Luterion and separating and culturing methods for the same

Abstract

To provide luterion which is a mitochondrial-like micro-material, and to provide separating and culturing methods for the same.SOLUTION: Provided is a method for isolating luterion, comprising the steps of: (a) filtering a condensate formed by cooling water vapor or gas obtained by boiling an extract of a plant or food, with a filter having a pore size of 0.8 to 1.2 μm; (b) centrifuging the filtered condensate; and (c) separating the luterion from the centrifuged supernatant by irradiation with IR light having a wavelength of 200 to 600 μm in order to gather luterion having mobility.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to luteolin, a microorganism similar to mitochondria, and a method for isolating and culturing the same. Regarding the method, specifically, the method uses a filter having a specific size of pores to filter Lutelium. A method for isolating luteolin having specific characteristics isolated by such a method, and The present invention relates to a culture method for propagating the same. [Background technology]

[0002] The inventors of the present application have discovered that microscopic substances present in excreted bodily fluids of patients or healthy individuals We will develop a method to effectively separate the ruthenium species and elucidate their properties. The above information was applied for as a patent on May 9, 2014 (WO2015 / 108246). It is used to diagnose disease by observing the properties of minute substances present in the body fluids already excreted by the patient. The company discovered that it is possible to predict and predict the effects of the aging process, and filed a patent on the discovery on January 14, 2014. (WO2015 / 005553).

[0003] Such lutelia are: (1) cells that have intermediate fusion properties between prokaryotic and eukaryotic cells; or cell analogs; (2) present in bodily fluids such as blood, semen, intestinal fluids, saliva, and cellular fluids; (3) Immunofluorescence assay using Janus green B and acrid Acridine Orange, and Rhodamine 123 (4) under optimal conditions (pH 7.2-7.4) showed the expression characteristics of genes from Beta-proteobacteria and Gamma-proteobacteria. (5) Under acidified conditions, it is a beta-proteobacterium. Expression characteristics of genes from eukaryotic cells as well as genes from rhabditis elegans and gamma-proteobacteria Although it shows sexuality, it mainly shows the homologue characteristics with Sterptophyta genes. (6) It grows from over 400 nm to over 2000 nm; under normal conditions, it is involved in the production of ATP. and (7) unlike mitochondria, exosomes are involved in a completely different set of cell or It is a cell analogue.

[0004] Such luteria are mainly found in the blood of animals, including humans, whereas luteria Lutelin, a microscopic substance found mainly in plants and foods, with a structure and function similar to that of lutein. (Luterion) was confirmed to exist.

[0005] Under such technical background, the inventors of the present application have been able to apply Lutelinone to clinical practice. As a result of our efforts to effectively isolate and cultivate plants or foods that are solvent-free, After adding and shaking, the vaporized gas was collected and then filtered and centrifuged. The ruthenium contained in the vaporized gas can be effectively separated, and the ruthenium separated from it The present invention has been made by confirming the possibility of culturing the terion under specific conditions and in a medium. Finished. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide Luteinium. Another object of the present invention is to extract Luteinium luteum using a filter having a specific pore size. The object of the present invention is to provide an effective separation method that can be applied to clinical practice. It is yet another object of the present invention to provide a method for efficiently culturing Luteinium. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention provides a lutetium having one or more of the following selected characteristics: Lion offers: (a) 50-800 nm, circular or elliptical, motile; (b) containing nucleic acids; (c) When stained with immunofluorescence, it shows a reaction similar to that of mitochondria; (d) exhibit fusion and / or fission ecological patterns; (e) In the absence of fusion, the mitochondria mature to a size of 500 nm and become DNA-containing mitochondria. They mature into structures similar to mitochondria in SEM or TEM electron micrographs. Show; (f) exhibits a different light response than exosomes; (g) Fission occurs when exposed to IR or under pressure; (h) expressing CD332, CD133, CD73, or CD39 as a surface antigen; (i) exhibits autofluorescence; (j) 200-400 nm in size and produce ATP; (k) double- or multi-membrane structures; (l) Adhesive; (m) Inhibition of telomerase activity in cancer cells; (n) Promoting telomerase activity in normal cells; (o) Possesses cell penetrating ability; and (p) Ability to penetrate the blood-brain barrier (BBB).

[0008] The present invention also provides a method for isolating Lutelin, comprising the steps of: (a) 0.8 g of condensate obtained by cooling a steam or gas-extracted extract of a plant or food filtering using a filter having pores of 1.2 μm; (b) centrifuging the filtered condensate; and (c) separating the luteolin from the centrifuged supernatant.

[0009] The present invention also provides a method for isolating Lutelin, comprising the steps of: (a) isolating Lutelin; The extract contains an antibody or aptamer that specifically binds to the luteinizing surface antigen. (b) adding the particles to induce binding between the Lutelin and the particles; and Recovering the bound lutherion.

[0010] The present invention also relates to a method for producing the above-mentioned luteolin by adding water and subjecting the luteolin to irradiation with IR light or pressure for 18 to 3 A method for culturing Lutetium is provided, comprising growing the Lutetium at 0°C.

[0011] The present invention also relates to a method for growing the Lutelinium erythrocyte in a sugar-containing medium under conditions of pH 5 to 9 and 18 to 30° C. The present invention provides a method for culturing Luteolin, comprising the step of growing the Luteolin. [Brief description of the drawings]

[0012] [Figure 1] This is a photograph of a Lutetium taken with a confocal laser scanning microscope (Zeiss), showing its size. [Diagram 2] The photographs show the presence or absence of coloring after staining lutellium with Mitotracker. [Diagram 3] The photographs show the presence or absence of color development after staining luteinium with Rhodamine 123. [Figure 4]Fluorescent / non-fluorescent photographs of Lutelinium are shown for comparison (A: Janus Green B Positive; B: Rhodamine 123 Positive; C: Mito-tracker Red Positive; D: Unstained). [Diagram 5] The results show the presence or absence of color development after the luteolin was stained with Rhodamine 123, Mitotracker Red, and Janus green B (A: Janus Green B Positive; B: Rhodamine 123 Positive; C: Mitotracker Red Positive; D: Unstained). [Figure 6] The size of the fluorescent stained area of ​​luteinium was measured to show the size of luteinium (50-800 nm) (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] Life cycling A of normal lutherion and life cycling B of mutated lutherion are shown. [Figure 8] This is a TEM electron microscope photograph confirming that Lutelinium has a double membrane structure. [Figure 9] This is an atomic microscope photograph confirming that luteolin contains nucleic acid inside. [Figure 10A] These are the results of a bioanalyzer analyzing whether or not RNA is contained inside the lutelion (L: Control; 1: 50 nm or less, 2: 50 to 100 nm, 3: 100 to 200 nm, 4: 200 to 400 nm). [Figure 10B] The total RNA of 200-400 nm luteolin is shown. [Figure 11]The results of induction of Lutelinium division under conditions of 5,000 psi and 10-20°C are shown. [Figure 12] The results of induction of Lutelinium division under conditions of 15,000 psi and 10-20°C are shown. [Figure 13] The results of induction of Lutelinium division under conditions of 35,000 psi and 10-20°C are shown. [Figure 14] The results of introducing lutherion into the nucleus are shown by fluorescent staining with red fluorescence of PKH26-1, PKH26-2, and PKH26-3. [Figure 15-16] This shows the results of a magnified observation that red fluorescently stained luteolin was introduced into the intracellular nucleus. [Figure 17] The results of oral gavage administration of fluorescently stained Lutelinium are shown. [Figure 18-19] The results of intraperitoneal injection of fluorescently stained luteinium are shown. [Figure 20] This shows telomerase activity in normal cells (fibroblast) and cancer cells (lung cancer, breast cancer, and colon cancer) treated with telomerase. [Figure 21] The figure shows the cell viability of normal cells (fibroblast) and various cancer cells (lung cancer, breast cancer, colon cancer, liver cancer, leukemia) depending on the concentration of luteolin treatment. [Figure 22] This shows the inhibition of cell proliferation in pancreatic cancer cell line (AsPC-1) treated with various sources of luteolin. [Diagram 23] The figure shows the inhibition of cell proliferation in pancreatic and lung cancer cell line (A549) treated with various sources of luteolin. [Figure 24] This shows the inhibition of cell proliferation in pancreatic breast cancer cell line (BT-20) treated with various sources of luteolin. [Diagram 25] This shows telomerase activity in normal cells (fibroblasts) treated with telomerase. [Figure 26]FIG. 1 is a schematic diagram of an ATP assay design to confirm the presence or absence of ATP production and inhibition by Luteinion treatment. [Figure 27] This shows the result of photographing Lutetium using a Carl Zeiss scanning electron microscope (SEM). [Figure 28] This shows the result of photographing a lutheran ion using a Bruker Fast Scan AFM atomic microscope. [Figure 29] The lutellium was stained with anti-CD39 antibody, Mitotracker, and DAPI, and the presence or absence of coloring is shown separately or in combination. [Diagram 30] The lutellium was stained with anti-CD73 antibody, Mitotracker, and Hoechst, and the presence or absence of coloring is shown separately or in combination. [Diagram 31] 1 shows the results of confirming whether Luteinium is positive for anti-CD332 antibody via fluorescence emission. [Diagram 32] The results of confirming whether or not Luterion was positive for anti-CD133 antibody through fluorescence emission are shown. The upper row shows the result of PE (Phycoerythrin) fluorescent staining, and the lower row shows the result of anti-FITC fluorescent staining. [Diagram 33] This shows the step of coating particles with carbon and carboxyl groups, on which antibodies that specifically bind to erythrocyte surface antigens are immobilized. [Diagram 34] 1 shows a step of separating, using a magnet, a complex formed by binding Luteinium and an antibody that specifically binds to the Luteinium surface antigen immobilized on magnetic particles. [Diagram 35]Photographs taken with a fluorescence microscope after isolating luteinium are shown. (a) shows a photograph taken with a fluorescence microscope after isolating luteinium using a nanofilter. (b) shows a photograph taken with a fluorescence microscope after isolating luteinium using a separation method that uses particles having antibodies fixed thereto that specifically bind to luteinium surface antigens. (c) shows a photograph taken with an electron microscope after isolating luteinium using a nanofilter. (d) shows a photograph taken with an electron microscope after isolating luteinium using a separation method that uses particles having antibodies fixed thereto that specifically bind to luteinium surface antigens. [Diagram 36] The figures show the amount of water extracted from Luterin, (a) shows the amount of water extracted from Luterin after it was separated using a nanofilter, and (b) shows the amount of water extracted from Luterin after it was separated using a separation method that utilizes particles immobilized with antibodies that specifically bind to Luterin surface antigens. [Figure 37] FIG. 1 is a schematic diagram illustrating the extraction steps of Lutelin. [Figure 38] This shows the decrease in ATP production in cancer cells due to treatment with luteolin. [Figure 39] This shows ATP production in normal cells (fibroblasts) treated with luteolin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Unless otherwise defined, all technical and scientific terms used herein are The terms have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods detailed below are consistent with the present invention. This is well known and widely used in the technical field.

[0014] The terms "luterial" and "luterion" as used herein "Dimers of 50 to 400 nm in diameter are present in all living organisms, including animals and plants. It is a nano-organism with a size of 100 mm, similar to a virus, and grows to 800-1200 nm when fused. However, mutants that exhibit genetic abnormalities and pathological phenomena at sizes greater than 1200 nm This is the name given by the inventor to the fine material classified as "particles of interest."

[0015] Luterials and luteolins are distinct in that they contain DNA and RNA and have the properties of motility and adhesion. It is different from exosomes and microvesicles. Mitochondria were stained with Janus green B and fluorescent The stains Rhodamine 123 and Mitotracker tracker, Acridine Orange, and D The color development was confirmed by the API, but the luteolin and luteal were also found in mitochondria. The staining was confirmed by the same dye as that used in the study (Figures 1 to 6). A membrane structure with an incomplete internal cristae structure. The structure of the mitochondria is similar to that of mitochondria (Fig. 7), and they are observed in the same laser wavelength range as mitochondria. "mitochondrial", "mitochondrial analogue" or "mitochondrial precursor" It may also be called "o-mitochondria."

[0016] Luterial refers to nano-organisms that exist within a host in an ecosystem, and is used by animals, including humans. In some cases, blood, saliva, lymphatic vessels, semen, vaginal fluid, breast milk (especially colostrum), umbilical cord blood, brain cells, They can reside in the spinal cord and bone marrow. On the other hand, Lutelinium can be used by the host as food. The term refers to nano-organisms that can survive on their own and are found primarily in plants and foods.

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

[0018] In one aspect, the present invention provides an isolated lutetium having one or more of the following selected characteristics: About Lion: (a) 50-800 nm, circular or elliptical, motile; (b) containing nucleic acids; (c) When stained with immunofluorescence, it shows a reaction similar to that of mitochondria; (d) exhibit fusion and / or fission ecological patterns; (e) In the absence of fusion, the mitochondria mature to a size of 500 nm and become DNA-containing mitochondria. They mature into structures similar to mitochondria in SEM or TEM electron micrographs. Show; (f) exhibits a different light response than exosomes; (g) Fission occurs when exposed to IR or under pressure; (h) expressing CD332, CD133, CD73, or CD39 as a surface antigen; (i) exhibits autofluorescence; (j) 200-400 nm in size and produce ATP; (k) double- or multi-membrane structures; (l) Adhesive; (m) Inhibition of telomerase activity in cancer cells; (n) Promoting telomerase activity in normal cells; (o) Possesses cell penetrating ability; and (p) Ability to penetrate the blood-brain barrier (BBB).

[0019] According to one embodiment of the present invention, Lutein is a living organism. ) from a level similar to a virus to about 500 nm (normal division stage 50-500 nm) The inventor has named this microscopic substance with a size of 800 nm or more (abnormal fusion stage) They are microvesicles in that they contain DNA and / or RNA and are motile. It is also distinguished from autofluorescence and Mitochondria are characterized by the fluorescent dye rhodamine 123 (Rh odamine123), Mitotracker, Acridine Oligonucleotide Acridine Orange, DAPI, and Janus Green B Coloring is confirmed by using US green B), but luteolin is the same as mitochondria. Since color development is confirmed by the staining agent, the staining agent can be used to correctly separate the luteolin. It is possible to check whether the above has been done (Figures 1 to 6).

[0020] In addition, RNA was detected in the lutein by DAPI and acridine orange (AO) staining. It can be confirmed that the DNA is included as well as the RNA. The level of excitation by the chromidine orange dye is 460 nm and emission is 650 nm. The DNA is stained orange at an excitation level of 502 nm and emission at 525 nm. The cells are stained green with DAPI, and the presence of DNA can be confirmed by the DAPI staining method. The lutein of the present invention can be stained by the above-mentioned staining method to detect RNA and DNA in the lutein. You can see that A is included.

[0021] In one embodiment of the present invention, the structure of Lutelinium was confirmed to have a bilayer or multilayer structure. It was confirmed that the lutein contains nucleic acids, especially RNA (Figure 8). It was confirmed (Figures 9 and 10).

[0022] Previously, the presence of lutein in plants or foods was not known, so It can be said that there is no technology for isolating or culturing lions. It is possible to provide a method for effectively isolating and culturing terions.

[0023] In another aspect, the present invention relates to a method for isolating Luteolin, comprising the steps of: ) The condensate obtained by cooling the steam or gas-extracted extract of plants or foods is 0.8 to 1 (b) filtering the filtered condensate using a filter having pores of 0.2 μm; (c) separating the luteolin from the centrifuged supernatant. This is the stage where...

[0024] First, the step (a) is a process for cooling and extracting a steam or gas-extracted extract of a plant or food. The obtained condensate is filtered using a filter having pores of 0.8 to 1.2 μm. be.

[0025] In one embodiment, the shaking extraction is carried out by adding a solvent to a plant or food and subjecting the plant or food to gas extraction at 50 to 90°C. The present invention can be carried out by intermittently bubbling the mixture while shaking it. The density of the luteria used in this study is less than 1, which is higher than that of fat and soil. It has a lower density than protein and can be isolated from plants or foods by steam distillation. It can be, but is not limited to, this.

[0026] When the mixture was shaken while intermittently bubbling with gas at 50-90°C, the is liberated in the form of steam or gas together with the gas, but when shaken with steam or gas, The boiling point of plants or foods containing lutein is lowered, and the decomposition and transformation of lutein is accelerated. This can prevent quality or damage.

[0027] Optionally, in step (a), shaking is performed for 8 to 10 hours, with shaking being performed every 2 to 3 hours for 20 to 30 minutes. An additional step of bubbling is performed to prevent the plant or food luteolin from clumping together. This can increase the efficiency of isolating Lutein.

[0028] The details of the extraction-filtration in the step (a) are shown in FIG. After adding a solvent, such as distilled water, to the food and extracting through shaking, about 100-250g, Preferably, the primary centrifugation is performed at 190 g to spin down and remove impurities, and then the mixture is centrifuged for about 1000 min. - Secondary centrifugation at 5000g, preferably about 3000g for several minutes to several tens of minutes (preferably the second After stabilization for a period of time (approximately 24 hours), the supernatant can be collected. After staining with D332 to confirm motility, the presence or absence of luteolin was first confirmed. After that, to obtain pure lutein, 100,000-150,000g of lutein is required. A third centrifugation step, preferably at 120,000 g, may be performed.

[0029] After that, the lower layer was filtered through a filter of about 800 nm and collected, and the plasma and other internal implants were Remove the pellet of cell debris such as exosomes. The pellet-free supernatant can be collected by centrifugation at 40,000 g or more. The supernatant was filtered through a 400 nm filter to collect the lower layer, which was then resuspended in CD332. Motility can be confirmed by staining the cells. The supernatant on top of the filter can be collected to collect the Lutelinium. Alternatively, it can be stored at a temperature of -90°C or lower.

[0030] In one embodiment, the plant may be a medicinal plant selected from Tables 1 to 4. Lyons are present in all plants and are not limited to the medicinal plants listed in Tables 1-4.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] In addition, it is expected that the luteolin contained in plants is distributed in large amounts in the stems of plants. It is preferable to isolate the luteolin by including

[0036] The condensate is a liquid that collects water vapor or gas vaporized by the shaking and then cools. The collected vaporized water vapor or gas can be separated from the lutherio. You can get ruthenium mixed with it, and you can get vaporized water vapor. Or if gas and luteolin are mixed, an additional separation step is required to obtain luteolin. It is possible.

[0037] Optionally, the method further includes irradiating the condensate obtained in step (a) with IR light. For example, IR light may be irradiated for 20 to 60 minutes, preferably 30 to 40 minutes, to obtain a lutetium-containing cellulose membrane. The on can be prevented from being deformed and the mobility can be prevented by IR light irradiation before performing step (c). By collecting luteolin having To do so.

[0038] The step (b) is carried out by filtering the obtained mixture using a filter having a pore size of 0.8 to 1.2 μm. The condensate is filtered through a filter with pores of 0.8 to 1.2 μm. The optimal size was derived by the inventor of the present application taking into account the major axis of the rutherion. The condensate obtained in step (a) is filtered to obtain a solution containing the desired luteolin. This can be done.

[0039] The step (c) is a step of centrifuging the filtered condensate. The centrifugation is performed at 1200 to 500° C., so that the luteolin of higher purity can be obtained. Repeated centrifugation at 0 rpm for 5-10 min. for improved purity. can also be adjusted and applied.

[0040] The step (c) is a step of separating the luteolin from the supernatant after centrifugation. For example, the centrifuged supernatant is irradiated with IR light having a wavelength of 200 to 600 um to detect the movement of the liquid. We isolated the luteinizing particles that aggregated with luteinizing properties, and identified the luteinizing surface antigens CD332 and CD1 33. Check for the presence or absence of binding to CD73 or CD39-recognizing ligands. This includes, but is not limited to, separating children.

[0041] Thus, even after isolating the luteolin through step (c), the isolated luteolin After particles are filtered through a filter with pores smaller than 50 nm, the part that is caught by the filter It is also possible to collect the luteolin and separate only the luteolin that is filtered. By using this method, fine particles other than ruthenium can be removed, and ruthenium having a size of 50 nm or more can be removed. You can get Therion.

[0042] Optionally, additional 200nm, 400nm, 600nm, 800nm ​​and 1000nm The filters were sequentially used to measure the wavelengths of 50-200 nm, 200-400 nm, and 4 Lutheran sizes: 00-600nm, 600-800nm, and 800-1000nm The luteolin of the present invention can be classified into two types: Observation is possible through a mirror, and the wavelengths are 200nm, 400nm, 600nm and 800nm, respectively. Using a filter of 50-200 nm (nascent stage) / 200-40 0nm (maturity phase) / 400-600nm (mitosis phase) / 600-800nm ​​(hypermitosis phase) It can be divided into two categories (Figures 27 and 28).

[0043] Hereinafter, the method may further include a step of storing the luteolin under specific pH and temperature conditions. For example, it can be stored under conditions of pH 7 or less and 0°C or less. It can be stored at pH 1 to 5 and at -90°C to 0°C. When stored at temperatures below 0°C under conditions H1-3, motile Lutelinium is preserved. It was confirmed that preserving lutein can improve the separation efficiency. do.

[0044] It may also include additional propagation steps, such as a fission induction step. Apply pressure of 5,000 psi (pound per square inch) or more. In one embodiment of the present invention, the cell division can be induced, particularly 25,000 to 35,000. It was confirmed that division was induced by applying pressure of 100 psi. ° C. and maintained at pH 1 to 3 as used during storage.

[0045] In another aspect, the present invention relates to a method for isolating Luteolin, comprising the steps of: (a) an antibody that specifically binds to a luteinizing agent surface antigen in an extract containing luteinizing agents; or adding particles having aptamers immobilized thereon to induce binding between Luteinion and the particles; and (b) recovering the luteolin bound to the particles.

[0046] In the present invention, a large amount of lutein is efficiently produced based on the expression of a specific antigen on the lutein surface. In order to efficiently separate the specific antigens, particles to which antibodies or aptamers against the specific antigens are immobilized are used. As a result, in one embodiment of the present invention, it was found that the time required for isolating Lutelin was shorter than that of the conventional method. It was confirmed that large amounts of luteolin could be isolated between the two.

[0047] The extract containing luteolin may be, for example, (i) a hot water extract of a plant or food, ii) a solvent extract of the plant or food, or (iii) the plant or food in the presence of a solvent antibiotic. It may also be condensation of gases evolved by heating the food.

[0048] The hot water extract is, for example, a product obtained by extracting physiologically active substances from plants using water at 40°C or higher. If hot water extraction is used, problems due to the toxicity of organic solvents may occur. The solvent extract is, for example, ether, methanol, etc. Solvents such as ethanol, hexane, etc. It may be extracted using

[0049] The condensate is made by adding distilled water to plants or food and refrigerating them at 50 to 90 degrees Celsius using gas. The mixture is shaken while bubbling intermittently to capture the water vapor or gas vaporized by the shaking. It may be a condensate obtained by collecting and then cooling.

[0050] The "Luteinizing surface antigen" according to the present invention refers to a hydrophobic surface antigen located in the lipid layer of the Luteinizing membrane. It is attached to the cell surface membrane via a carboxy-terminal domain that contains cyclic amino acids and is It is possible to exert a biological antigen effect, for example, CD39 (Ectonucleoside triphosphate diphosphohydrolase 1;ENTPD 1), CD73 (Ecto-5'nucleotidase; NT5E), HBsAg, SLC3A2, CD109, LY9, CD332, CD133 or CD53. Preferably, it may be CD332, CD133, CD39 or CD73.

[0051] "Antibody" means a protein molecule that specifically binds to an antigenic site. The present invention uses particles immobilized on a substance capable of specifically binding to the luteinizing surface antigen. However, an antibody that specifically binds to the antigen can be used. The body may be, for example, administered an anti-CD332 antibody, an anti-CD133 antibody, an anti-CD39 antibody or an anti- The antibody may be a CD73 antibody. The antibody may be a commercially available product. Instead of such antibodies, aptamers that bind to the surface-expressed antigens can also be used. good.

[0052] "Particle" means a particle that, by its nature, has magnetic, luminescent, electrostatic and ionic properties. and those that have a micro or nano size depending on the size. There are no particular limitations as long as it can separate lutein through binding with an antibody. For example, magnetic particles, silica particles, quantum dot particles, glass particles, polymer particles, fiber particles and fluorescent particles, and the particles have magnetic and fluorescent properties. At the same time, the particles include those in which quantum dots and gold or silver particles are bonded to magnetic particles. can be done.

[0053] The particles are, for example, magnetic particles, The magnetic particles refer to particles that are magnetic and move in response to a magnetic field. The magnetic particles may be, for example, a metal material. ), magnetic material or magnetic alloy The magnetic particles are particles that are magnetic, but in the present invention, However, the magnetic particles are not limited thereto, and may be particles that are not magnetic in themselves, but may have magnetic properties. Alternatively, the metal particles may be metal particles that are attracted by a magnetic force. Iron, aluminum, cobalt, nickel, manganese, tin, zinc, cadmium, magnesium, copper any one selected from the group consisting of oxides of barium, lithium, or yttrium; Among these, iron is preferably selected. The particles are micrometers in size. When the particle size is 100 nm, it becomes ferromagnetic, but when the particle size is small, it becomes supermagnetic. Such particles are easy to synthesize and their size can be easily controlled. For example, the size is 1 to 1,000 nm, preferably 10 to 1,000 nm, more preferably Preferably, particles having a size of 10 to 500 nm, more preferably 10 to 100 nm, are used. It is possible.

[0054] In some cases, the magnetic particles are modified to maintain their dispersibility and safety. It can be modified, for example by coating it with carbon.

[0055] Additionally, it may further comprise functional groups suitable for antibody or aptamer binding. The functional group may be, for example, an amide group, an ester group, or the like. oup), amine group, carboxyl group group), thiol group (SH), epoxy group, phenyl Phenyl group, sulphone group, a Alkoxy group, aldehyde group p) may be selected from the group consisting of a ketone group. The amine group (NH2) is a monoamine, diamine, ), triamine, ethylenediamine ine or diethylenetriamine This is also fine.

[0056] In the step of recovering the luteolin bound to the particles, when the particles are magnetic particles, Step (b) is to apply magnetism externally to recover the magnetic nanoparticles to which luteolin is bound. It is characterized by:

[0057] The method of collecting the magnetic nanoparticles to which the luteolin is bound by applying the magnetic material and then recovering the magnetic nanoparticles includes: A magnetic material having an antibody or an aptamer that binds to the luteinium and the luteinium surface antigen immobilized thereon After mixing and reacting the sample containing the particles, the mixture is placed on a magnet or a magnetic activated cell separator (Ma Using gnatic-activated cell sorting (MACS) After collection, the supernatant was removed and the cells were resuspended in a buffer solution, and then the agglutination film and the capture film were applied. The antibody-magnetic particle complex was then filtered through a membrane to confirm the presence of the antibody-magnetic particle complex. Cut.

[0058] In the present invention, when the particles are fluorescent particles, the step (b) is carried out using a fluorescence-based cell sorter. The method is characterized in that the particles to which Lutein is bound are recovered using the above method.

[0059] The method using the fluorescence-based cell sorter is to recover cells according to the type and degree of fluorescence. A method for classifying minute substances of various shapes, in which fluorescent cells are selected for quantitative analysis and classification. A "fluidic system" that allows the liquid to flow through the pipes, and a "flow control system" that controls the flow The control system is equipped with an optical system to observe the flow of minute substances. The optical system converts the optical signals of the optical system into electrical signals and processes them. The fluorescence activated cell sorter is a system that characterizes various cells. It is a more efficient classification and collection device, and is useful in the life sciences (zoology, botany, microbiology, agriculture, water science, etc.) This device is widely used in the fields of industry, academia, forestry, and medical research. The fluorescent substance used in the experiment can be any fluorescent substance that can be used for biological imaging. The fluorescent material may be, but is not limited to, rhodamine and its derivatives, fluorescein, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, Lithrosin and its derivatives, eosin and its derivatives, and 4-acetamido-4'-iso and thiocyanatostilbene-2,2'-disulfonic acid. More specific examples of the fluorescent substance are as follows: Rhodamine and its derivatives: 6-carboxy-X-rhodamine (ROX), 6-carbo Xyrhodamine (R6G), Lissamine 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 -Rhodamine (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-dichlorotriazin-2-yl)aminofluorescein (DTA F), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JO E), fluorescein, fluorescein isothiocyanate, QFITC ​​(XRITC), Luoresamine, IR144, IR1446, Malachite Green Isothiocyanate, 4 -Methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosa Phytophthylamine, phenol red, B-phycoerythrin, o-phthalaldehyde; coumarin and and its derivatives: Coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120 ), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosigna, 4'-6-diamidino-2-phenylindole (DAPI), 5',5''-dibromo Pyrogallol-sulfonphthalein (Bromopyro-gallol Red), 7 -Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin diede Triamine pentaacetate, 4-(4'-diisothiocyanato dehydro-styryl 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 ester, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-Amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfide N-(4-anilino-1-naphthyl) methyl ester (LuciferYellow VS) Brilliant Yellow; Pyrene and its Derivatives: Pyrene, Pyrenebutyrate, Succinimidyl 1-pyrenebutyrate, Reac tive Red 4(Cibacron Brilliant Red 3B-A); Erythrosine and its derivatives: Erythrosine B, Erythrosine isothiocyanate, Erythrosine thiocyanate;Eosin and its derivatives;Eosin, Eosin isothiocyanate;4-Athidium Cetoamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid.

[0060] In the present invention, when the particles are electrostatically charged particles, the step (b) is carried out by discharging the particles with a non-uniform electric charge. By creating a field, the particles are forced to form dipole moments, and the ruthenium is held together by electrostatic attraction. The method is characterized in that the removed ionic particles are collected.

[0061] The non-uniform electric field is formed to cause the particles to form a dipole moment, which causes the particles to move together by electrostatic attraction. The method for recovering the ionic particles with the terion bonded thereto is to use an electric Particle classification device (Differential mobility analyzer; DMA) Electric particle classifiers use the difference in particle mobility caused by electrostatic forces to separate particles. This is a device that classifies particles, and is also called a differential mobility analyzer or a differential electrostatic classifier. Specifically, electrical particle classifiers exploit the fact that the moving speed of charged particles is a function of particle size. By using the method, poly-disperse particles are converted into mono-disperse particles of the desired diameter. This equipment separates (no-disperse) particles.

[0062] In the present invention, the particles are ionic particles, for example, anionic or cationic particles. In the case where the step (b) is performed, the ionic particles to which the ruthenium is bound by electrostatic attraction are formed. The child can be retrieved.

[0063] The method for recovering fiber particles to which luteolin is bound by electrostatic attraction comprises the steps of: Via a differential mobility analyzer (DMA) This is possible. The present invention further includes a step of separating only the luteolin from the luteolin bound to the particles. It is possible to do so.

[0064] The particle-bound luteolin was added with a BSA / PBS buffer solution and incubated at 25°C. After incubation, the BSA is attached to the antibody using magnetic or ionic properties. Only the particles with BSA adsorbed on them are separated. PBS was added again to the particles to which 1,2-dichlorophenyl phosphate had been adsorbed, and the particles were incubated. ) to detach the particles and isolate only the luteolin.

[0065] In yet another aspect, the present invention provides a method for the preparation of a luteinized rice product by adding water to the luteinized rice product and irradiating the luteinized rice product with IR light or The present invention relates to a method for culturing L. lutellium, which comprises a step of growing the L. lutellium under pressure at 18 to 30°C.

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

[0067] In yet another aspect, the present invention relates to a method for culturing Luteolin in a sugar-containing medium at a pH of 5 to 9 (during storage). A method for culturing Lutelon including a step of growing the Lutelon under conditions of pH 1 to 3 and 18 to 30°C. Regarding.

[0068] In one embodiment, the sugar is rhamnose, glucose, e), galactose, fructose or It may be xylose, and preferably glucose. In one embodiment of the invention, glucose is added to a medium containing glucose, for example, a DMEM medium or a blood medium. Add 1 to 10% by weight of ethanol, preferably 2 to 8% by weight, and incubate at pH 7 and 20° C. It was confirmed that Lutein can be cultured under optimal conditions to maintain the conditions. The number of lysosomes was determined by the expression of lysosome-specific surface antigens CD332, CD133, CD73, or Count the number of CD39-containing lutenoids and those stained with Mitotracker Red. I checked and confirmed it.

[0069] The size of the luteolin before culture may be 50 to 200 nm. The size of the Lutelinium cultured by the culture method may be 300 to 500 nm. At this time, observe under a microscope to ensure that the size of the luteolin does not exceed 500 nm. After the cultivation is completed, the cells are sorted by size and stored at -80℃. It can be stored under nitrogen or above 0°C, and a preservative can be added during storage.

[0070] Lutelinium cultured as described above grew to the desired size (50 It can be stored at a low temperature (less than 0.1 nm) and can be effectively used in the treatment of diseases using luteolin. can.

[0071] Specifically, the Luteinizing agent of the present invention can be introduced into the nucleus of a cell. The cells were stained with red fluorescence to confirm whether the luteolin was introduced into the nucleus. We confirmed that luteolin was introduced into the nuclei of cells stained with DAPI.

[0072] In addition, the luteinium of the present invention is fluorescently stained and the fluorescently stained luteinium is intraperitoneally injected. The drug was administered intraperitoneally (IP) and by oral gavage. The drug was administered orally and the results were consistent at least 3 hours after administration. It was confirmed that the drug crossed the BBB within 5 minutes after intraperitoneal injection. By utilizing the above properties, the Lutelinone of the present invention can be used to treat degenerative brain diseases by breaking down the blood-brain barrier. This overcomes the limitations of drugs that could not be used as therapeutic agents because they could not pass through the bloodstream. It can be used as a therapeutic agent for degenerative brain diseases that can pass through the blood-brain barrier, or ... It can also be used as a drug delivery agent to enable passage of the blood-brain barrier.

[0073] In addition, the telomerase of the present invention inhibits telomerase activity in cancer cells and inhibits telomerase activity in normal cells. has no effect or enhances telomerase activity, effectively inhibiting only the proliferation of cancer cells It has an anti-cancer effect.

[0074] On the other hand, in normal cells, telomerase expression was increased by Luteinin treatment, and telomere length was increased. The amount of telomerase in normal cells was increased, and luteolin showed anti-aging activity by enhancing telomerase activity. We were able to confirm that this was the case.

[0075] In the present invention, "normal cells" refer to cells that have increased telomerase activity along with cancer cells and are in a state of infinite proliferation. By this we mean cells that undergo the normal aging process rather than cells that have a proliferative phenotype.

[0076] The term "telomerase" as used herein refers to the enzyme that binds telomeres to the ends of telomeres. ric) means a ribonucleoprotein that catalyzes the addition of repeats. Telomeres are the ends of chromosomes. They are long expansions of repeat sequences that cap the ends of chromosomes and are thought to stabilize chromosomes. In humans, telomeres are typically 7-10 kb in length and consist of multiple repeats of the sequence -TTAGGG-. Telomerase is not expressed in most adult cells, and telomere length is determined by a continuous protozoan structure. After a certain number of cell replications, telomeres gradually shorten and The cells then enter a terminal decay stage, which causes the cells to senesce. The enzyme is inactive in somatic cells but active in 90% of cancer cells, and is involved in telomerase inhibition. The agents may be useful in fighting cancer.

[0077] Furthermore, treatment of healthy human fibroblasts with luteolin significantly reduced the telomerase activity of the normal human cells. It was confirmed that the expression of enzymes increased and ATP production increased. Confirmation that telon can suppress aging by increasing telomerase activity in normal cells. Thus, the telomerase gene of the present invention is capable of enhancing telomerase expression and / or or for use in the treatment of a disease or condition susceptible to being affected by telomerase activity, and increasing telomerase activity in cells or tissues of a patient in need thereof. It is possible.

[0078] "Diseases or disorders associated with aging" refers to tumor formation and malignant development of cancer, myocardial infarction (intracranial paralysis), ), cerebellar infarction, stroke, Parkinson's disease, heart failure, atherosclerosis, hypertension, white Cataracts, age-related vision loss, sarcopenia, osteoarthritis, osteoporosis, bone marrow loss, multiple sclerosis, Sjogren's syndrome, rheumatoid arthritis, immunosuppression, diabetes, idiopathic pulmonary fibrosis, and and neurodegenerative diseases, Alzheimer's disease, Huntington's disease, and testosterone, estrogen Disorders resulting from decreased production of hormones, growth hormone, IGF-I, or energy means...

[0079] "Anti-aging effects" include increased mitochondrial biogenesis and function, decreased R At the OS level, extended life span of senescent cells and postmitotic cells such as neuronal cells, tumors Prevention of aging-related phenomena such as tumor formation, malignant progression of cancer, cerebellar infarction and myocardial infarction It means the present type.

[0080] Mitochondria, the powerhouses of the cell, are responsible for most of the respiration. It is the active oxygen consuming area where all cells carry out their metabolic activity. It provides the energy needed for all other activities (Boveris A et al. , Biochem.J.134:707-716, 2973). Reactive substances such as O2·- or OH· generated during the electron transport process Oxygen species (ROS) destroy mitochondrial structure and components, This induces changes in respiration rate and oxidative phosphorylation, which ultimately determines cellular metabolism. This affects the production and NADH / NAD+ ratio of many higher animals. , and is used for ATP synthesis in mitochondria, and energy metabolism that occurs in mitochondria Its action is said to be closely related to aging (Lee JW et al., J Ko rean Med Assoc.52(10):1007-19, 2009).

[0081] We confirmed that ATP production increased in normal cells by treatment with luteolin. The term "preventing a disease or condition associated with aging" means reducing the occurrence of the disease or condition. This suggests that age-related diseases may be slowed or reversed.

[0082] As used herein, the term "senescence" or "senescent cells" refers to telomere dysfunction, D Mitosis, which can be induced by NA damage or oncogene activation The cell cycle arrest in the cleaved cells is shown. In budding yeast, telomere dysfunction also leads to Senescent cells are arrested in the G2 / M phase of the cell cycle. In mammalian cells, senescence The cells are arrested in the outer non-dividing phase of the cell cycle, G0. Senescence in cells was observed under a microscope, with no increase in the number of β-galactosyltransferases for 10 days after passage. The term refers to cells that exhibit positive staining for galactosidase.

[0083] As used herein, the term "post-mitotic cells" refers to non-dividing cells outside the cell cycle. The cells are arrested in the G0 phase, which is the mitotic phase, but perform their main functions for the life of the remaining organism. After mitosis, cells are divided into neuronal cells, cardiac muscle cells, and muscle cells. cells, such as the parenchymal cells of the liver and kidney. Some cell types in certain organisms enter the G0 phase semi-permanently and only differentiate again under very special circumstances. These types of cells can be induced to begin splitting. At some point after somatic cell division they are considered cells.

[0084] The aging-related disease or symptom is a mitochondrial dysfunction, a telomere dysfunction, an aging Cell degeneration and age-dependent cell loss, or mitochondrial It is associated with doria degeneration or a state of cell cycle arrest in postmitotic cells.

[0085] In one embodiment, the telomeres interact with the telomerase enzyme to promote proliferation of the telomerase in tissues or cells of an individual. Stimulate and / or increase telomerase expression and / or activity in cells. In some cases, such activity may be reduced or absent, thereby contributing to the individual's disease or This leads to the increase or development of a disease or syndrome associated with the condition. , in particular by increasing telomerase expression and / or activity in healthy tissues. thereby extending the life span of the individual and maintaining the health of the individual, and / or By "treatment" is meant clinical treatment, including any embodiment encompassed by that term, and / or It may include diagnostic fields. EXAMPLES

[0086] The present invention will now be described in more detail with reference to the following examples. These examples are merely intended to illustrate the present invention in more detail. Those skilled in the art will appreciate that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. This is self-evident to a person of ordinary skill in the art.

[0087] Example 1: Isolation of Lutelinium (1) Luteinizing agent 2 x 100g of lacquer (Rhus verniciflua stokes), a medicinal plant Cut to 0-30 times the size, i.e., to fit the size of a 2-3 liter container and place in the container. The weight is 500-800g, which is 5-8 times the weight of the plant (preferably 6 times the weight of the plant, i.e. 600g). g) After pouring the distilled water into the container, boil it at 80℃ or less. Boil it for about 8 hours. However, the plant's luterium was allowed to solidify by bubbling with oxygen for 20-30 minutes every 3 hours. The vaporized water vapor generated during the extraction after bubbling was discharged into the flask. The collected water vapor was cooled and condensed to obtain a condensate, which was then irradiated with IR light (wavelength: The ruthenium oxide is then irradiated with ultraviolet light (approximately 3 to 1000 μm, preferably 200 to 600 μm) for 1 to 2 hours. This process is shown diagrammatically in Figure 37. .

[0088] (2) Separation by centrifugation and filtration The condensate collected in (1) was spun down by primary centrifugation at 190 g to remove impurities. Then, the mixture is centrifuged at 3000 g for a second time and allowed to stabilize for several minutes to several tens of minutes (preferably within two hours). After incubation, the supernatant was collected. The motility was confirmed by staining for CD332, and then the The presence or absence of luteolin was first confirmed. 000g and centrifuged a third time.

[0089] The lower layer is filtered through a filter of about 800 nm and collected to extract plasma and other internal plants or After removing the exosomes and other cell debris pellets, the cells were incubated at 140,000 The supernatant was collected after centrifugation at ≥0 g and pellet-free. The lower layer was collected by filtration through a 1.0 mm filter, and then stained again for CD332 to confirm motility. The mixture was filtered through a 500 nm filter, and the supernatant on top of the filter was collected. Lutelinus luteum was collected and stored at pH 1 or at temperatures below -90°C.

[0090] Through the above process, luteolin with a long diameter of 50 to 800 nm can be obtained. Observation and confirmation were possible through a field microscope or a confocal microscope. The size of the nuclei varies from 50 to 200 nm (development stage) to 200 to 400 nm (mature stage) to 400 The tissue was divided into ~600 nm (mitotic phase) and 600~800 nm (hypermitotic phase). In a similar manner, luteolin was extracted from the medicinal plants listed in Tables 1 to 4, namely, Angelica sinensis, Rhus bark, and Kiwifruit. was obtained.

[0091] (3) Isolation using surface antigen-specific antibodies 3-1) Identification of Luteinizing Surface Antigens Luteinizing leucine isolated from plants was treated with anti-CD39 antibody (sc-18766, Santa Clara, CA). ruz Biotechnology), anti-CD73 antibody (sc-25603, San ta Cruz Biotechnology) or anti-CD332 antibody (BS-06 75R, Bioss Inc.) and anti-FITC were added to observe whether color development occurred. (Blue fluorescence: CD133 / 1-VIOBRIGHT-FITC manufactured). Red fluorescence: PE (Phycoerythrin: Manufactured by Miltenyi Bitech GmbH) The cells were stained with Mitotracker, DAPI, and Hoec. After staining with hst, the presence or absence of color development was observed under a fluorescent microscope.

[0092] As a result, the lutelia were stained with Mitotracker and DAPI or Hoechst. On anti-CD39 antibody, anti-CD73 antibody, anti-CD133 antibody or anti-CD332 antibody By confirming that the antibody binds, CD39, CD73, CD133 or C It was confirmed that D332 is a surface antigen of Luteinium (Figures 29 to 32). ).

[0093] 3-2) Production of antibody-immobilized particles Step 1. Preparation of carbon-coated iron (Fe) magnetic nanoparticles Iron acetylacetonate hydrate (0.5 mmol) in 10 mL of octyl ether The metal precursor solution was prepared by stirring the mixture in an Erlenmeyer flask containing the metal oxide. The metal precursor solution was irradiated with ultrasonic waves at 20 kHz (50%) for 10 minutes using a fluorine atomizer. As ultrasonic waves are irradiated onto the solution, the initial orange color changes to dark brown over time. The above changes were observed because the magnetic nanoparticles of iron oxide (Fe2O3) were This means that the iron oxide magnetic nanoparticles were successfully prepared. The magnetic nanoparticles were precipitated by adding 100 ml of ethanol, and then centrifuged to separate the magnetic nanoparticles. The particles and the supernatant were separated, and the supernatant was removed. The washing process was repeated three times. After that, the magnetic nanoparticles were dried at a temperature of 50° C. for 12 hours to obtain oxidized nanoparticles with a size of 300 nm. The iron oxide magnetic nanoparticles were then heated at 600° C. for 3 hours in a sintered state. The carbon-coated iron-based magnetic nanoparticles were obtained by heat treatment under an Ar atmosphere.

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

[0095] Step 3: Preparation of antibody-immobilized particles Anti-CD39 antibody, anti-CD73 antibody, anti-CD133 antibody or anti-CD332 antibody was reacted with a thiol-reactive reagent to form a thiol. The iron magnetic nanoparticles having the functional group introduced therein are reacted with the antibody having the thiol group to produce the iron The antibody was attached to magnetic nanoparticles. The particle with the antibody immobilized is shown in the schematic diagram (Figure 1). (33 rear).

[0096] 3-3) Isolation of Luteolin from Plants or Foods 100-200ul plant extract and 5ul CD39 antibody-iron magnetic nanoparticles or CD7 3. Place the antibody-iron magnetic nanoparticles in a beaker and allow them to bind for 30 minutes, then place them in a magnetic separator for 1-2 min. After leaving it for 2 minutes, the Luterio-magnetic nanoparticles were collected, the supernatant was discarded, and the nanoparticles were washed. The iron magnetic nanoparticles were combined with 0.033wt% BSA (Bovine Serum). Add albumin) / PBS buffer solution and incubate at 25°C for 1 hour ( After incubation, the BSA-adsorbed iron nanoparticles were removed using a magnet. The BSA-adsorbed iron magnetic nanoparticles were then separated and a certain amount of PBS was added again to the iron magnetic nanoparticles to separate the ink. The adsorbed BSA was desorbed via incubation. P-640 spectrofluorometer (JASCO) Quantitative analysis was performed using a standard calibration curve method at 280 nm (emission slit: 0.5 nm). , absorption slit: 0.5 nm). The on-separation step is shown diagrammatically (Figure 34).

[0097] 3-4) Confirmation of isolated Luteolin (3) Lutelin isolated in the above 3-3) and (2) Lutelin isolated by filtration Anti-FITC antibodies that bind to CD39, CD73, CD133, or CD332 The presence or absence of color development was observed by binding the antibody (Figure 35 (a), (b), (c), and (d)). The extraction yields of the two separation methods were compared using a modified cell sorter.

[0098] As a result, when the nanofiltering method was used to separate the luteolin number was 1.5× 10 8 cells / ml, whereas CD39, CD73, CD133 or CD332 surface antigens When separated using particles that bind to 8 Pieces / ml We confirmed that the number of luteolins increased by more than four times compared to the conventional filtering method (Figure 3 6(a) and (b)).

[0099] [Table 5]

[0100] As shown in Table 5, when using the nano-filtering method, the size of 50 to 400 nm is The final extraction rate of the luteolin of this invention is 40-45%, and the loss rate is 50-60%. Antibody-iron nanoparticles that bind to CD39, CD73, CD133 or CD332 surface antigens When using the molecular separation method, the final extraction rate of luteolin with a size of 50 to 400 nm is 90 to 100%. It was confirmed that the extraction rate and efficiency were significantly improved with a loss rate of less than 10% and a high extraction rate of over 95%. I was able to do it.

[0101] From these results, the present invention is more effective than the conventional method for separating luteolin using a nanofilter. Separation using particles immobilized with antibodies or aptamers that bind to the lutein surface antigens of It was confirmed that the method increases the final extraction rate and reduces the loss rate during the separation process. Ta.

[0102] (4) Identifying the differences between plant mitochondria and luteolin As shown in Table 6, plant mitochondria are expressed by CD332, CD39, CD73, and CD133. Not only do they not express any of the surface antigens CD326 and CD326, When centrifuged at g or higher, mitochondria are torn apart, but luteolin still retains its shape. It was also confirmed that the pressure required to induce fission, for example, 35,000 psi or more, When α-aminobutyric acid was applied, mitochondria were torn apart, but luteolin remained intact. It was confirmed that.

[0103] [Table 6]

[0104] Example 2: Preservation of Lutelin Motile luteinizing factor receptors expressing CD39, CD73, CD133, or CD332 The number of luteolins containing surface antigens was confirmed by storing them. luteolins were collected by IR irradiation. Confirm that the antibody was added to the luteinizing agent by using anti-CD332, anti-CD39, anti-CD133 or anti-C D73 antibody was bound to the mitochondria, which were colored by binding anti-FITC. The number of stained Luteolins was counted using a tracker. The number of lutellium was counted by storing it at 0°C or below and -90°C. The results are shown in Tables 7 and 8 below, based on the H and temperature conditions (+ indicates the number of counted luteolins). (indicating the number).

[0105] [Table 7]

[0106] [Table 8]

[0107] According to Tables 7 and 8, when kept at pH 1-3 and -90-0℃ for 1-3 months, , it can be confirmed that conservation can be maintained without affecting the number of luteolin.

[0108] Example 3: Pressure-induced division of Luteinocytes The luteolin isolated in Example 1 was subjected to French press (French press) as shown in Table 9. Pressure was applied via the ess. The effect of temperature on the induction of Lutelinium division was also examined. (+ indicates degree of division).

[0109] [Table 9]

[0110] According to Table 9 and Figures 11 to 13, pH 1 to 3, 25,000 to 35,000 psi, Confirm that division is induced and proliferation of Lutein is achieved at temperatures between 10 and 20°C. It is possible.

[0111] Example 4: Characteristics of Lutelinium (1) Structure Among the Lutelinium obtained in Example 1, Lutelinium having a size of about 50 to 400 nm was Confocal Laser Scanning Microscope roscope (Zeiss), transmission electron microscope (Transmission Electron Microscope ( Scanning Electron Microscope, Scanning Electron Microscope Atomic Force Microscope The images were taken with a Leica TCS-SP8 confocal scanner and Similar to mitochondria, the mitochondrion is a membrane structure with a double or multiple membranes. It has a structure in which the liste structure has not been completed and is similar to mitochondria. It was confirmed that the same laser wavelength range was observed. The morphology was circular or elliptical. It was possible to observe that (Figure 6).

[0112] Furthermore, the results of photographing the lutelin isolated in Example 1 using a TEM electron microscope are shown in FIG. As shown, it was confirmed that the membrane had a bilayer or multilayer structure.

[0113] (2) Dyeing properties Among the Lutelinium obtained in Example 1, Lutelinium having a size of about 50 to 800 nm was Mito-tracker, Rhodamine 123 123), and the color development after staining with Janus green B. As a result, Luterion was also found to be sensitive to Mitotracker, Rhodamine 123, and Janus currant. It was confirmed that color development was observed with tan B (see Figures 2, 3 and 5).

[0114] (3) Autofluorescence Among the Lutelinium obtained in Example 1, Lutelinium having a size of about 50 to 800 nm was The light reaction was confirmed by fluorescent photography (see FIG. 4).

[0115] (4) Analysis of RNA content in Lutein The atomic microscope photograph of the 200-400 nm luteolin separated in Example 1 is shown in FIG. As shown, we hypothesize that luteolin contains nucleic acids such as RNA and DNA. can be done.

[0116] Total RNA and DNA were isolated from the 200-400 nm luteolin isolated in Example 1. To achieve this, we used the QIAGEN AllPrep DNA / RNA immuno kit: Cat 80284) and then purified using the Experion RNA (DNA) S Quantification was performed using a tdSens (Bio-Rad) chip.

[0117] The lutein was collected by centrifugation (8000 g, 1 hour 30 minutes) and then digested with the kit. Liquid solution RLT plus (Guanidine isothiocycanate, det Add 3.5 ul of beta-mercaptoethanol to 50 ul of eluents and measure for 20 The lutein was dissolved by passing the solution through a syringe with a gauge needle 5 to 10 times. The solution buffer was transferred to the AllPrep DNA spin column and then centrifuged (≥8000 g, 15 sec) and the DNA on the column and the buffer containing the RNA that passed through the column were They were separated by fur.

[0118] First, add 350ul of 70% ethanol to the same volume of the buffer that has passed through the column. After mixing thoroughly, 700 ul of the mixture was applied to an RNease MinElute spin column. The buffer that passed through the column was removed by centrifugation (≧8000 g, 15 seconds). Add 700ul of RW1, 500ul of RPE buffer and 80% ethanol. The column was washed stepwise using the centrifuge (≥800 0g, 15 seconds) were carried out under the same conditions. After the -free solution was placed in the column, it was centrifuged (≧8000 g, 60 seconds) to separate the lutein. RNA was isolated.

[0119] Genomic DNA was attached to the AllPrep DNA spin column in the first step. The DNA was washed with 500 ul AW1 and 500 ul AW2 buffers. All centrifugation speeds and times (≧8000 g, 15 sec) used were RN The separation was carried out under the same conditions as in the A separation process. 50 ul of EB buffer was added to the column, and then the chamber was After leaving it at room temperature for 2 to 5 minutes, centrifuge (≧8000g, 60 seconds) to separate the lutein DNA. Experion RNA (DNA) StdSens (Bio-Rad) chip As shown in Figures 10A and 10B, RNA was detected in the lutein. As shown in Figure 10C, it was confirmed that the granules not only contained α- and β-terminal fragments, but also contained DNA. In particular, the 200-400 nm luteolin contains RNA and DNA. I was able to confirm that it was.

[0120] Example 5: Cultivation of Luteolin (1) Among the lutellium obtained in Example 1, the size of the lutellium is about 50 to 200 nm. After adding PBS and irradiating with IR light, the cells were incubated at 18-30°C for about 3 hours. The size of the luteolin was checked under a microscope at approximately one-hour intervals starting immediately after irradiation. After incubation, the size of the luteolin, which was about 200 nm before incubation, grew to about 500 nm. It was confirmed that adding water to Lutelinone and irradiating it with IR light produced When cultured at 18-30°C, the size of the bacteria can grow to about 500 nm. It was found that...

[0121] (2) Lutelinium obtained in Example 1 having a size of about 400 to 800 nm PBS was added to the medium, and the medium was exposed to IR light and then incubated at 18-30°C for approximately 3 hours. The size and condition of the lutherions were observed under a microscope at approximately one-hour intervals starting immediately after irradiation. After ~6 hours, the luteolin, which had a size of about 400-800 nm before incubation, did not grow and divided. It was confirmed that this was a fission.

[0122] (3) Using a medium (DMEM or blood medium) containing 5% glucose, Lutelinus luteum was cultured at pH 7 and 20°C. Lutelinus luteum was observed to gather under IR irradiation. In addition, we used anti-CD332, anti-CD73, anti-CD133, and anti-CD39 antibodies against lutein. The antibody was combined with anti-FITC and color development was confirmed. The results are shown in Table 10 below. (+ indicates the relative ratio of the number of luteolins).

[0123] [Table 10]

[0124] Example 6: Confirmation of intracellular introduction Lutelinium was stained with red fluorescence for PKH26-1, PKH26-2, and PKH26-3. The cells were incubated with A549 arsenic lung cancer cells (ATCC♯: CCL-185) for 24 hours. The cells were fixed and their morphology was observed under a microscope. The results are shown in Figures 14 to 16. showed.

[0125] Referring to FIG. 14, PKH26-1, PKH26-2, and PKH26-3 were stained with red fluorescence. Confirm that the stained luteolin is introduced into the nucleus of cells stained with DAPI. The enlarged images in Figures 15 and 16 show that luteolin was introduced into the intracellular nucleus. It is possible to confirm more specifically that

[0126] Example 7: Confirmation of BBB (blood-brain barrier) penetration Mice were injected intraperitoneally (IP) and orally via gavage with PKH26-fluorescently stained luteolin. We administered the drug by the same method to confirm whether the administered Luteinizing agent crossed the BBB. According to the study, it is necessary to confirm that the drug crosses the BBB at least 3 hours after oral gavage. can.

[0127] In addition, Figure 18 shows that the drug crosses the BBB within 5 minutes of intraperitoneal injection. According to Figure 19, even 24 hours after intraperitoneal injection, the heart, lungs, spleen, liver, and pancreas were still healthy. It can be seen that it is distributed in the kidneys, testes, abdomen and viscera.

[0128] Example 8: Confirmation of anti-cancer (telomere / telomerase activity) effect of Lutelin (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 cell / ml onto a 60 mm plate, After about 12 hours, the culture was treated with the Lutelinium isolated in Example 1 at a concentration of 50 μg / ml. The cell culture was performed using antibiotic PSF (antibiotic-antimycotic). The incubation was carried out in a 37℃, 5% CO2 incubator with 1% FBS DM without FBS. Cells were harvested 48 hours after inoculation using EM medium and telomerase activity was measured. .

[0129] TRAP analysis (TRAPeze® Telomerase Detect The telomerase activity was analyzed using the Luteinion Kit (Millipore). The treatment increased the telomere counts of cancer cells (NCl-H1975, MDA-MA-468, WiDr). It was confirmed that the expression and activity of the enzyme were decreased (FIG. 20B).

[0130] In addition, the same experiment and analysis was performed on normal fibroblast cells, and the results were as follows: In human normal cells, telomerase expression and activity were decreased compared to the control group (non-treated group). It was confirmed that the concentration of β-glucose was increased (Fig. 20A).

[0131] (2) Inhibition of cancer cell proliferation by treatment with luteolin Various human cancer cell lines (lung cancer (NCI-H1975), colon cancer (WiDr), breast cancer (MD) A-MB-486), liver cancer (HCC38), leukemia (AGH-77) and normal cells To measure the cytotoxicity of Luteinizing agent against cancer cells in a fibroblast line, M A TT analysis was performed.

[0132] 5×10 4 cells / ml (8×10 3 The cell suspension (well / well) was plated in a 96-well plate. l) Inoculate 100 ul into each well of a flat-bottom microtiter plate and After 4 hours of incubation, the medium was replaced with one containing various concentrations of luteolin. After 48 hours of incubation, each well was filled with 10-fold diluted non-soluble yellow MTT solution. (3-(4,5-dimethylthiazole-2-yl)-2,5-diphe Add 100 ul of nyltetrazolium bromide solution. Incubate the mixture at 37°C and 5% carbon dioxide to allow formazan crystals to form. After about 4 hours, the excess medium was removed, and the water-insoluble β- 200 ul of DMSO was added to each well to dissolve the formazan, and then the The absorbance was analyzed at 595 nm using a plate reader.

[0133] The absorbance of the control group that was not treated with luteolin was set at 100%, and the absorbance of the cancer cells at each concentration was The results showed that it reduced the survival rate of cancer cells in a concentration-dependent manner. was not cytotoxic (Figure 21).

[0134] In addition to the above cancer cell lines, AsPC-1 pancreatic cancer cell line, A549 lung cancer cell line, BT We confirmed that various sources of luteolin have an inhibitory effect on cancer cell proliferation in the -20 breast cancer cell line. The rate measurement was performed in the same manner as in the MTT analysis method described above, and the results showed that the various types of leukocytes isolated by the method of Example 1 were Therion exhibited a cytostatic effect on cancer cells (FIGS. 22 to 24).

[0135] Therefore, the luteolin of the present invention can suppress the proliferation of cancer cells and prevent or treat cancer. I found out that it is possible.

[0136] Example 9: Increased telomerase activity in normal cells of Lutein Normal cells (Fibroblast) 1×10 6 Cells / ml in each 60mm plate After about 12 hours, the Lutelon isolated in Example 1 was inoculated at a concentration of 50 μg / ml. The cells were cultured in the presence of the antibiotic PSF (antibiotic antimycotic The cells were cultured at 37℃ in 5% CO2 using 1% FBS DMEM medium under conditions without otics. The cells were harvested 48 hours after inoculation and telomerase activity was measured.

[0137] TRAPeze® Telomerase Detection Kit The telomerase activity was analyzed by TRAP analysis using the Millipore Telomerase expression and activity in normal fibroblasts treated with lysine. It was confirmed that the amount of oxidized cellulose increased (Figure 25).

[0138] Example 10: Effect of Luteolin on ATP production ATP production in cancer cells and normal fibroblast cells treated with luteolin To confirm the increase, we used three cancer cell lines, A549, WTM266-4, and AsPC-1. and normal cells (MRC-5) 1×10 6 Cells / ml in a 60mm plate of standard DMEM medium After inoculation and incubation for one day, the antibiotic PSF (antibiotic-antimycotic factor) was administered. In the absence of nutrient starvation (serum starvation), 0.1% FBS DMEM medium was used. The cells were then cultured again for one day under conditions of 0.5 M starvation. After treatment with 50 μg / ml of ATP, the cells were incubated for 0, 10, 30, and 120 minutes. The ATP production was measured (Figure 26). The ATP measurement kit was purchased from Abcam (Cambridge, MA). The product was purchased from Co., Ltd. (USA) and used.

[0139] As a result, it was confirmed that ATP production was significantly increased in normal cells treated with Lutein. In addition, it was confirmed that ATP production was decreased in cancer cells treated with luteolin. (Figure 38).

[0140] Therefore, the Lutelinone of the present invention increases the energy production of normal cells and suppresses aging. It was also found that it can reduce the energy production of cancer cells, making it suitable for cancer treatment. This allows the use of cancer cell-specific anticancer drugs without affecting normal cells. Treatment may be considered. [Industrial Applicability]

[0141] According to the present invention, it is possible to effectively separate luteolin, which is a microscopic substance present in plants or foods. The isolated luteolin can then be cultured to grow to a certain size. This can be utilized in the development of various applications for the prevention and treatment of diseases.

[0142] Although the specific parts of the present invention have been described in detail above, those skilled in the art will be able to easily understand the present invention. For the purposes of the present invention, such specific descriptions are merely preferred embodiments. It is clear that the scope of the present invention is not limited. It can be said that the term "common practice" is defined by the claims and their equivalents.

Claims

1. A luteinium with one or more of the following characteristics selected from the following: (a) 50-800 nm, original to elliptical, motile; (b) containing nucleic acid; (c) When stained with immunofluorescence, it shows a reaction similar to that of mitochondria; (d) exhibiting fusion and / or fission ecology; (e) In the absence of fusion, they mature to a size of 500 nm and become DNA-containing mitochondria. They mature into structures similar to mitochondria in SEM or TEM electron micrographs. Showing; (f) exhibits a different light response than exosomes; (g) Fission occurs when exposed to IR or when pressure is applied; (h) expressing CD332, CD133, CD73 or CD39 as a surface antigen; (i) exhibits autofluorescence; (j) 200-400 nm in size and produces ATP; (k) bimembrane or multimembrane structures; (l) adhesive; (m) suppression of telomerase activity in cancer cells; (n) promoting telomerase activation in normal cells; (o) Possesses cell penetrating ability; and (p) Ability to penetrate the blood-brain barrier (BBB).

2. Rhodamine 123, Mitotracker ker), Acridine Orange, DAPI, and Janus green B. The present invention relates to a method for producing a dye composition comprising the steps of: Lutetium luteum.

3. A method for isolating Lutelin, comprising the steps of: (a) 0.8% by weight of the condensate obtained by cooling the steam or gas agitation extract of a plant or food product; filtering using a filter having pores of 1.2 μm; (b) centrifuging the filtered condensate; and (c) separating the luteolin from the centrifuged supernatant.

4. 4. The method according to claim 3, wherein the plant is a medicinal plant selected from Tables 1 to 4. Separation method.

5. The plant is selected from the group consisting of angelica, lacquer bark and kiwi. The method of claim 3.

6. The shaking extraction in step (a) is carried out by adding a solvent to a plant or food and subjecting the plant or food to a gas atmosphere at 50 to 90° C. The method is characterized in that the method proceeds by intermittently bubbling and shaking the mixture. Item 3. The method for separating the nucleophile according to item 3.

7. The shaking extraction was carried out by shaking for 8 to 10 hours and bubbling for 20 to 30 minutes every 2 to 3 hours.

7. The method according to claim 6, characterized in that the lutein of the plant or food is prevented from clumping. The separation method described above.

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

9. The centrifugation is preferably performed repeatedly at 1200 to 5000 rpm for 5 to 10 minutes. The method for separating according to claim 3.

10. The step (c) is to irradiate the centrifuged supernatant with IR light having a wavelength of 200 to 600 um. Separating the Luteinizing leukocyte particles that are motile and aggregate when exposed to radiation, or isolating the Luteinizing leukocyte surface antigens The presence or absence of binding to a binding substance that recognizes CD39, CD73, CD133, or CD332 is confirmed.

4. The method for separating luteolin particles according to claim 3, further comprising the steps of: recognizing the luteolin particles;

11. The separated luteolin particles were filtered through a 50 nm filter, and the 4. The method of claim 3, further comprising the step of collecting the isolated portions.

12. 200 nm, 400 nm, 600 nm, 800 nm and 1000 nm filters By using them in sequence, 50-200 nm, 200-400 nm, 400-600 nm, Classification of luteolin into two sizes: 600-800 nm and 800-1000 nm. The method of claim 11 further comprising:

13. The method according to claim 1, further comprising the step of fixing the mixture at a pH of 7 or less and at a temperature of 0° C. or less. Item 3. The method for separating the nucleophile according to item 3.

14. The method of claim 1, further comprising the step of fixing at pH 1 to 5 and −90° C. to 0° C. Item 3. The method for separating the nucleophile according to item 3.

15. 25,000~35,000psi (pound per square inch) 4. The method according to claim 3, further comprising the step of inducing division by applying the pressure. Separation method.

16. The method further comprises the step of inducing proliferation at pH 1 to 3 and 10 to 20° C. The method for separating according to claim 3.

17. A method for isolating Lutelin, comprising the steps of: (a) administering to an extract containing lutein an antibody that specifically binds to a lutein surface antigen, or adding particles having aptamers immobilized thereon to induce binding between the luteinizing enzymes and the particles; and (b) recovering the luteolin bound to the particles.

18. (c) further comprising a step of separating only the lutelins from the lutelins bound to the particles. The method for isolating lutein as described in claim 17, characterized by:

19. The extract containing the luteolin is a plant extract or a food extract. The method for isolating lutein as described in claim 17.

20. The extract containing the luteolin is (i) a hot water extract of a plant or food; (iii) by heating the plant or food in the presence of a solvent The method for separating luteolin according to claim 17, characterized in that the condensate is a gas generated by the separation of luteolin. Law.

21. 21. The method of claim 20, wherein the condensate is produced by the following steps: Law: (a) Add a solvent to a plant or food, and bubble the mixture intermittently with gas at 50-90°C. shaking while mixing; and (b) collecting the water vapor or gas vaporized by the shaking, and then cooling the collected liquid to obtain a condensate; The acquisition stage.

22. The luteinizing surface antigen is CD39, CD73, CD133 or CD332. The method for isolating luteinium as described in claim 17.

23. The particles include magnetic particles, silica particles, quantum dot particles, glass particles, polymer particles, and fibers.

18. The method of claim 17, characterized in that the particle is selected from the group consisting of particles and fluorescent particles. How to isolate Lutetium.

24. The magnetic particles may be of iron, aluminum, cobalt, nickel, manganese, tin, zinc, cadmium, or zinc. It is composed of oxides of tungsten, magnesium, copper, barium, lithium or yttrium. The method for isolating luteinium described in claim 23, characterized in that it is selected from the group consisting of:

25. The magnetic particles are double coated with carbon and functional groups for antibody or aptamer binding. The method for isolating luteinium as described in claim 23, characterized in that it is subjected to filtration.

26. The functional group may be an amide group, an ester group, or a combination of both. oup), amine group and carboxyl group 26. The method according to claim 25, characterized in that the compound is selected from the group consisting of How to separate therion.

27. 18. The method according to claim 17, wherein the particle size is 10 to 1,000 nm. Method for isolating Lutetium.

28. The particles are magnetic nanoparticles, and the step (b) is a step of applying a magnetic force from the outside to the ruthenium.

18. The method according to claim 17, further comprising collecting and recovering the magnetic nanoparticles to which the magnetic nanoparticles are bound. How to isolate Lutetium.

29. The particles are fluorescent particles, and step (b) is a step of sorting the particles using a fluorescence-based cell sorter. The method according to claim 17, further comprising recovering fluorescent particles to which the luteinizing agent is bound. How to separate the

30. The particles are electrostatically charged particles, and the step (b) comprises forming a non-uniform electric field to The method according to claim 17, characterized in that the particles to which the luteolin is bound are collected by electrostatic attraction. The method for isolating Luteinium is described above.

31. The particles are ionic particles, and step (b) is a step of electrostatically attracting the particles to the electrolyte. The method for separating luteolin according to claim 17, characterized in that the particles to which the luteolin is bound are recovered. method.

32. The Lutelinium according to claim 1 is added with water and heated for 18 to 30 minutes under IR light irradiation or pressure. A method for culturing Lutetium, comprising the step of growing the Lutetium at 37°C.

33. The luteinium of claim 1 is grown in a sugar-containing medium at pH 5 to 9 and 18 to 30° C. A method for culturing Lutetium, comprising the step of propagating the same.

34. The sugars include rhamnose, glucose, galactose, and glycerol. Galactose, fructose or xylose ( The culture method according to claim 33, characterized in that the lysate is xylose.

35. The size of the luteolin before and after the culture was 50-200 nm and 300 nm, respectively. The culture method according to claim 32 or 33, characterized in that the particle size is 500 nm or less.

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