electron Microscope (Scanning Electron Microscope) (on Microscope), Atomic Force Microscope Images were taken using a scope and a confocal scanner (Leica TCS-SP8), and the luteal function was then used. Like mitochondria, ON also has a membrane structure with a double or multilayer membrane, and its internal structure is It has a structure that does not complete the cristae structure, and mitochondria and It was confirmed that it could be observed within the same laser wavelength range. Furthermore, its shape was circular or elliptical. We were able to observe that this was the case (Figure 6).

[0112] Furthermore, the luterins separated in Example 1 were imaged using a TEM electron microscope, and the result is shown in Figure 8. As shown, it was confirmed that the structure had a bilayer or multilayer structure.

[0113] (2) Dyeing characteristics Among the rutheions obtained in Example 1, rutheions having a size of approximately 50 to 800 nm were selected. Mito-tracker, Rhodamine 123 123) After staining with Janus Green B, the color development was observed. I observed nothingness. As a result, I also took luteolion, mitotracker, rhodamine 123, and janus gooseberry. We confirmed that color development was observed using ¾B (see Figures 2, 3, and 5).

[0114] (3) Autofluorescence Among the rutheions obtained in Example 1, rutheions having a size of approximately 50-800 nm are The photoreaction was confirmed by fluorescence photography (see Figure 4).

[0115] (4) Analysis of whether luterion contains RNA Atomic microscopy results of 200-400 nm luteions separated in Example 1 are shown in Figure 9. As indicated, it is presumed that luterions contain nucleic acids such as RNA and DNA. It is possible.

[0116] Total RNA and DNA were separated from the 200-400 nm luteions isolated in Example 1. To do this, QIAGEN kit (AllPrep DNA / RNA icro kit: After isolation using Cat 80284, Experion RNA (DNA) S Quantitative analysis was performed using the tdSens (Bio-Rad) chip.

[0117] After recovering the luterion by centrifugation (8000g, 1 hour 30 minutes), the kit was decomposed slowly. Liquid solution RLT plus (Guanidine isothiocycanate, det Add 3.5 ul of beta-mercaptoethanol to 50 ul of ergents to make 20 gels. The luteion was dissolved by passing it through a syringe with a needle 5 to 10 times. After transferring the decomposition buffer to the AllPrep DNA spin column, centrifuge (≧8000) (g, 15 seconds) The batch containing the DNA caught in the column and the RNA that passed through the column They were separated by fur.

[0118] First, add the same volume of 350 µl of 70% ethanol to the buffer that has passed through the column. After mixing well, apply 700 µl of the mixture to an RNease MinElute spin column. The mixture was then transferred to a separate container and centrifuged (≥8000g, 15 seconds) to remove the buffer that had passed through the column. Each of the following is mixed with 700µl RW1, 500µl RPE buffer and 80% ethanol. The column was washed in stages using the following method. All centrifugation (≧800) used above The experiment (0g, 15 seconds) proceeded under the same conditions. To obtain RNA, 14µl RNeasy - After placing the free solution into the column, centrifuge (≧8000g, 60 seconds) to obtain luteol. RNA was isolated.

[0119] The genomic DNA is attached to the AllPrep DNA spin column in the first step described above. The DNA was washed using 500µl AW1 and 500µl AW2 buffers, respectively. Purified. All centrifugal separation (≥8000g, 15 seconds) speeds and times used were RN The process proceeded under the same conditions as the A separation process. After adding 50 µl of EB buffer to the column, the chamber After leaving it at warm temperature for 2-5 minutes, luteinic DNA is separated by centrifugation (≧8000g, 60 seconds). Experion RNA (DNA) StdSens (Bio-Rad) chip. The quantitative results obtained using this method show that RNA was present in the luteion, as shown in Figures 10A and 10B. In addition to containing [unclear], we confirmed that it also contains DNA, as shown in Figure 10C. This was possible. In particular, RNA and DNA are contained in lutein of 200-400 nm. We were able to confirm that they were there.

[0120] Example 5: Culture of luterion (1) Among the rutheions obtained in Example 1, rutheions with a size of approximately 50-200 nm After adding PBS and irradiating with IR light, the cells were cultured at 18-30°C for approximately 3 hours. The size of the luteions was checked under a microscope at approximately one-hour intervals immediately after irradiation. (Approximately 1-6 hours) Furthermore, the luteion, which was approximately 200 nm in size before culturing, has grown to approximately 500 nm. This confirmed that water was added to luterion and irradiated with IR light. When cultured at 18-30°C, it can be grown to a size of approximately 500 nm. It was discovered that...

[0121] (2) Ruterions with a size of approximately 400-800 nm obtained in Example 1 PBS was added and irradiated with IR light, then incubated at 18-30°C for approximately 3 hours. The size and condition of the luteions were examined under a microscope at approximately one-hour intervals immediately after irradiation. After approximately 6 hours, the luteion, which was about 400-800 nm in size before culturing, did not grow but instead divided. (fission) was confirmed.

[0122] (3) Using a culture medium (DMEM or blood medium) supplemented with 5% glucose, Lutherions were cultured under pH 7 and 20°C conditions. Irradiation with IR confirmed the accumulation of lutein. It was confirmed that luterion has anti-CD332, anti-CD73, anti-CD133, and anti-CD39 Lutherion and Mitotracca, whose coloration was confirmed by combining the bodies and then binding with anti-FITC. The number of stained rutheions was counted after staining with -. The results are shown in Table 10 below. (The + indicates the relative ratio of the luterion numbers).

[0123] [Table 10]

[0124] Example 6: Confirmation of intracellular introduction status Lutherions were fluorescently stained with PKH26-1, PKH26-2, and PKH26-3 red fluorescent dyes. Then, A549 arsenic cell lung cancer cells (ATCC#:CCL-185) and 24-hour incubation The cells were fixed and their morphology was observed under a microscope. The results are shown in Figures 14 to 16. I showed it.

[0125] Referring to Figure 14, staining with PKH26-1, PKH26-2, and PKH26-3 red fluorescence. It was confirmed that the colored luteion was introduced into the cell nucleus stained with DAPI. Upon examining the enlarged images in Figures 15 and 16, it was confirmed that luteolins were introduced into the intracellular nucleus. This allows for more concrete confirmation of the facts.

[0126] Example 7: Confirmation of whether or not blood-brain barrier (BBB) ​​permeation occurs. Mice were given luteion, fluorescently stained with PKH26, via intraperitoneal injection (IP) and oral gastric tube. The administered luteon was checked to see if it could cross the blood-brain barrier (BBB) ​​after being administered using the prescribed method. (Figure 17) According to the study, it is necessary to confirm that the drug crosses the blood-brain barrier (BBB) ​​at least 3 hours after oral gastric tube administration. can.

[0127] Furthermore, as shown in Figure 18, it is important to confirm that the drug crosses the blood-brain barrier (BBB) ​​within 5 minutes of intraperitoneal injection. This can occur, and according to Figure 19, even 24 hours after intraperitoneal injection, the heart, lungs, spleen, liver, and pancreas It can be confirmed that it is distributed throughout the kidneys, testes, abdomen, and viscera.

[0128] Example 8: Confirmation of the anti-cancer (telomere / telomerase activity) effect of luterion. (1) Inhibition of telomerase activity in cancer cells Normal cells (Fibroblast) and cancer cells (NCl-H1975, MDA-MA) -468, WiDr) 1 × 10 6 After inoculating each 60mm plate with cells / ml, After approximately 12 hours, the lutein isolated in Example 1 was treated with a concentration of 50 μg / ml and cultured. The cell culture was performed using the antibiotic PSF (antibiotic-antimycotic). Under no conditions, the procedure was carried out in a 5% CO2 incubator at 37°C with 1% FBS conditions, and 1% FBS DM was performed. Cells were harvested 48 hours after inoculation using EM medium, and telomerase activity was measured. .

[0129] TRAP analysis (TRAPeze® Telomerase Detect) Analysis of telomerase activity using the ion Kit (Millipore) revealed that luteion The treatment altered the telomere size of cancer cells (NCl-H1975, MDA-MA-468, WiDr). We were able to confirm a decrease in both enzyme expression and activity (Figure 20B).

[0130] Furthermore, when the same experiment and analysis were performed on fibroblasts, which are normal cells, the results showed that In normal human cells, telomerase expression and activity were higher compared to the control group (untreated lutein group). We were able to confirm that it was increasing (Figure 20A).

[0131] (2) Suppression of cancer cell proliferation by luterion treatment Various human cancer cell lines (lung cancer (NCI-H1975), colorectal cancer (WiDr), breast cancer (MD)) A-MB-486), liver cancer (HCC38), leukemia (AGH-77), and normal cells To measure the cytotoxicity of lutein against cancer cells using the strain (fibroblast) M A TT analysis was conducted.

[0132] 5 x 10 4 cells / ml (8×10 3 Cell suspension in 96 wells (well) l) Inoculate 100 ul into each well of a microtiter plate with a flat bottom, and 2 After culturing for 4 hours, the culture medium was replaced with one containing various concentrations of lutein, and then... The cells were incubated for 48 hours. After that, each well was filled with a 10-fold diluted, non-water-soluble yellow MTT culture medium. (3-(4,5-dimethylthiazole-2-yl)-2,5-diphe Add 100 µl of nyltetrazolium bromide solution. To form formazan crystals, an incubation chamber with a temperature of 37°C and a carbon dioxide concentration of 5% was used. It was stored in a medium. After about 4 hours, after removing the excess medium, the insoluble cells formed inside the cells were examined. After adding 200 µl of DMSO to each well to dissolve the formazan, Mike Absorbance was analyzed at 595 nm using a photometer reader.

[0133] The absorbance of the control group, which was not treated with luterion, was set to 100%, and the cancer cells at each concentration were compared. When the survival rate was measured, it was found that it reduced the survival rate of cancer cells in a concentration-dependent manner. On the other hand, in normal cells... It was not cytotoxic (Figure 21).

[0134] Furthermore, in addition to the aforementioned cancer cell lines, there are also the AsPC-1 pancreatic cancer cell line, the A549 lung cancer cell line, and BT. -20 In breast cancer cell lines, the inhibitory effect of lutein derived from various sources on cancer cell proliferation was confirmed. The rate measurement was performed in the same manner as the MTT analysis method described above, and the various origins of the separated ions were obtained using the method of Example 1. Therion showed an inhibitory effect on cell proliferation against cancer cells (Figures 22 to 24).

[0135] Therefore, the luteion of the present invention suppresses the proliferation of cancer cells and prevents or treats cancer. It was discovered that this is possible.

[0136] Example 9: Increased telomerase activity in normal cells of lutein. Normal cells (Fibroblast) 1×10 6 Each cell / ml is plated in a 60mm plate. After inoculation, approximately 12 hours later, the luteion isolated in Example 1 was administered at a concentration of 50 μg / ml. The cells were treated and cultured. Cell culture was performed using the antibiotic PSF (antibiotic-antimyc). Under conditions without otics, culture in 1% FBS DMEM medium at 37°C with 5% CO2. The experiment was conducted using a cell culture system. Cells were harvested 48 hours after inoculation, and telomerase activity was measured.

[0137] TRAPeze® Telomerase Detection Kit TRAP analysis using Millipore showed that telomerase activity was analyzed, Rion treatment improves telomerase expression and activity in normal fibroblast cells. We were able to confirm that it increases (Figure 25).

[0138] Example 10: Effect of luterion on ATP production ATP production in cancer cells and fibroblast-normal cells induced by luterion treatment. To confirm the increase, three types of cancer cells, A549, WTM266-4 and AsPC-1, Normal cells (MRC-5) 1 × 10 6 Cells / ml in a 60mm plate of general DMEM medium After inoculation and incubation for 1 day, the antibiotic PSF (antibiotic-antimyco) is administered. Nutritional starvation (seru) was performed using 0.1% FBS DMEM medium under conditions without tic. The cells were cultured again for 1 day in a state of starvation. Next, on the 3rd day, the cells were separated according to Example 1. After treating with luteion at a concentration of 50 μg / ml, ATP was administered at 0, 10, 30, and 120 minutes. The generation was measured (Figure 26). The ATP measurement kit was Abcam (Cambridge, M I purchased and used it from company A (USA).

[0139] As a result, it was confirmed that ATP production was significantly increased in normal cells treated with luterion. (Figure 39). Furthermore, it was confirmed that ATP production decreased in cancer cells treated with luterion. (Figure 38)

[0140] Therefore, the luterion of the present invention increases energy production in normal cells and suppresses aging. It was found that this is possible. Furthermore, it reduces the energy production of cancer cells, making it suitable for cancer treatment. This allows for cancer cell-specific anticancer treatment without affecting normal cells. Treatment can be considered. [Industrial applicability]

[0141] According to the present invention, luteion, a microparticle present in plants or food, can be effectively separated. The separated luteon can be cultured to grow to a certain size. This allows for the development of various applications for the prevention and treatment of diseases.

[0142] The above describes in detail specific parts of the present invention, but a person with ordinary knowledge in the industry may not be able to do so. For this invention, such specific descriptions are merely preferred embodiments, and thus the present invention It is clear that the scope is not limited. Therefore, the substantial scope of the present invention It can be said that this is defined by the claims and their equivalents.

Claims

1. Lutherion possessing one or more of the following characteristics: (a) Original or elliptical in shape, 50–800 nm in length, and mobile; (b) containing nucleic acid; (c) Shows a reaction similar to that of mitochondria during immunochemical fluorescence staining; (d) Exhibiting fusion and / or fission ecological patterns; (e) If fusion does not occur, the mitochondria mature to a size of 500 nm and contain DNA-like mitochondria. It matures into a rear structure that resembles mitochondria in SEM or TEM electron microscopy images. To indicate; (f) Exhibits a different photoreaction than exosomes; (g) Fission occurs during IR irradiation or pressurization; (h) Expressing CD332, CD133, CD73, or CD39 as a surface antigen; (i) Exhibits autofluorescence; (j) Produces ATP at a size of 200-400 nm; (k) Bilayer or multilayer structure; (l) Adhesive; (m) Inhibition of telomerase activity in cancer cells; (n) Promoting telomerase activation in normal cells; (o) Possesses cell penetrating ability; and (p) Possesses the ability to penetrate the blood-brain barrier (BBB).

2. Rhodamine 123, Mitotracker ker), Acridine Orange, DAPI, and Select one from the group consisting of Janus green B. The above staining agent has the characteristic of being stained positively when used in the present invention. The luterion described.

3. The method for separating luteion, including the following steps: (a) 0.8 The step involves filtering using a filter with voids of approximately 1.2 μm; (b) the step of centrifuging the filtered condensate; and (c) The step of separating luteion from the supernatant liquid obtained by centrifugation.

4. The plant is a medicinal plant selected from Tables 1 to 4, as described in claim 3. Separation method.

5. The aforementioned plants are selected from a group consisting of Angelica sinensis, Porcelain bark, and Kiwifruit. The separation method according to claim 3.

6. The shaking extraction in step (a) above involves adding a solvent to the plant or food and heating it at 50-90°C until it becomes a gas. The claim is characterized by proceeding in a manner that involves intermittently bubbling and shaking using [a specific method / tool]. The separation method described in item 3.

7. The aforementioned shaking extraction involves shaking for 8 to 10 hours, followed by bubbling for 20 to 30 minutes every 2 to 3 hours. The present invention is characterized in that it prevents the lutein of plants or food from clumping together. Method of separating the contents.

8. The method further includes the step of irradiating the condensate obtained in step (a) with IR light. The separation method according to claim 3.

9. The aforementioned centrifugal separation is performed repeatedly at 1200 to 5000 rpm for 5 to 10 minutes. The separation method described in claim 3, characterized by the feature.

10. Step (c) involves irradiating the centrifuged supernatant with IR light with a wavelength of 200 to 600 μm. Separating luteion particles that are attracted to the surface and exhibit mobility, or luteion surface antigens. Confirm whether or not binding occurs with a conjugate that recognizes CD39, CD73, CD133, or CD332. The separation method according to claim 3, characterized in that it identifies and separates luteion particles.

11. The separated luteion particles are filtered through a 50 nm filter, and then the particles that pass through the filter... The separation method according to claim 3, further comprising the step of collecting the removed parts.

12. Filters of 200nm, 400nm, 600nm, 800nm ​​and 1000nm They are used sequentially, for 50-200 nm, 200-400 nm, and 400-600 nm respectively. The classification of rutheons into two sizes: 600-800 nm and 800-1000 nm. The separation method according to claim 11, further characterized by including the following:

13. Claim further comprising the step of fixing under conditions of pH 7 or lower and 0°C or lower. The separation method described in item 3.

14. Claim further comprising the step of fixing at pH 1 to 5 and -90°C to 0°C. The separation method described in item 3.

15. 25,000~35,000psi (pound per square inch) The method according to claim 3, further comprising the step of inducing fission by applying the above pressure. Separation method.

16. The invention further includes a step of inducing cell division and propagation at a pH of 1 to 3 and a temperature of 10 to 20°C. The separation method described in claim 3, characterized by the feature.

17. The method for separating luteion, including the following steps: (a) An extract containing luterion, with an antibody that specifically binds to the luterion surface antigen or A step in which particles immobilized with aptamers are added to induce binding between luteion and particles; and (b) A step of recovering the luteion bound to the particles.

18. (c) Further comprising the step of separating only the luteions from the luteions bound to the particles. A method for separating luteion according to claim 17, characterized by the following:

19. The extract containing luterion is characterized by being a plant extract or a food extract. The method for separating luteion according to claim 17.

20. The extract containing luterion is (i) a hot water extract of a plant or food, (ii) a plant Solvent extract of a substance or food, or (iii) heating of a plant or food in the presence of a solvent The method for separating luteion according to claim 17, characterized in that it is a condensate of the gas generated. Law.

21. The separation method according to claim 20, characterized in that the condensate is produced through the following steps. Law: (a) Add a solvent to a plant or food and intermittently bubble it using gas at 50-90°C. The stage of shaking while rubbing; and (b) After collecting the water vapor or gas vaporized by the shaking, cool it to make a condensate. The acquisition stage.

22. The luterion surface antigen is CD39, CD73, CD133, or CD332. The method for separating luteion according to feature 17.

23. The aforementioned particles include magnetic particles, silica particles, quantum dot particles, glass particles, polymer particles, and fibers. The claim in 17, characterized by being selected from the group consisting of particles and fluorescent particles. Method for separating luterions.

24. The aforementioned magnetic particles are iron, aluminum, cobalt, nickel, manganese, tin, zinc, and cadmium. It is composed of oxides of magnesium, copper, barium, lithium, or yttrium. A method for separating luteion according to claim 23, characterized in that it is selected from the group.

25. The magnetic particles are double-coated with carbon and functional groups for antibody or aptamer bonding. The method for separating luteols according to claim 23, characterized by being quenched.

26. The aforementioned functional groups are amide groups and ester groups. (oup), amine group and carboxyl group The L according to claim 25, characterized in that it is selected from the group composed of l group). The method for separating Therion.

27. The particle size is 10 to 1,000 nm, as described in claim 17. The method of separating the luteion.

28. The particles are magnetic nanoparticles, and step (b) is performed by applying an external magnetic force to the luteor. The method described in 17, characterized in that magnetic nanoparticles to which the ions are bonded are collected and then recovered. Method for separating luterions.

29. The particles are fluorescent particles, and step (b) is performed using a fluorescence-based cell sorter. The Luterio according to claim 17, characterized in that fluorescent particles to which therion is bound are recovered. Method of separating n.

30. The aforementioned particles are electrostatically charged particles, and step (b) creates a non-uniform electric field. The method described in 17, which involves recovering particles to which luteions are bound by electrostatic attraction. The method of separating the luteion shown.

31. The particles are ionic particles, and step (b) utilizes electrostatic attraction to lutell The separation of luteion according to claim 17, characterized by recovering particles to which ON is bound. method.

32. The luteion described in claim 1 is mixed with water and subjected to IR light irradiation or pressurization for 18 to 30 minutes. A method for culturing luteolion, including a step of growing it at °C.

33. The luteion described in claim 1 is grown in a sugar-containing medium under conditions of pH 5 to 9 and 18 to 30°C. A method for culturing luteolion, including the propagation stage.

34. The aforementioned sugars are rhamnose, glucose, and galac. Galactose, fructose, or xylose The culture method according to claim 33, characterized in that it is xylose.

35. The sizes of the luteions before and after culture were 50-200 nm and 300 nm, respectively. The culture method according to claim 32 or 33, characterized in that the wavelength is ~500 nm.

Images