Cellular vesicle staining agent and Anti-inflammatory agent

Abstract

The purpose of the present invention is to provide a novel naturally derived cellular vesicle staining agent that can easily fluorescently stain cellular vesicles and that is safe even when introduced into the body, and to provide a cellular vesicle staining agent having anti-inflammatory activity. The purpose of the present invention is also to provide a method for producing a cellular vesicle staining agent, whereby the cellular vesicle staining agent can be easily produced. Another purpose of the present invention is to provide a method for detecting cellular vesicles using the cellular vesicle staining agent. The present invention provides: a cellular vesicle staining agent comprising chlorophyll or a chlorophyll derivative; an anti-inflammatory agent comprising chlorophyll or a chlorophyll derivative as an active ingredient; a method for producing a cellular vesicle staining agent, the method comprising a step for extracting a cellular vesicle staining agent from a photosynthetic organism; and a method for detecting a cellular vesicle in a sample or in a body using the cellular vesicle staining agent.

Description

Cellular vesicle staining agents and anti-inflammatory agents

[0001] This invention relates to a cellular vesicle staining agent and an anti-inflammatory agent.

[0002] Cellular vesicles include extracellular vesicles (EVs), which are currently attracting attention. EVs are noteworthy for their hormonal effects on target cells. In particular, cancer cells are known to secrete large amounts of exosomes, inducing angiogenesis and immunosuppression, thereby promoting cancer growth and metastasis. Conversely, the ability of exosomes to embed substances within them has been proposed for use in drug delivery systems that specifically deliver highly side-effective anticancer drugs to cancer cells. Furthermore, it has been suggested that exosomes may enhance the signaling pathway for the removal of β-amyloid, a causative agent of dementia. In addition, viral release and infection are said to have many similarities to exosome metabolism. For these reasons, methods for quantifying exosomes are being developed.

[0003] Patent Document 1 discloses a fluorescent staining agent for staining cellular vesicles and a method for measuring cellular vesicles using a plate reader or the like. Patent Document 2 discloses a fluorescent staining agent for staining cellular vesicles and a method for measuring cellular vesicles using a high-performance liquid chromatography gel filtration system or the like.

[0004] International Publication No. 2022-163446, International Publication No. 2024-053316

[0005] Patent documents 1 and 2 disclose a system for easily separating and detecting cellular vesicles and impurities using a fluorescent dye. However, because the fluorescent staining agent, which is an organically synthesized, non-natural compound, is used to detect cellular vesicles, there were concerns about safety when introducing it into the body, even if it could be used for testing.

[0006] The present invention has been made in view of the above problems, and provides a novel natural-derived cellular vesicle stain that can easily fluorescently stain cellular vesicles and is safe even when introduced into the body, and also provides a cellular vesicle stain having anti-inflammatory activity. Another object is to provide a method for producing a cellular vesicle stain that can easily produce the cellular vesicle stain. Furthermore, an object is to provide a method for detecting cellular vesicles using the cellular vesicle stain.

[0007] That is, the present invention includes the following inventions. [Invention 1] A stain for fluorescently staining cellular vesicles, wherein the stain is chlorophyll and / or a chlorophyll derivative. A cellular vesicle stain characterized by the above. [Invention 2] The cellular vesicle stain according to Invention 1, wherein the chlorophyll and chlorophyll derivatives are represented by the following general formula (1). (In formula (1), R1 represents CH ３ , CHCH ２ , CH ２ CH ３ , CHO, CH ２ OH, COOCH ３ . R2 represents CHCH ２ , CH ３ , CHO, CH ２ OH, COOCH ３ . n represents H / H (uncoordinated), Mg, Zn, Fe, Cu, Ni, Co.) [Invention 3] The cellular vesicle stain according to Invention 2, wherein the chlorophyll and chlorophyll derivatives are represented by the following general formula (2). (In formula (2), R1 represents CH ３ , CHO, CH ２ OH. R2 represents CHCH ２ . n represents H / H (uncoordinated), Mg, Zn, Fe.) [Invention 4] The cellular vesicle stain according to Invention 2, wherein the chlorophyll and chlorophyll derivatives are represented by the following general formula (3). (In formula (3), R1 represents CH ３ , CHCH ２ , CH ２ CH ３ , CHO, CH ２OH, COOCH ３ This represents R2, CHCH ２ ,CH ３ , CHO, CH ２ OH, COOCH ３ (This represents...) [Invention 5] The cellular vesicle staining agent according to Invention 4, characterized in that the chlorophyll and chlorophyll derivative are represented by the following general formula (4). (In equation (4), R1 is CH ３ , CHO, CH ２ It represents OH. R2 is CHCH ２ (This represents...) [Invention 6] The cellular vesicle staining agent according to Invention 4, characterized in that the chlorophyll and chlorophyll derivative are represented by the following general formula (5). (In equation (5), R1 is CH ２ It represents OH. R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH, COOCH ３ (This represents.) [Invention 7] The cellular vesicle staining agent according to Invention 6, characterized in that the chlorophyll and chlorophyll derivative are 7-hydroxymethylpheophytin represented by the following general formula (6). (In equation (6), R1 is CH ２ It represents OH. R2 is CHCH ２[Invention 8] A cellular vesicle staining agent according to any one of Inventions 1 to 7, characterized in that the cellular vesicle staining agent has an anti-inflammatory effect. [Invention 9] A topical skin preparation characterized by containing the anti-inflammatory agent according to Invention 8. [Invention 10] A method for producing a cellular vesicle staining agent according to any one of Inventions 1 to 7, comprising an extraction step of extracting the cellular vesicle staining agent from photosynthetic organisms. [Invention 11] A method for producing a cellular vesicle staining agent according to any one of Inventions 1 to 7, comprising an extraction step of extracting the cellular vesicle staining agent from algae. [Invention 12] A method for producing a cellular vesicle staining agent according to Invention 10, further comprising a heat treatment step. [Invention 13] A method for producing a cellular vesicle staining agent according to Invention 11, further comprising a heat treatment step. [Invention 14] A method for producing a cellular vesicle staining agent according to Invention 10, further comprising an acid treatment step. [Invention 15] A method for producing a cellular vesicle stain according to Invention 11, further comprising an acid treatment step. [Invention 16] A method for detecting cellular vesicles, comprising: a staining step of selecting one of the cellular vesicle stains according to Inventions 1 to 7 and bringing it into contact with a sample to stain the cellular vesicles in the sample; and a detection step of detecting the stained cellular vesicles in the sample. [Invention 17] A method for detecting cellular vesicles, comprising: a staining step of selecting one of the cellular vesicle stains according to Inventions 1 to 7 and introducing it into the body to stain the cellular vesicles in the body; and a detection step of detecting the stained cellular vesicles in a sample obtained from the body.

[0008] Furthermore, the present invention includes the following inventions: [Invention 18] A cellular vesicle staining agent containing chlorophyll or a chlorophyll derivative. [Invention 19] The cellular vesicle staining agent according to Invention 18, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (1). (In equation (1), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH or COOCH ３ And R2 is CHCH ２,CH ３ , CHO, CH ２ OH or COOCH ３ The chlorophyll or chlorophyll derivative is a compound represented by the following general formula (2), wherein n is H / H (uncoordinated), Mg, Zn, Fe, Cu, Ni, or Co.) [Invention 20] The cellular vesicle staining agent according to Invention 19, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (2). (In equation (2), R1 is CH ３ , CHO or CH ２ OH is OH, and R2 is CHCH ２ The formula is such that n is H / H (uncoordinated), Mg, Zn, or Fe.) [Invention 21] The cellular vesicle staining agent according to Invention 19, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (3). (In equation (3), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH or COOCH ３ And R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３ (Invention 22) The cellular vesicle staining agent according to Invention 21, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (4). (In equation (4), R1 is CH ３ , CHO or CH ２ OH is OH, and R2 is CHCH ２ (Invention 23) The cellular vesicle staining agent according to Invention 21, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (5). (In equation (5), R1 is CH ２ OH is OH, and R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３[Invention 24] The cellular vesicle staining agent according to Invention 18, wherein the chlorophyll or chlorophyll derivative is chlorophyll a, chlorophyll b, or 7-hydroxymethylpheophytin. [Invention 25] The cellular vesicle staining agent according to Invention 24, wherein the chlorophyll or chlorophyll derivative is 7-hydroxymethylpheophytin. [Invention 26] An anti-inflammatory agent containing chlorophyll or a chlorophyll derivative represented by the following general formula (1) as an active ingredient. (In equation (1), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH or COOCH ３ And R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３ The formula is H / H (uncoordinated), Mg, Zn, Fe, Cu, Ni, or Co.) [Invention 27] The anti-inflammatory agent according to Invention 26, wherein the chlorophyll or chlorophyll derivative is 7-hydroxymethylpheophytin.

[0009] In another embodiment, the present invention includes a therapeutic or prophylactic agent for inflammatory diseases that contains chlorophyll or a chlorophyll derivative represented by the general formula (1) as an active ingredient.

[0010] In yet another embodiment, the present invention includes a pharmaceutical composition for treating or preventing inflammatory diseases, comprising chlorophyll or a chlorophyll derivative represented by the general formula (1) and a pharmacokinetically acceptable carrier.

[0011] In yet another embodiment, the present invention includes chlorophyll or a chlorophyll derivative represented by the general formula (1) for use in the treatment or prevention of inflammatory diseases.

[0012] In yet another embodiment, the present invention includes the use of chlorophyll or a chlorophyll derivative represented by the general formula (1) for the treatment or prevention of inflammatory diseases.

[0013] In yet another aspect, the present invention includes the use of chlorophyll or a chlorophyll derivative represented by the general formula (1) in the manufacture of a pharmaceutical product for treating or preventing an inflammatory disease.

[0014] In yet another aspect, the present invention includes a method for treating or preventing an inflammatory disease, comprising administering a therapeutically effective amount of chlorophyll or a chlorophyll derivative represented by the general formula (1) to a subject in need thereof.

[0015] This application claims priority to Japanese Patent Application No. 2025-026511, filed on 21 February 2025, and encompasses the contents described in the specification of said patent application.

[0016] The cellular vesicle staining agent according to the present invention is chlorophyll and chlorophyll derivatives contained in photosynthetic organisms, and can be efficiently obtained from photosynthetic organisms. Since the chlorophyll and chlorophyll derivatives also have anti-inflammatory properties, they can be used as low-cost and highly safe staining agents for cellular vesicles, and can also be used in food and beverage compositions or pharmaceutical compositions that utilize their anti-inflammatory properties.

[0017] The method for producing cellular vesicle staining agents according to the present invention allows for extraction from photosynthetic organisms, and the yield can be increased simply by heat treatment or acid treatment, thus enabling the easy and low-cost production of cellular vesicle staining agents.

[0018] The method for detecting cellular vesicles according to the present invention allows for the staining of cellular vesicles with simple operations.

[0019] Figure 1A is a graph showing the results of analyzing Euglena-derived cellular vesicles (heat-treated at 120°C) with and without fluorescence staining using a high-performance liquid chromatography gel filtration system (solid line: unstained, dashed line: GIF-2276 stained). Figure 1B is a graph showing the results of analyzing the difference in fluorescence intensity of cellular vesicles due to different heat-treatment conditions using a high-performance liquid chromatography gel filtration system (solid line: 120°C, dotted line: 90°C, dashed line: 60°C, dashed line: room temperature). Figure 2 is a photograph showing the difference in autofluorescence intensity of cellular vesicles due to different heat-treatment conditions. Figure 3 is the result of identifying the cellular vesicle staining agent of the present invention using an HPLC-MS system. Figure 4 is a graph showing the staining results of milk-derived cellular vesicles with chlorophyll derivatives (Euglena probe fraction), chlorophyll a, and chlorophyll b (solid line: Euglena probe fraction, dotted line: chlorophyll a, dashed line: chlorophyll b). Figure 5 is a graph showing the staining results of milk-derived cellular vesicles with acid-treated chlorophyll b (dashed line: control, solid line: acid treatment, dashed line: zinc acetate treatment, dotted line: zinc acetate treatment after acid treatment). Figure 6A is a graph evaluating the anti-inflammatory activity of chlorophyll a and chlorophyll derivatives (Euglena probe fraction) by the decrease in nitric oxide (NO) levels, and Figure 6B is a graph evaluating the anti-inflammatory activity of chlorophyll a and chlorophyll derivatives (Euglena probe fraction) by the decrease in IL-6 levels. Figure 7 is a graph showing that chlorophyll b efficiently labels cellular vesicles in vivo (solid line: 5 minutes, dashed line: 10 minutes, dotted line: 30 minutes, faint solid line: 60 minutes).

[0020] In order to solve the above problems, the present inventors conducted extensive research and, as a result, discovered that chlorophyll and chlorophyll derivatives contained in photosynthetic organisms such as plants, algae, and photosynthetic microorganisms efficiently stain cellular vesicles and their analogous vesicles, thus completing the present invention. The cellular vesicles described herein include not only intracellular vesicles and extracellular vesicles (EVs) naturally secreted from cells, but also analogous vesicles composed of lipid and protein complexes produced by artificial treatments such as heat treatment.

[0021] (Embodiment of the Cellular Vesicle Staining Agent) The cellular vesicle staining agent according to this embodiment will be described below. The cellular vesicle staining agent according to this embodiment is chlorophyll and / or a chlorophyll derivative, and the compound efficiently fluorescently stains cellular vesicles.

[0022] Chlorophyll and / or chlorophyll derivatives can generally be extracted from photosynthetic organisms. Photosynthetic organisms are organisms that perform photosynthesis, and include, for example, plants, algae, and photosynthetic bacteria. Algae are a general term for organisms that mainly live on land, excluding mosses, ferns, and seed plants, and include cyanobacteria, which are true bacteria, as well as eukaryotes that are single-celled (diatoms, yellow-green algae, dinoflagellates, etc.) and multicellular organisms such as seaweed (red algae, brown algae, green algae, etc.).

[0023] The chlorophyll in this embodiment includes not only the chlorophyll found in plants and algae that perform oxygen-evolving photosynthesis, but also bacteriochlorophyll found in photosynthetic bacteria that perform oxygen-non-evolving photosynthesis. The tetrapyrrole ring in the structure of chlorophyll may be porphyrin, chlorin, or bacteriochlorin, all of which are in different unsaturated states of the pyrrole ring.

[0024] Chlorophyll derivatives according to this embodiment include, for example, pheophytin, which is a substance obtained by removing the coordinating metal from chlorophyll; chlorophyllide, which is obtained by removing the side chain phytol from chlorophyll; pheophorbide, which is obtained by removing both the coordinating metal and phytol from chlorophyll; and chlorophyll in which another metal is coordinated instead of magnesium.

[0025] The compound according to this embodiment is characterized by being a compound represented by the following formula (1).

[0026]

[0027] The above compound may be at least one selected from chlorophyll, pheophytin, chlorophyllide, and pheophorbide.

[0028] The metal coordinated to the above compound may be any metal that can coordinate. For example, it is at least one selected from magnesium, zinc, iron, copper, nickel, cobalt, tin, and manganese, and preferably magnesium, zinc, iron, copper, nickel, cobalt.

[0029] In the above formula (1), R1 is CH ３ , CHCH ２ , CH ２ CH ３ , CHO, CH ２ OH, or COOCH ３ , and R2 is CHCH ２ , CH ３ , CHO, CH ２ OH, or COOCH ３ . And n is H / H (non-coordinated), Mg, Zn, Fe, Cu, Ni, or Co. That is, R1 may be CH ３ , may be CHCH ２ , may be CH ２ CH ３ , may be CHO may be CH ２ OH, or may be COOCH ３ . Also, R2 may be CHCH ２ , may be CH ３ , may be CHO, may be CH ２ OH, or may be COOCH ３ . Also, n may be H without metal coordination, may be magnesium, may be zinc, may be iron, may be copper, may be nickel, or may be cobalt.

[0030] The compound according to this embodiment is characterized by being a compound represented by the following formula (2).

[0031] [[ID=5!]]

[0032] In the above formula (2), R1 is CH ３ , CHO, or CH ２ OH, and R2 is CHCH ２And n is H / H (uncoordinated), Mg, Zn, or Fe. That is, R1 is CH ３ It may be CHO, and it may be CH ２ OH may also be used. Also, R2 is CHCH ２ Furthermore, n may be H without metallic coordination, magnesium, zinc, or iron.

[0033] The compound according to this embodiment is characterized by being a compound represented by the following formula (3).

[0034]

[0035] In the above formula (3), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH, or COOCH ３ And R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH, or COOCH ３ Therefore, R1 is CH ３ It may also be CHCH ２ It may also be CH ２ CH ３ It may be CHO, and it may be CH ２ It may also be OH, COOCH ３ It may also be CHCH. ２ It may also be CH ３ It may be CHO, and it may be CH ２ It may also be OH, COOCH ３ That's fine.

[0036] The compound according to this embodiment is characterized by being a compound represented by the following formula (4).

[0037]

[0038] In the above formula (4), R1 is CH ３ , CHO, or CH ２ It is OH. R2 is CHCH.２ Therefore, R1 is CH ３ It may be CHO, and it may be CH ２ OH may also be used. Also, R2 is CHCH ２ That is the case.

[0039] The compound according to this embodiment is characterized by being a compound represented by the following formula (5).

[0040]

[0041] In the above formula (5), R1 is CH ２ It is OH. R2 is CHCH. ２ ,CH ３ , CHO, CH ２ OH, or COOCH ３ Therefore, R1 is CH ２ It is OH. Also, R2 is CHCH. ２ It may also be CH ３ It may be CHO, and it may be CH ２ It may also be OH, COOCH ３ That's fine.

[0042] The compound according to this embodiment is characterized by being a compound represented by the following formula (6).

[0043]

[0044] In the above formula (6), R1 is CH ２ It is OH. R2 is CHCH. ２ That is, the compound in question is 7-hydroxymethylpheophytin.

[0045] [Anti-inflammatory agent] The compound of this embodiment has anti-inflammatory properties and can therefore be suitably used as an anti-inflammatory agent. It is becoming increasingly clear that chronic inflammation causes damage and dysfunction to biological tissues and is a cause of inflammatory diseases. The compound of this embodiment can suppress chronic inflammation and can be used not only for pharmaceutical purposes but also for non-pharmaceutical purposes. It can be suitably used in food and beverages aimed at patients with inflammatory diseases and healthy individuals who are conscious of preventing inflammatory diseases.

[0046] Inflammatory diseases are not particularly limited to diseases that involve a local reaction to damage to living tissues, and include systemic inflammatory diseases and infectious diseases, as well as localized inflammatory diseases and infectious diseases. Examples include arteriosclerosis, diabetes, obesity, Alzheimer's disease, autoimmune diseases (multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, pernicious anemia, etc.), allergic diseases (hay fever, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic gastroenteritis, food allergies, drug allergies, atopic dermatitis, urticaria, hemolytic anemia, thrombocytopenic purpura, acute glomerulonephritis, collagen disease, serum sickness, viral hepatitis, etc.), inflammatory bowel disease (ulcerative colitis, Crohn's disease, etc.), cancer (colon cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, etc.), infectious diseases (HIV, influenza virus, norovirus, rotavirus infections, etc.), and inflammatory skin diseases (psoriasis, eczema, epidermolysis bullosa, alopecia areata, acne, pruritus, etc.).

[0047] [Skin Protectant] The skin protectant contains the anti-inflammatory agent of the present invention as an active ingredient and is provided in combination with a pharmaceutically acceptable base material or carrier. The skin protectant may also contain, to the extent that it is pharmaceutically acceptable, additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure adjusters, pH adjusters, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, humectants, colorants, fragrances, thickeners, bactericides, cell activators, and anti-inflammatory agents as appropriate.

[0048] The proportion of anti-inflammatory agents in skin protectants is set appropriately according to their effective amount and the form of the skin protectant, but for example, it is 0.000001 to 1% by mass, preferably 0.000001 to 0.01% by mass, and more preferably 0.000003 to 0.01% by mass, relative to the total amount of the skin protectant.

[0049] [Cosmetics] Cosmetics are provided with the anti-inflammatory agent of the present invention as an active ingredient, in combination with pharmaceutically acceptable base materials and carriers. To the extent that is pharmaceutically acceptable, the cosmetics may also contain as appropriate additives such as excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure adjusters, pH adjusters, emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, humectants, colorants, fragrances, thickeners, bactericides, and cell activators.

[0050] Specifically, cosmetics include skincare cosmetics such as lotions, creams, cleansers, masks, oils, massage creams, serums, cleansers, deodorants, hand creams, and lip balms; makeup cosmetics such as makeup bases, face powders, liquid foundations, oil-based foundations, blushes, eyeshadows, mascaras, eyeliners, eyebrow products, and lipsticks; antiperspirants, sunscreen lotions and creams for UV protection, and topical skin preparations, and also include those used as quasi-drugs.

[0051] The proportion of anti-inflammatory agents in cosmetics is set appropriately according to their effective amount and the form of the cosmetic, but for example, it is 0.000001 to 1% by mass, preferably 0.000001 to 0.01% by mass, and more preferably 0.000003 to 0.01% by mass, relative to the total amount of the cosmetic.

[0052] [Food and Beverages] Food and beverages can be formulated with the anti-inflammatory agent of the present invention as an active ingredient. These foods and beverages can also be used for skin protection and wrinkle improvement. Furthermore, these foods and beverages can be provided as functional beverages or functional foods. Examples of functional foods include foods for specified health uses, foods with nutritional function claims, foods with functional claims, foods for the elderly, and health supplements (balanced nutrition foods, supplements).

[0053] Examples of food products include grains, potatoes, fish and shellfish, meat, eggs, oils and fats, dairy products, vegetables, beans, fruits, sugars, seaweed, confectionery, seasonings, and processed foods.

[0054] Processed foods are not particularly limited, but examples include: processed seafood products such as chikuwa and kamaboko; processed meat products such as ham and sausage; confectionery such as cookies, biscuits, snacks, chocolate and cakes; noodles such as soba, udon, fresh noodles, Chinese noodles and pasta; bread such as sliced ​​bread and sweet buns; fermented processed foods such as natto and miso; soy products such as tofu and okara; pickles such as asazuke and nukazuke; canned seafood, processed meat, vegetables and fruits; dairy products such as butter, margarine, yogurt, cheese and milk; and frozen desserts such as ice cream and sherbet.

[0055] Beverages are not particularly limited, but examples include roasted and dried tea leaves as a substitute for tea leaves, fruit juices, vegetable juices, flavored beverages, and fruit drinks for dilution; carbonated beverages; coffee, coffee beverages, coffee-flavored soft drinks, cocoa beverages, black tea, green tea, matcha, oolong tea, barley tea, hojicha, and other beverages; vinegar drinks; sports drinks and other soft drinks; milk; dairy beverages; dairy beverages; lactic acid beverages; lactic acid bacteria beverages; soy milk and processed soy milk; alcoholic beverages such as beer, sake, shochu, liqueurs, and wine; non-alcoholic beverages; and nutritional beverages containing taurine, royal jelly, amino acids, vitamins, minerals, iron, etc.

[0056] [Pharmaceuticals and Quasi-drugs] Pharmaceuticals and quasi-drugs may be formulated with the cellular vesicle staining agent and / or anti-inflammatory agent of the present invention as active ingredients. Pharmaceuticals and quasi-drugs may be formulated into appropriate forms and administered to humans or animals in any dosage form. The dosage form is not particularly limited, but examples include oral, transdermal, enteral, transmucosal, and injection.

[0057] Pharmaceuticals and quasi-drugs can be formulated with the cellular vesicle staining agent and / or anti-inflammatory agent of the present invention and administered to patients. Pharmaceuticals and quasi-drugs can be formulated into appropriate forms and administered to humans or animals in any dosage form. The dosage form is not particularly limited, but examples include oral, transdermal, enteral, transmucosal, and injection.

[0058] Dosage forms administered orally include, for example, pills, powders, tablets, granules, capsules, syrups, and liquids.

[0059] When administered parenterally, examples include intravenous injection, intramuscular injection, transdermal absorption, inhalation, suppositories, eye drops, nasal sprays, etc.

[0060] These various formulations can be manufactured by conventional methods using pharmaceutically acceptable carriers such as commonly used excipients, disintegrants, binders, lubricants, colorants, and diluents.

[0061] Examples of excipients include lactose, corn starch, glucose, sorbitol, calcium carbonate, kaolin, and crystalline cellulose.

[0062] Examples of disintegrants include starch, sodium alginate, carboxymethylcellulose, calcium carboxymethylcellulose, calcium carmellose, sodium croscarmellose, sodium carboxymethyl starch, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose.

[0063] Examples of binders include dimethylcellulose, glucose solution, starch solution, gelatin solution, bound cellulose, sucrose, D-mannitol, dextrin, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl starch, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone.

[0064] Examples of lubricants include talc, magnesium stearate, calcium stearate, colloidal silica, borax, and polyethylene glycol.

[0065] (Method for producing cellular vesicle staining agent) The method for producing cellular vesicle staining agent includes at least an extraction step of first extracting the cellular vesicle staining agent from photosynthetic organisms, and a purification step of purifying the cellular vesicle staining agent from the obtained extract. Furthermore, a heat treatment step may be performed when preparing the sample of photosynthetic organisms (including dried and pulverized samples), and an acid treatment step may be performed on the photosynthetic organisms (including dried and pulverized samples) and / or the extracted cellular vesicle staining agent.

[0066] [Preparation of Samples of Single-Cell Algae such as Chlorella and Euglena] When using single-cell algae such as Chlorella and Euglena as samples for extraction of cellular vesicle stains, the preparation can be carried out by any method that allows for the extraction of the cellular vesicle stain from the single-cell algae. For example, the culture solution of the single-cell algae can be separated by centrifugation, filtration, or sedimentation, and the raw single-cell algae can be used as is. The culture solution of the single-cell algae may be washed with water or physiological saline, or it may be used in a dispersed state in a liquid such as water. In this embodiment, dried algal bodies obtained by freeze-drying or spray-drying the single-cell algae can also be used. Furthermore, extracts obtained by sonication or mechanical processing such as homogenization of the single-cell algal bodies may also be used. If the single-cell algae such as Chlorella and Euglena are used industrially, commercially available fine powders of Chlorella, etc., or extracts thereof can be suitably used.

[0067] [Preparation of Samples of Multicellular Algae such as Seaweed] When using multicellular algae such as seaweed as samples for extraction of cellular vesicle staining agents, any method that can extract the cellular vesicle staining agent from kelp is acceptable for preparation. For example, dried seaweed can be used, and then pulverized using a pulverizer. Drying can be done by sun-drying or by using a commonly used drying machine. Furthermore, extracts obtained by mechanical processing such as ultrasonic treatment or homogenization of the seaweed can also be used. If the algae are industrially used, such as those of the Laminariaceae family, commercially available fine powders of kelp can be suitably used.

[0068] [Preparation of Samples of Multicellular Organisms such as Land Plants] When using multicellular organisms such as land plants as samples for extraction of cellular vesicle stains, any method that allows for the extraction of cellular vesicle stains from land plants is acceptable for preparation. For example, land plants can be dried and then pulverized using a pulverizer. Furthermore, extracts obtained by mechanical processing such as ultrasonic treatment or homogenization of the plant may also be used. If the plant is used industrially, such as tea leaves, commercially available fine powders of tea leaves can be suitably used.

[0069] [Preparation of Sample by Heat Treatment] The heat treatment conditions for the obtained sample are not particularly limited as long as a suitable yield of heat-treated extract can be obtained. For example, when water is used as the extraction solvent, the temperature is 20 to 120°C, preferably 60 to 120°C, and more preferably 100 to 120°C. The extraction time is not particularly limited as long as a suitable yield of extract and reconstituted product can be obtained. For example, it is 1 to 60 minutes, preferably 10 to 20 minutes. The amount of solvent added is not particularly limited as long as a suitable yield of extract can be obtained. For example, in the case of algae, 1 to 50 parts by mass, preferably 5 to 20 parts by mass, of the extraction solvent is added per 1 part by mass of dry powder.

[0070] [Preparation of Samples by Acid Treatment] The acid treatment conditions for the obtained samples are not particularly limited in terms of the type of acid and pH, as long as a suitable yield of acid-treated extract can be obtained. For example, hydrochloric acid with a pH of 5 to 2 is used. Acid treatment can be performed not only during sample preparation, but also on the cellular vesicle stain extracted in the extraction process described below. Zinc acetate treatment may also be performed after acid treatment. Zinc acetate treatment is preferable because it concentrates the cellular vesicle stain.

[0071] [Extraction Step of Cellular Vesicle Staining Agent] The cellular vesicle staining agent according to this embodiment is extracted from the prepared sample using an organic solvent (extraction step). Examples of organic solvents used for extraction include lower alcohols such as methanol, ethanol, and butyl alcohol; ketones such as acetone and methyl ethyl ketone; and esters such as ethyl acetate and butyl acetate. Preferably, the organic solvent is acetone, methanol, ethanol, aqueous methanol solution, aqueous ethanol solution, hexane, and ethyl acetate, and more preferably, acetone, methanol, or aqueous methanol solution. The organic solvent may be used alone or in combination of two or more.

[0072] The above organic solvent may be hydrated by adding water to make an aqueous organic solvent. The organic solvent used in the aqueous organic solvent is not particularly limited, but is preferably a lower alcohol, and more preferably methanol. The proportion of the organic solvent in the aqueous organic solvent is preferably 30% by mass or more, more preferably 50 to 99.9% by mass, and particularly preferably 70 to 99.9% by mass.

[0073] The method for extracting the cellular vesicle stain with an organic solvent is not particularly limited, but examples include adding 1 to 50 parts by weight, preferably 5 to 20 parts by weight, of the above organic solvent or aqueous organic solvent to 1 part by weight of the dried powder of the obtained sample, extracting the cellular vesicle stain while standing or stirring at an extraction temperature of 5 to 60°C, preferably 10 to 40°C, for an extraction time of 5 to 120 hours, preferably 8 to 48 hours, and then removing the solids using a centrifuge or the like.

[0074] [Purification process for cellular vesicle staining agent] The extracted cellular vesicle staining agent can be purified by purification treatment. Purification treatment can be carried out using silica gel, liquid-liquid partitioning, ion exchange resin, adsorption chromatography, partition chromatography, or size exclusion chromatography.

[0075] (Method for detecting cellular vesicles) The method for detecting cellular vesicles according to this embodiment includes at least a staining step of staining cellular vesicles using a cellular vesicle staining agent according to this embodiment, and a detection step of detecting stained cellular vesicles in a sample, and may also include an evaluation step of evaluating the sample after the detection step.

[0076] The staining step involves fluorescently staining the cellular vesicles with the cellular vesicle staining agent according to this embodiment. For example, when staining intracellular vesicles, the cellular vesicle staining agent can be added to the cultured cells. Alternatively, the cultured cells may be collected, the cellular vesicle staining agent can be added to the collected cells, and then the cells can be spread on a dish or the like. When staining extracellular vesicles such as exosomes, the cellular vesicle staining agent can be added to a solution containing extracellular vesicles.

[0077] In the staining process, the amount of cellular vesicle staining agent added may be such that the final concentration after addition is in the range of 0.01 μM to 100 μM, and preferably in the range of 0.1 μM to 1 μM.

[0078] The detection step involves irradiating the sample with excitation light corresponding to the fluorescent group of the cellular vesicle stain according to this embodiment, and detecting the fluorescence emitted from the cellular vesicle stain bound to the cellular vesicles in the sample. For the irradiation of the excitation light, the same irradiation means as for general fluorescence detection can be used; for example, a predetermined wavelength can be selected as needed from the laser light source provided by the fluorescence microscope. Fluorescence detection may be performed from the microscope tube of the fluorescence microscope, or images captured by a camera or the like installed in the fluorescence microscope may be displayed on a display means such as a monitor.

[0079] Furthermore, the method for detecting cellular vesicles may include an evaluation step for evaluating the sample. In the evaluation step, the sample is evaluated using the cellular vesicles detected by the cellular vesicle staining agent according to this embodiment. Examples of sample types include cosmetics, pharmaceuticals, and food samples. In addition, it is possible to evaluate the degree of cell differentiation by detecting intracellular vesicles, and to evaluate diseases, etc., by detecting exosomes in body fluids.

[0080] For example, when evaluating cosmetics or pharmaceuticals, cellular vesicles can be pre-labeled with the cellular vesicle staining agent according to this embodiment. By detecting and observing the uptake of the labeled cellular vesicles into cells in response to the presence of cosmetics or pharmaceuticals, the cosmetics or pharmaceuticals can be evaluated. For instance, pigmentation and associated skin diseases can be evaluated by observing the uptake of melanosomes, which are vesicles that accumulate melanin, into keratinocytes. Furthermore, foods can be evaluated by measuring the cellular vesicles contained in them. For example, the uptake efficiency of miRNAs within vesicles that affect intestinal function can be used to predict food function.

[0081] When evaluating pharmaceuticals or quasi-drugs, cellular vesicles can be pre-labeled with the cellular vesicle staining agent according to this embodiment, and then the labeled cellular vesicles can be introduced into the body for detection and observation to evaluate the pharmaceuticals or quasi-drugs. Alternatively, the cellular vesicle staining agent can be introduced into the body, and cellular vesicles present in the body can be detected and observed to evaluate the pharmaceuticals or quasi-drugs. The cellular vesicle staining agent according to this embodiment can be formulated into an appropriate form and introduced into humans or animals in any administration method. The administration method is not particularly limited, but examples include oral, transdermal, enteral, transmucosal, and injection.

[0082] The present invention will be described in more detail and specifically below with reference to examples, but the present invention is not limited to the following examples.

[0083] [Evaluation of Autofluorescence of Cellular Vesicles Derived from Euglena] The inventors have been developing methods for detecting cellular vesicles because they possess various functions. In the process, they discovered that cellular vesicles of algae, which are photosynthetic organisms, may emit autofluorescence, and conducted the following tests. Figure 1A is a graph showing the results of analyzing cellular vesicles stained with the fluorescent dye GIF-2276 and those that were not stained using a high-performance liquid chromatography gel filtration system. Figure 1B is a graph showing the results of analyzing the difference in fluorescence intensity of cellular vesicles due to different heat treatment conditions using a high-performance liquid chromatography gel filtration system. Figure 2 is a photograph showing the difference in autofluorescence intensity of cellular vesicles due to different heat treatment conditions.

[0084] "Extraction of cellular vesicles from Euglena culture medium" Euglena (Euglena gracilis G.A. Klebs (NIES-47) strain) was obtained as a photosynthetic organism from the National Institute for Environmental Studies. The culture medium used was KH medium (pH 3.5), and the cells were cultured at approximately 30°C for 7 days. The culture was carried out under conditions of direct sunlight near a window, and irradiated from a distance of 10 cm using a SUKITEN LED floodlight 100W. For the purification of cellular vesicles, Euglena cells and culture medium (total 500 mL) were heat-treated at 60°C, 90°C, and 120°C for 20 minutes, and then centrifuged at 8,000 g for 1 hour to collect the supernatant. The supernatant was filtered through a 0.1 μm (90 mm diameter) PES (polyethersulfone: Thermo Fisher) membrane, and the cellular vesicles were collected on a 0.08 μm, 90 mm polycarbonate track etch (PCTE) filter (GVS, Hessen, Germany). After washing three times with 20 mL of phosphate-buffered saline (PBS), the vesicles were collected in 10 mL of PBS. The collected material was sterilized using a 0.22 μm PES syringe filter (GVS).

[0085] "Evaluation of cellular vesicles extracted by heat treatment" Each type of cellular vesicle (at room temperature, 60°C, 90°C, and 120°C) was separated by gel filtration, and the cellular vesicles were detected by fluorescence. Detection was performed using a high-performance liquid chromatogram system (SCL-10A vp) from Shimadzu Corporation equipped with two tandem fluorophotometers (RF-10A xl). Gel filtration resin (EVSecond resin, GL Sciences) was packed into a stainless steel column (φ8 mm × 200 mm: Senshu Science Co., Ltd.). Fluorescence was detected using tandem-connected RF-10A vp fluorophotometers (Shimadzu) at excitation 470 nm / emission 530 nm. Each recovered sample was separated using 10 μL of buffer solution (10 mM Tris-HCl (pH 6.8), 0.5 mM EDTA, 50 mM NaCl) at a flow rate of 1 mL / min. For comparison, samples with surface proteins fluorescently labeled with 20 μM GIF-2276 (see PCT / JP2023 / 28846) were also separated. Labeling with GIF-2276 was performed at 80°C for 5 minutes.

[0086] "Visual observation of cellular vesicles" 0.5 ml of purified cellular vesicles, which were heat-treated at 60°C, 90°C, and 120°C for 20 minutes, were placed in an Eppendorf tube and irradiated with ultraviolet (UV) black light (395 nm, DARKBEAM).

[0087] As shown in Figure 1A, it was demonstrated that cellular vesicles possess autofluorescence even without the use of fluorescent staining agents for cellular vesicles. As shown in Figure 1B, the fluorescence intensity increased with increasing heat treatment temperature. As shown in Figure 2, it was visually confirmed that the intensity of red autofluorescence increased with increasing heat treatment temperature. From the above results, it became clear that heat treatment of Euglena cells increases the amount of fluorescent compounds that exhibit affinity for cellular vesicles.

[0088] [Isolation and Identification of Cellular Vesicle Staining Agents] Chlorophyll is known to have red fluorescence, but since chlorophyll a and b are likely to decompose when subjected to heat treatment, there is a possibility that fluorescent substances other than chlorophyll a and b are generated. Therefore, we attempted to identify these substances. Figure 3 shows the results of identifying the cellular vesicle staining agents of the present invention using an HPLC-MS system.

[0089] Euglena cells (20 g) were extracted with 100 mL of chloroform:methanol (3:1). After evaporation, the extract was dissolved in 45 mL of methanol and 5 mL of H2O2 was added. ２ O was added. The extract was applied to a C18 column (1 mL: SEP cartridge) (GL Sciences). After washing with 100 mL of 90% methanol, it was eluted with 20 mL of 100% methanol. After evaporating the ethanol, the eluate was dissolved in 20 mL of chloroform and applied to a silica gel column (approximately 100 mL) (Fujifilm WAKO). Chlorophyll was eluted in 20 mL portions using chloroform:methanol (99:1). Fraction 12 from the silica gel column was used as the Euglena probe fraction. The eluate was concentrated by evaporation and then re-purified using a C18 spin column (GL Sciences).

[0090] The purified Euglena probe fraction was detected in negative ion mode using HPLC-MS (UPLC / ESI-TOF-MS) Xevo QTof (Waters). For comparison, similar analyses were also performed using chlorophyll a (Cayman Chemical) and chlorophyll b (Merck) derived from spinach.

[0091] The identification results in Figure 3 revealed that the compound contained in the Euglena probe fraction is 7-hydroxymethylpheophytin, which is a chlorophyll derivative that lacks Mg and has a hydroxyl group substituted at the 7-position, compared to chlorophyll a and b.

[0092] [Evaluation of Cellular Vesicle Staining by Cellular Vesicle Staining Agents for Milk-Derived Cellular Vesicles] The following tests were conducted to confirm whether cellular vesicle staining agents, which are chlorophyll and chlorophyll derivatives, stain not only cellular vesicles derived from Euglena obtained from the same cells, but also cellular vesicles from other sources. Figure 4 is a graph showing the staining results of milk-derived cellular vesicles with chlorophyll derivatives, chlorophyll a, and chlorophyll b.

[0093] "Preparation of Milk-Derived Cellular Vesicles" 1 L of commercially available pasteurized milk (Seki Milk or Takanashi) was centrifuged at 8000 rpm for 30 minutes, and the supernatant was filtered through a coffee filter. Next, acetic acid was added to a concentration of 1%, and the mixture was centrifuged again at 8000 rpm for 30 minutes. The supernatant was filtered again through a coffee filter. The filtered liquid was filtered through a 0.45 μm filter (Sartorius), and the filtrate was filtered through a 0.1 μm filter (Sartorius). The milk-derived cellular vesicles on the 0.1 μm filter were washed three times with 5 mL of phosphate buffer, and then collected with 1 mL of phosphate buffer. 10 μL of the purified milk-derived cellular vesicles were diluted with 40 μL of phosphate buffer.

[0094] "Staining Evaluation of Milk-Derived Cellular Vesicles" Euglena probe fraction (chlorophyll derivative), spinach-derived chlorophyll a (Cayman Chemical), and chlorophyll b (Merck) were added to a milk-derived cellular vesicle preparation solution to a final concentration of 1 μM. After 12 hours, 10 μL of the reaction product was separated by gel filtration, and cellular vesicles were detected by fluorescence. Detection was performed using a high-performance liquid chromatogram system (SCL-10A vp) from Shimadzu Corporation equipped with two tandem fluorometers (RF-10A xl). Gel filtration resin (EVSecond resin, GL Sciences) was packed into a stainless steel column (φ8 mm × 200 mm: Senshu Science Co., Ltd.). Fluorescence was detected using a tandem-connected RF-10Avp fluorometer (Shimadzu) at excitation 415 nm / emission 470 nm. Cellular vesicles derived from milk were separated using a buffer solution (10 mM Tris-HCl (pH 6.8), 0.5 mM EDTA, 50 mM NaCl) at a flow rate of 1 mL / min.

[0095] As shown in Figure 4, the Euglena probe fraction (chlorophyll derivative), chlorophyll a, and chlorophyll b all labeled milk-derived cellular vesicles, but the Euglena probe fraction (chlorophyll derivative) was found to label milk-derived cellular vesicles with particularly high efficiency.

[0096] [Staining Evaluation of Milk-Derived Cellular Vesicles by Acid Treatment of Chlorophyll and Chlorophyll Derivatives] It is known that under acidic conditions, Mg is released from the porphyrin ring of chlorophyll, and that Zn coordinates in place of Mg when treated with zinc acetate. Therefore, chlorophyll derivatives and Zn-coordinated chlorophyll were artificially created by acid treatment, and the staining evaluation of milk-derived cellular vesicles was performed. The staining evaluation of milk-derived cellular vesicles by acid-treated chlorophyll and chlorophyll derivatives was performed on chlorophyll b samples, acid-treated chlorophyll b samples (hereinafter referred to as acid-treated samples), chlorophyll b samples treated with zinc acetate after acid treatment (hereinafter referred to as acid-treated zinc acetate samples), and chlorophyll b samples treated with zinc acetate (hereinafter referred to as zinc acetate samples). Figure 5 is a graph showing the staining results of milk-derived cellular vesicles by acid-treated chlorophyll b.

[0097] "Acid Treatment of Chlorophyll and Chlorophyll Derivatives" A chlorophyll mixture (manufactured by Tokyo Chemical Industry Co., Ltd.), mainly chlorophyll b, was used. For acid treatment, 100 μg of the chlorophyll mixture was added to 1 mL of methanol and exposed to acidic conditions of 0.01 NHCl for 10 minutes. After adding 100 μl of distilled water, the mixture was applied to a C18 spin column (manufactured by GL Sciences Inc.), the column was washed with 90% methanol, and then eluted with 100% methanol and dried.

[0098] For samples treated with chlorophyll b after acid treatment, zinc acetate treatment was performed. The acid-treated chlorophyll b samples were further treated with zinc acetate to a final concentration of 1 mM, and then dried. For the zinc acetate-treated chlorophyll b samples, 100 μg of the chlorophyll mixture was added to 1 mL of methanol and exposed to 1 mM zinc acetate acidic conditions for 10 minutes. After adding 100 μl of distilled water, the mixture was applied to a C18 spin column (GL Sciences), washed with 90% methanol, and then eluted with 100% methanol and dried.

[0099] "Staining Evaluation of Milk-Derived Cellular Vesicles" Acid-treated samples of the above chlorophyll and chlorophyll derivatives were stained and evaluated using the same method as described in "Staining Evaluation of Milk-Derived Cellular Vesicles".

[0100] As shown in Figure 5, all samples labeled milk-derived cellular vesicles, but the acid-treated sample and the acid-treated followed by zinc acetate treatment labeled them very efficiently. Furthermore, although the reason is unclear, the acid-treated followed by zinc acetate treatment showed an effect of concentrating the Euglena probe fraction (chlorophyll derivative) (data not shown).

[0101] [Evaluation of the anti-inflammatory effect of cellular vesicle staining agents] Cellular vesicles derived from Euglena are known to possess anti-inflammatory activity (see Japanese Patent Application No. 2023-199229). Therefore, the anti-inflammatory activity of the Euglena probe fraction (chlorophyll derivative) described above was investigated. Figure 6A is a graph evaluating the anti-inflammatory activity of chlorophyll a and chlorophyll derivatives by the decrease in nitric oxide (NO) levels, and Figure 6B is a graph evaluating the anti-inflammatory activity of chlorophyll a and chlorophyll derivatives by the decrease in IL-6 levels. The evaluation methods described below are explained below.

[0102] Anti-inflammatory activity was evaluated by the reduction in nitric oxide (NO) levels induced by lipopolysaccharide (Fujifilm Wako Co., Ltd.) and the reduction in IL-6 levels, a type of cytokine associated with inflammation. Mouse macrophage RAW264.7 cells were cultured in 96-well plates in RPMI medium (Fujifilm Wako Co., Ltd.) with 10% fetal bovine serum and 1× antibiotic medium. After 12 hours, 500 ng / mL of lipopolysaccharide (LPS) was added, and simultaneously, chlorophyll a and Euglena probe fractions were added at concentrations of 0.3 μg / mL, 1.0 μg / mL, and 10 μg / mL, respectively. After 24 hours of incubation, the concentration of NO accumulated in the medium was measured. NO concentration was determined by mixing 100 μL of supernatant with 100 μL of Griess reagent [0.1% sulfanilamide, 1% N-(1-naphthyl)ethylenediamine [Merck], 0.625% (w / v) phosphoric acid solution] and incubating at room temperature for 20 minutes. Next, the absorbance at 560 nm was measured using a Glomax Multi Detection System (Promega). The level of IL-6 in the culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) kit (BioLegend).

[0103] As shown in Figures 6A and 6B, chlorophyll a and chlorophyll derivatives suppressed NO production and IL-6 expression. Furthermore, chlorophyll derivatives suppressed both NO production and IL-6 expression more efficiently than chlorophyll a.

[0104] [Administration of chlorophyll b to mice] We investigated whether chlorophyll could efficiently label cellular vesicles in vivo. Figure 7 is a graph showing that chlorophyll b efficiently labels cellular vesicles in vivo. The evaluation methods are described below.

[0105] Mice were orally administered 10 μg of chlorophyll b (Merck) (diluted in 200 μL of 10% Tween 80). After 5, 10, 30, and 60 minutes, 10 μL of blood was collected from the tail vein, centrifuged (10000 g, 10 minutes), and 4 μL of plasma was recovered. This plasma was then diluted to 1 / 10 with 36 μL of PBS. After centrifugation, 10 μL was stained and evaluated using the same method as described in "Staining Evaluation of Milk-Derived Cellular Vesicles".

[0106] As shown in Figure 7, chlorophyll was absorbed from the mouse stomach and effectively labeled cellular vesicles in the blood.

[0107] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A cellular vesicle staining agent containing chlorophyll or a chlorophyll derivative.

2. The chlorophyll or chlorophyll derivative is a compound represented by the following general formula (1), and the cellular vesicle staining agent according to claim 1. (In formula (1), R1 is CH ３ , CHCH ２ , CH ２ CH ３ , CHO, CH ２ OH or COOCH [[ID=IS]] ３ ; R2 is CHCH ２ , CH ３ , CHO, CH ２ OH or COOCH ３ ; and n is H / H (uncoordinated), Mg, Zn, Fe, Cu, Ni or Co.).

3. The cellular vesicle staining agent according to claim 2, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (2). (In equation (2), R1 is CH ３ , CHO or CH ２ OH is OH, and R2 is CHCH ２ (where n is H / H (uncoordinated), Mg, Zn, or Fe.) 4. The cellular vesicle staining agent according to claim 2, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (3). (In equation (3), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH or COOCH ３ And R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３ (That is the case.) 5. The cellular vesicle staining agent according to claim 4, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (4). (In equation (4), R1 is CH ３ , CHO or CH ２ OH is OH, and R2 is CHCH ２ (That is the case.) 6. The cellular vesicle staining agent according to claim 4, wherein the chlorophyll or chlorophyll derivative is a compound represented by the following general formula (5). (In equation (5), R1 is CH ２ OH is OH, and R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３ (That is the case.) 7. The cellular vesicle staining agent according to claim 1, wherein the chlorophyll or chlorophyll derivative is chlorophyll a, chlorophyll b, or 7-hydroxymethylpheophytin.

8. The cellular vesicle staining agent according to claim 7, wherein the chlorophyll or chlorophyll derivative is 7-hydroxymethylpheophytin.

9. An anti-inflammatory agent containing chlorophyll or a chlorophyll derivative represented by the following general formula (1) as an active ingredient. (In equation (1), R1 is CH ３ ienCHCH ２ ,CH ２ CH ３ , CHO, CH ２ OH or COOCH ３ And R2 is CHCH ２ ,CH ３ , CHO, CH ２ OH or COOCH ３ (where n is H / H (uncoordinated), Mg, Zn, Fe, Cu, Ni, or Co.) 10. The anti-inflammatory agent according to claim 9, wherein the chlorophyll or chlorophyll derivative is 7-hydroxymethylpheophytin.

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