KR100641968B1 - Physiologically active extract - Google Patents

Abstract

A bioactive extract containing an ethyl acetate soluble component of an indigo plant, obtained by dipping a raw indigo plant in an organic solvent used for extraction.

Ethyl acetate soluble components include 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone, kaempferol, 3,5,7,4'-tetrahydroxy-6-methoxy- Flavones, gallic acid, caffeic acid, indirubin, pheophorbide a, and methyl pheophorbide a.

Description

Bioactive Extracts {PHYSIOLOGICALLY ACTIVE EXTRACT}

1 is an infrared absorption spectrum of 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone as compound 2. FIG.

2 is an infrared absorption spectrum of kaempferol, compound 3.

3 is an infrared absorption spectrum of 3,5,7,4'-tetrahydroxy-6-methoxy-flavone as compound 4. FIG.

4 is an infrared absorption spectrum of caffeic acid as compound 6. FIG.

FIG. 5 is an infrared absorption spectrum of 3- (1,3-dihydro-3-oxo-2H-indole-2-ylidene) -1,3-dihydro-2H-indol-2-one as compound 7. FIG.

The present invention relates to a novel bioactive extract derived from a plant, and more particularly, to a bioactive extract containing an ethyl acetate soluble component derived from a raw indigo plant, a manufacturing method and a use thereof.

Indigo plant or Polygonum tinctorium Lour is an annual plant belonging to the polygonum family classified as a dicotyledonous plant. It is native to South Vietnam. This plant was introduced in Japan from China, along with dyeing technology, as a plant for dark blue dyeing seven centuries ago, and is currently cultivated in Tokushima Prefecture, Japan. In ancient times, people thought that leaves and seeds of spines contained useful bioactive ingredients and that the leaves and seeds of spines were used as herbal medicines in the sun. Nippon-Yakuso-Zensho (Encyclopedia of Japanese Medicinal Plants), edited by Mizuo Mizuno, published on February 22, 1995 by Shin-Nihon-Hoki-Shuppan Publisher, Tokyo, Japan, pp. 5-7)] was known to have only anti-inflammatory, antipyretic and detoxification effects. This physiological action can only be expected if it is used in the form of decoction prepared by dipping the leaves and seeds in hot water.

In the present situation where there is practically no discomfort, food, or attention, most people, including young people, are remarkably concerned about their health, more specifically, herbal medicines that are readily available every day without a doctor's prescription. This can be seen from the fact that various literatures on various herbal medicines have been published and that various kinds of health foods and supplements are flooded in grocery stores and pharmacies. In general, herbal medicines have the advantage of having a gentle action and fewer side effects, but the drawback is that each individual has a different sensitivity. The establishment of new herbal medicines with various physiological activities is greatly required to meet the needs of consumers.

 In view of the above circumstances, an object of the present invention is to provide a novel biologically active extract exhibiting various physiological actions derived from plants.

It is also an object of the present invention to provide a method for producing the biologically active extract.

It is also an object of the present invention to provide a bioactive composition containing a bioactive extract.

The present inventors have diligently studied, and the novel bioactive extracts or extracts derived from the extract obtained by extracting the ethyl acetate soluble component by immersing the fresh side in an organic solvent have antibacterial, antiviral and anti-tumor effects on mammals and humans. It has been shown to exhibit a variety of physiological effects including radical scavenging, cell necrosis, cytokine production, cytokine production, and nitric oxide synthase. The inventors have also found that the extract can be safely used in foods, cosmetics and medicines for humans since it has substantially no side effects in mammals and humans.

This invention solves the 1st subject of this invention by providing the bioactive extract containing the ethyl acetate soluble component from a raw indigo plant.

The present invention provides a second object of the present invention, in the method for producing a bioactive extract containing an ethyl acetate soluble component derived from a raw indigo plant, extracting ethyl acetate soluble component from the side by dipping the side in an organic solvent. It is solved by providing a method for producing a novel bioactive extract containing ethyl acetate soluble components derived from the side characterized in that the step of extracting, and extracting the extract.

This invention solves the 3rd subject of this invention by providing the bioactive composition containing an ethyl acetate soluble component.

As mentioned earlier, it is well known that the extraction of leaves and seeds from the sun-dried leaves with anti-inflammatory, antipyretic and detoxification effects. It is surprising that the ethyl acetate soluble component obtained by treating the side directly with an organic solvent exerts the various physiological effects described above. The present invention is based on this finding.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A preferred embodiment of the present invention relates to an indigo plant belonging to a perennial plant belonging to the dicotyledonous plant whose biological name is Polygonum tinctorium. As used herein, the term "raw indigo plant" refers to a fresh raw that is not substantially dried or decayed. If it is in such a state, it can be used in this invention in any way regardless of the kind and form of the whole plant body and the specific part of its leaf, stem, and a seed. Most preferably, fresh side of the side, especially those obtained from plants before the fruiting phase, is used.

The physiologically active extract of the present invention is prepared by dipping raw greens in an organic solvent to extract ethyl acetate soluble components from plants, and extracting the extracts. Some or all of the raw side is washed with water to remove impurities, further cut as needed to be powdered and / or pressed, and then optionally heated in an appropriate organic solvent in an amount of 1 to 100 times the volume for the raw side. It is immersed for 0.1 to 100 hours under conditions. Organic solvents used in the present invention include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, methyl ether, ethyl ether, tetrahydrofuran, chloroform, n-hexane, acetone, ethyl acetate Hydrophilic and hydrophobic organic solvents; You may use combining these organic solvents according to a use.

In the case where the bioactive extract of the present invention is used without substantially purifying, it is preferable to use a mixture of water and ethanol or methanol even if the extraction efficiency of the ethyl acetate soluble component is rather reduced.

The physiologically active extract thus obtained may be used as it is. Usually, the type of organic solvent and the final extract of the extract used after further treatment by filtration, separation, fractional precipitation, decantation, and / or centrifugation remove impurities. Depending on the application, the treatment is carried out to remove the organic solvent.

Depending on the type of organic solvent, the physiologically active extract of the present invention may be selected from the group consisting of 6,12-dihydro-6,12-dioxoindolo [2,1-b] quinazoline as ethyl acetate soluble component; 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone; Camphorol; 3,5,7,4'-tetrahydroxy-6-methoxy-flavone; Gallic acid; Caffeic acid; 3- (1,3-dihydro-3-oxo-2H-indol-2-ylidene) -1,3-dihydro-2H-indol-2-one; [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid) ; And / or [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21 (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-propinepropane Acid methyl ester is contained 0.01% or more in terms of solids. These components effectively exert antimicrobial activity, antiviral action, antitumor action, radical scavenging action, cytotoxicity and / or cytokine production regulation or inhibition in mammals and humans. For example, when the biologically active extract of the present invention is used as a medicine in need of a relatively highly purified form, the ethyl acetate soluble component must be separated from the extract in advance by methods generally used for purification of indole derivatives and flavone compounds. Examples of the purification method include salting out, dialysis, filtration, concentration, liquid separation, fractional precipitation, decantation, liquid chromatography, gas chromatography, high performance liquid chromatography, and crystallization. If necessary, these methods may be appropriately used in combination. The degree of physiology of each of the ethyl acetate soluble components is different and the range of action is also different. When used in various fields including foods that can use each component without separating them into individual components, it is preferable to add them in the required products as they are in the same ratio as long as they are contained in the extract. This physiologically active ingredient has the drawback that its physiological action varies when used in this form. When these ingredients are added to medicines such as injections, one or more of the ingredients may be arbitrarily used after or without separation depending on the disease susceptible to the ingredients. The above methods are merely preferred examples for preparing the bioactive extracts of the present invention and do not limit the bioactive extracts of the present invention as long as the extract contains an ethyl acetate soluble component.

The bioactive extract containing the ethyl acetate soluble component of the present invention has at least one or more of the following properties, and exhibits various biological activities for mammals and humans.

(1) inhibits the growth of Gram-positive and Gram-negative bacteria, including Helicobacter pylori, which causes gastritis, gastric ulcer, duodenal ulcer and gastric cancer;

(2) inhibits the growth of influenza virus, bullous stomatitis virus, herpes simplex virus, head virus and cytomegalovirus;

(3) inhibits the growth of tumor cells of refractory tumors, including leukemia cells, gastric cancer cells and lung cancer cells;

(4) It is derived from free radicals and fat peroxide, and captures radicals that cause various diseases related to lifestyles such as malignant tumor, myocardial infarction, cerebral hemorrhage, rheumatism, lifestyle disease (adult disease), kidney failure, stress, and aging.

(5) Acts on normal and abnormal B-cells, T cells, neurons, epithelial cells of the digestive tract, liver cells of the digestive tract, vascular endothelial cells, skin cells, etc. Regulating and treating / preventing various diseases of the digestive, circulatory, eye, ear, nose, neck, skin, nerves and bones;

(6) Regulates the production of cytokines, including interferon-γ and interleukin 10, by immune cells. It determines equilibrium in vivo between type 1 helper T cells (Th1) and type 2 helper T cells (Th2) to regulate equilibrium in steady-state conditions, such as autoimmune diseases, liver failure, kidney failure, pancreatic disorders, etc. Related to treatment / prevention of diseases and diseases related to transplant-host response.

(7) inhibits the expression of nitric oxide synthase by cells in vivo, caused by cytokines and bacterial endotoxins, and also inhibits the production of nitric oxide, thereby autoimmune diseases, allergic diseases, inflammatory diseases, malignant Treating / preventing various diseases such as tumors, kidney disorders and lung disorders,

The bioactive extract of the present invention may contain an angiogenesis inhibitor which is closely related to tumor growth in vivo. The physiological action (1) to (6) described above can be confirmed for various methods of the following experiments. For example, expression inhibition of nitric oxide synthase is common in mouse peritoneal macrophages or macrophages-derived cell systems induced when cultured in the presence of interferon-γ or lipopolysaccharide, according to conventional methods using antibodies against enzymes. This can be confirmed by testing the effect of the addition of a bioactive extract of the present invention on the expression of "induction-type nitrogen monoxide synthetic enzymes," an enzyme called "INOS." Previous studies, such as the Griess method on the level of nitric oxide, have shown that there is an inhibitory effect on the production of nitrogen monoxide by inhibiting the expression of these enzymes. Exerts an inhibitory effect on angiogenesis deeply related to proliferation. Various disorders of tissues in vivo due to gram-negative bacteria and fungi and inflammation caused by various viruses, contact with or infiltration of various proteins, organic compounds and metals of the organisms, their infiltration, and tumors Since it also acts against disorders and regulates functions in vivo, the bioactive extract of the present invention inhibits inflammation by inhibiting the production of inflammatory cytokines, including interferon-γ and interleukin-1. The bioactive extract of has the property of improving sleep disorders caused when various diseases occur.Therefore, the bioactive extract of the present invention is used in the fields of food, cosmetics and pharmaceuticals, such as antibacterial activity, antiviral action, antitumor action, Radical scavenging, cellular necrosis control, cytokine production regulation, cytokine It can be advantageously used as a herbal medicine that exerts the effect of inhibiting production, inhibiting angiogenesis, improving sleep disorders, regulating functions in vivo and / or inhibiting expression of nitric oxide synthase.

Due to these properties, the bioactive extract of the present invention is a herbal medicine that exerts antimicrobial activity, antiviral action, antitumor action, radical scavenging action and / or cellular necrosis suppression in healthy and sick or wounded individuals. It can be advantageously used in the fields of food, cosmetics and pharmaceuticals.

The use of each of the above-described fields may be used in the food field in combination with one or more materials and / or ingredients used in the general foodstuff for facilitating absorption of the extract. Examples of materials and / or ingredients include water, alcohols, starches, proteins, fibers, sugars, lipids, fatty acids, vitamins, minerals, flavors, pigments, sweeteners, seasonings, spices, and preservatives.

The resulting mixture can be formulated into the required dosage forms such as liquids, suspensions, creams, pastes, jellies, powders, granules and other desired shapes depending on the actual use of the food. For use in such foods, the bioactive extract of the present invention is used in an amount of 0.01 w / w% or more, preferably 0.1 w / w% or more.

Examples of foods to which the present invention can be advantageously applied include soy sauce, powdered soy sauce, miso (soybean paste), hunmatsu miso (powdered miso), moromi (refined liquor), hiso (refined soy sauce), furikake (fish seasoning) , Mayonnaise, dressing, vinegar, Sanbai sake (sugar, soy, vinegar source), hunmatsu sushi water (powdered vinegar for sushi), tentsuyu (Japanese fried food source), mentsuyu (Japanese burmese) Sauce), Sauce, Ketchup, Yaguniku No Tare (Japanese BBQ), Curry Roo, Chuka no Moto (Instant Chinese Mix), Instant Stew Mix, Instant Soup Mix, Dashi No Moto (Instant Stock Mix) , Various seasonings such as complex seasoning, mirin (sweetened liquor), shinmirin (synthetic mirin), table sugar, coffee sugar; Senbei (rice crackers), Arare (cuboid rice cakes), okoshi (cake with millet and rice), fried dough cake, kyuhi (starch paste), mochi (rice paste), manju (red bean jam dumplings), Uiro ( Sweet jelly), Ann (red bean jam), Yocan (sweet red bean jelly), Mizu yokan (soft azuki red bean jelly), king yoku (a type of yocan), jelly, pao de castella, amedama (Japanese toffee), etc. Japanese sweets; Confectionery such as biscuits, crackers, cookies, pies, puddings, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy, gum jelly and the like; Ice creams such as ice cream, popsicles, and sherbets; Syrups such as korimitsu (sugar syrup for shaved ice); Spreads and pastes such as butter cream, custard cream, flower paste, pine nut paste, and fruit paste; Processed foods of fruits and vegetables, such as jam, marmalade, syrup zuke (fruit pickles), and toka (sweets); Processed grain foods such as bread, noodles, fried rice, artificial meat; Pickles and pickled products such as pukujin zuke (red radish pickle), beta lazuke (a type of whole radish pickle), senmai zuke (a kind of sliced radish pickle), rakyo zuke (pickled green onion), and the like; Premixes for pickling and pickling products such as Takuang Zuke no Moto (premix for pickled radishes) and Hakusai Zuke No Moto (premix for cabbage peeling); Meat products such as ham and sausage; Fish meat products such as fish ham, fish sausage, fish paste, chikuwa (fried fish paste) and fried fish paste; Various delicacies such as sea urchin squid, squid salted fish, processed kelp, dried squid fish and dried puffer fish seasoned with mirin; Boiled foods prepared with agricultural products, livestock products, and marine products; Everyday dishes such as boiled foods, baked goods, fried foods, fried foods, steamed foods, and dishes dressed with a source; Frozen foods such as fried shrimps, croquettes, Xiao-mai, gyoza (fried or steamed dumplings with minced pork), harumaki (a type of Chinese dish), hamburger steak, meatballs, fish hamburgers, and fish balls; Ham burger, meat bowl, red bean rice, boiled rice with beef or chicken, brown rice porridge, curry, meat soup, demi-glace sauce, thick soup, clear soup, stew, Japanese steamed rice, Happosai (Chinese vegetables) Sorts), retort foods such as boiled beans, grilled chicken, steamed crucibles, boiled chestnuts and boiled vegetables; Egg and milk products such as Kinshi-Tamago (steamed egg rolls), milk drinks and butter cheeses; Canned and bottled products of meat, fish meat, fruits, vegetables, etc .; Alcohols such as synthetic liquor, liquor and wine shochu; Soft drinks such as coffee, cocoa, juice, green tea, black tea, oolong tea, mineral drink, soda, sour milk drink and lactic acid bacteria drink; Instant foods such as instant pudding mixes, instant hot-cate mixes, instant juices, beef kuseki-shiruko (an instant mix of rice cakes and azuki red bean porridge), and instant soup soup mixes.

Since the biologically active extract of the present invention has a property of capturing radicals generated in vivo, it may be advantageously used for health foods and health supplements related to the prevention of diseases related to lifestyle or geriatric disease, carcinogenesis, and aging. In addition to food for humans, the extract of the present invention can also be used for feeds and foods for animals such as livestock, poultry, bees, silkworms and fish.

In the field of cosmetics, the bioactive extract of the present invention is, for example, an oily base, a water-soluble base, a perfume, a pigment, a dye, a cooling agent, a humectant, a emollient, an emulsifier, a gelling agent, a viscosity increasing agent, a softening agent, a solubilizer, a surfactant, a foam stabilizer. You may use together with various components which make administration of an extract easy, such as a transparent agent, an antioxidant, a fatty excess, an antiseptic, a film forming agent, and a propellant.

The extract of the present invention is mixed with one or more drugs such as vitamins, amino acids, peptides, hormones, extracts, vasodilators, blood circulation improvers, cell activators, antimicrobial agents, anti-inflammatory agents, anti-galvanogenesis preventive agents, astringent agents, skin function enhancers, and keratin softeners. It can also be used. The resulting mixture can be processed into the form of a solution, an emulsion, a cream, a paste, a powder, granules, or any other desired shaped solid. Depending on the use, the bioactive composition of the present invention as a cosmetic usually contains at least 0.005 w / w%, preferably at least 0.05 w / w% of the bioactive extract of the present invention.

Examples of cosmetics to which the bioactive extract of the present invention can be advantageously applied include wool and hair growth promoters, pomades, hair sticks, hair oils, hair creams, hair solids, hair liquids, hair set lotions, hair styling gels, hair water greases. Hair cosmetics such as hair blow, hair aerosol, perm hair liquid, hair dye; Cleaning cosmetics such as hair and body shampoos, hair rinses, hair washing soaps, makeup soaps and cleansing foams; Skin cosmetics such as lotion, cream, emulsion, lotion, pack, foundation, lip stick, rouge, eyeliner, mascara, eyeshadow, eyebrow pencil, nail polish, powder; Oral cosmetics such as toothpaste powder, moisturizing toothpaste, general toothpaste, tooth cleanser, medicinal toothpaste, cachou, and gargle; And other cosmetics such as sunscreens, shaving cosmetics, bath cosmetics, fragrances, colon perfumes, armpit deodorants, baby powders, eye lotions, and bleaching creams.

In the case of skin and hair cosmetics, the bioactive extract of the present invention contains about 0.001 w / w% to about 10-glucosyl bioflavonoids such as α-glucosyl rutin, α-glucosyl hesperidin and α-glucosyl naringin. The addition of w / w% easily exerts the effects of the physiologically active extract of the present invention by supplementing the skin with nutrients and promoting metabolism in vivo.

When moisturizing saccharides or sugar alcohols such as maltose, trehalose and maltitol are added as moisturizing agents, preferably 1 w / w% or less, the skin, scalp and / or hair are adequately moistened. Biologically active extracts readily exert their effects.

In the field of pharmaceuticals, the physiologically active extracts of the present invention are used for all diseases susceptible to the ethyl acetate soluble component of the present invention, including bacterial diseases, fungal diseases, viral diseases, malignant tumors, hyperlipidemia, ischemic heart diseases, such as the digestive system. It can be advantageously used to treat and / or prevent diseases, circulatory diseases, urinary / genital diseases, immune diseases, cranial nerve diseases, eye diseases, skin diseases, otolaryngology diseases and the like.

Examples of such various diseases susceptible to the bioactive extract of the present invention include bacterial corneal ulcer, bacterial conjunctivitis, bacterial food poisoning, septic shock, endotoxin shock, bacterial endocarditis, bacterial meningitis, bacterial pneumonia, bacterial aneurysm, bacterial cerebral aneurysm, etc. Bacterial diseases; Viral diseases such as fungal meningitis, fungal corneal ulcers, fungal skin diseases, candidiasis, ringworm; Viral diseases such as viral gastroenteritis, viral hepatitis, viral bronchitis, viral colitis, viral myocarditis, viral meningitis, viral enteritis, viral encephalitis, viral pneumonia, AIDS; Solid malignancies such as renal cell carcinoma, mycelial sarcoma, and chronic granulomas; Colon cancer, colorectal cancer, colon and rectal carcinoma, stomach cancer, thyroid cancer, tongue cancer, bladder cancer, ciliary cancer, liver cancer, prostate cancer, uterine cancer, pharyngeal cancer, lung cancer, breast cancer, malignant melanoma, Kaposi's sarcoma, brain tumor, neuroblastoma, ovarian tumor, Hematopoietic malignancies such as testicular tumors, pancreatic tumors, kidney cancers, adrenal tumors, hemangioendothelioma, adult T cell leukemia (ATL), chronic myeloid leukemia (CML), malignant lymphomas; Active Chronic Hepatitis, Atrophic Gastritis, Autoimmune Hemolytic Anemia, Vasedou's Disease, Behcet's Syndrome, Crohn's Disease, CRST Syndrome, Cold Agglomerate Hemolytic Anemia, Idiopathic Ulcerative Colitis, Good Pasture Syndrome, Hyperthyroidism, Chronic Thyroiditis, Alveolaritis, Glomerulonephritis, idiopathic thrombocytopenic purpura, juvenile diabetes, insulin dependent diabetes mellitus, leukopenia, multiple sclerosis, myasthenia gravis, paroxysmal hemoglobinuria, pernicious anemia, multiple nodular arteritis, multiple myositis, primary biliary cirrhosis, rheumatic fever, Autoimmune diseases such as rheumatoid arthritis, Sjogren's syndrome, sympathetic ophthalmitis, progressive systemic sclerosis, wedgener granulomatosis, asthma, irritable dermatitis, bronchial asthma, graft-host disease, allergic rhinitis, hay fever and bee venom allergy, allergic Diseases and viral diseases; Liver diseases such as alcoholic hepatitis, addictive hepatitis, viral cirrhosis, alcoholic cirrhosis, addictive cirrhosis, biliary cirrhosis, fatty liver, liver tumor and hepatic vascular disorders; Gallbladder / biliary diseases such as cholangitis, cholecystitis, primary sclerotic cholangitis, gallbladder tumor and cholangiocarcinoma; Pancreatic diseases such as acute pancreatitis, chronic pancreatitis, pancreatic insufficiency, pancreatic cancer and pancreatic cysts; Anemia, anemia heart disease, cerebral anemia, basal migraine, abnormal aneurysm at the bottom of the brain, cerebral hemorrhage, basal aneurysm, atherosclerosis, vascular endothelial disorder, insulin independent diabetes, intestinal vascular occlusion; Circulatory system diseases such as superior vascular artery syndrome; Nervous system diseases such as Parkinson's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, Alzheimer's disease, dementia, cerebrovascular dementia, AIDS dementia, encephalomyelitis; Digestive system diseases such as peptic ulcer, peptic ulcer, intestinal polyp, intestinal adhesion, bowel stiffness, gastric ulcer; Sleep disorders caused by mental diseases, central nervous system inhibitors, habitual alcohol, respiratory disorders, etc .; And side effects associated with hypnotic drug administration.

In the pharmaceutical field, the bioactive extract of the present invention may be prepared by the following components commonly used in this field, namely anesthetics, hypnotic sedatives, anti-anxiety agents, antiepileptic agents, antipyretic analgesics, stimulants, wake amines, antiparkinsonants, Psychoneuropathy, central nervous system, skeletal muscle relaxant, autonomic nervous system, anticonvulsant, eye drops, ear, nose drops, anticoagulant, cardiovascular, antiarrhythmic, diuretic, hypotensive, vasoconstrictor, coronary vasodilator, peripheral vasodilator, hypertrophy Liposemia, respiratory stimulant, antitussive expectorant, bronchodilator, allergic drug, anti-diarrhea agent, formal medicine, peptic ulcer drug, peptic antacid, antacid, biliary excretory, pituitary hormone, salivary gland hormone, thyroid hormone, antithyroid drug, protein Anabolic steroids, corticosteroids, androgens, estrogens, progesterones, mixed hormones, urinary / reproductive drugs, anal medicine, surgical Fungicides / disinfectants, wound protectants, purulent disease external preparations, painkillers, antiperspirants, astringents, anti-inflammatory agents, parasitic skin diseases external preparations, emollients, caustics, dental / oral medicines, vitamins, mineral preparations, supplements, hemostatic agents, anticoagulants, hepatic diseases Medicines, antidote, habitual addicts, gout medications, enzymes, diabetics, antiedemas, antihistamines, stimulants, antibiotics, chemotherapeutic agents, biological agents, antiparasitic drugs, antiprotozoal agents, various pharmaceuticals, X-ray contrast agents and An effective amount of a diagnostic agent can be added and used.

Moreover, extracts, elixirs, capsules, granules, pills, eye ointments, suspensions, emulsions, plasters, suppositories, powders, spirits, tablets, syrups, infusions, decoctions, injectables, tinctures Eye drops, troche, ointment, poultice, aromatic water, liniment, lemonade, fluid extract, lotion, nasal drop, nasal spray Or at least one of lower airway inhalants, sustained-release eye drops, oral mucosal patches, and enema. Depending on the application and the route of administration or frequency of administration, the dosage of the bioactive extract of the present invention is usually from 0.01 to 100 mg per adult per day.

The bioactive extract of the present invention is blended, kneaded, dissolved, immersed, sprayed, applied, infused, or the like, and the required amount is formulated into the desired composition before completion of the processing process. Compounds contained in the physiologically active extract of the present invention, that is, 6,12-dihydro-6,12-dioxoindolo [2,1-b] quinazoline; 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone; Camphorol;

3,5,7,4'-tetrahydroxy-6-methoxy-flavone; Gallic acid; Caffeic acid; 3- (1,3-dihydro-3-oxo-2H-indol-2-ylidene) -1,3-dihydro-2H-indol-2-one; [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid) ; And / or [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-phosphine Propanoic acid methyl ester and the like are known compounds, and their synthesis methods are also known. If the content of the ethyl acetate soluble component in the bioactive extract is below the required level, those prepared separately can be supplemented with this additive.

As described above, the physiologically active extract of the present invention has the above-mentioned good action, so that not only in the above-mentioned field, but also antimicrobial agent, antiviral agent, antitumor agent, radical scavenger, cellular necrosis inhibitor, cytokine production inhibitor and nitrogen monoxide synthesis It can be used effectively in other fields, such as an enzyme expression inhibitor.

For example, when used as an antimicrobial agent, the bioactive extract of the present invention can be advantageously used in each of the above-mentioned fields, and also can be advantageously used for pasteurized antimicrobial compositions for general daily products. If necessary in these compositions, the biologically active extracts of the present invention may be prepared using other antimicrobial agents such as propolis, ε-polysine, benzoic acid, paraoxybutyl benzoate, sodium benzoate, glycine, potassium sorbate, myconazole, ketoconazole and ethanol. , A fragrance, a pigment, a surfactant, a buffer, a metal, a metal salt, and the like can be dissolved in an appropriate amount of a solvent or a diluent and used in combination. These pasteurized antimicrobial compositions can be applied to various products such as office equipment, clothing, furniture, toys, electrical appliances, bedding, and stationery products by the method of applying, spraying, mixing, kneading, dissolving, injecting or dipping prior to the completion of product manufacturing. Can be used. In any product, the bioactive extract of the present invention exerts antimicrobial action of the required.

In the following experiments will be described the physiological action of the bioactive extract of the present invention.

Experiment 1: Preparation of Bioactive Extract

30 kg of raw ground cultivated in Aki City, Shimane, Japan, was harvested and powdered in July, and extracted three times at room temperature with 30 to 60 liters of ethyl acetate for one extraction. The obtained extracts were collected together, filtered through a filter paper, and the filtrate was recovered, and then evaporated to remove ethyl acetate and dried to obtain 168 g of an extract containing ethyl acetate soluble components derived from the side.

Experiment 2: Separation of Ethyl Acetate Soluble Component

The extract obtained by the method of Experiment 1 was suspended in 50 v / v% methanol aqueous solution, and the obtained solution was divided into 8 parts, and then 1700 ml of "FS-1830" (Adsorption Chromatography, sold by Japan Organo, Japan). Solvent gel) and 60 v / v%, 70 v / v%, 80 v / v% and 90 v / v% aqueous methanol solution, methanol and ethyl acetate were used as the eluent in this order. Elution was carried out from the column by feeding in equal amounts.

The first and later fractions eluted from the column with 80 v / v% aqueous methanol solution are called fraction 2 and fraction 1, respectively; The later fraction eluted from the column with 60 v / v% aqueous methanol solution is called fraction 3; The later fraction eluted from the column with 90 v / v% aqueous methanol solution was referred to as fraction 4; The first and later fractions eluted from the column with methanol were fractions 5 and 6, respectively, and these fractions were each evaporated to remove the solvent and dried to obtain a solid.

In 5.2 g of solid obtained from fraction 1, 2.6 g was suspended in 30 ml of methanol and filtered off with suction to separate the soluble fraction and the insoluble fraction (876 mg). The soluble fraction was added to a column filled with 1350 ml of silica gel and eluted from the column by a straight slope of the solvent system of methanol and chloroform with a stepwise increase from methanol concentration of 5 v / v% to 100 v / v%, 450 ml of eluted fraction. Was recovered. The fourth fraction was evaporated to remove the solvent to give a solid, which was then suspended in 2 ml of methanol and the suspension was washed with suction under filtration to give 19.4 mg of the crystals of compound 1. The above insoluble fraction was dissolved in an appropriate amount of methanol and left at room temperature to obtain 278 mg of yellow needles of Compound 2.

10.7 g of solid obtained from fraction 2 were suspended in 40 ml of methanol, and this solution was added to a column filled with 1,520 ml of "SEPHADEX LH-20" (gel for adsorption chromatography from Pharmacia LKB Biotechnology AB, Sweden) and methanol 190 ml of the eluate was recovered while eluting from the column. For each of the 15th and 18th fractions, the solvent was removed by evaporation and dried to give a solid. 400 mg of solids from the 18th fraction were completely dissolved in about 210 ml of a solvent system of methanol and water (= 5: 2 volume ratio). The solution was filtered through a 0.22 μm membrane filter, and the filtrate was left at room temperature for 3 days to precipitate crystals. The resulting mixture was filtered with a conventional filter paper to recover 89.3 mg of the crystals of compound 3. 500 mg of the solid obtained in the 15th fraction was suspended in 10 ml of methanol and filtered to recover insoluble matters, and then 250 ml of methanol was added thereto and completely dissolved in methanol. The solution was filtered through a 0.22 μm membrane filter, left at room temperature for 7 days to precipitate crystals, and then filtered through a filter paper commonly used to obtain 252.3 mg of the crystals of Compound 4.

11.8 g of solid obtained from fraction 3 were suspended in 40 ml of methanol, and the solution was added to a column packed with 1680 ml of "SEPHADEX LH-20" (gel for adsorption chromatography from Pharmacia LKB Biotechnology AB, Sweden), 560 ml of an elution fraction was collect | recovered, eluting from a column with methanol. The fourth fraction was evaporated to remove the solvent and dried to give a solid. This solid was dissolved in 10 ml of methanol, and this solution was added to a column packed with 480 ml of "FS-1830" (gel for adsorption chromatography sold by Japan Organo, Japan). 480 ml of 20, 30, 40, 50, 60 and 70 v / v% methanol aqueous solutions were added, respectively, and again eluted with 960 ml of methanol. The eluate was fractionated to obtain 240 ml of fractions, and the fifth fraction was evaporated to remove the solvent and dried to obtain 150 mg of solids.

The obtained solid was dissolved in 300 ml of ethyl acetate and the solution was filtered through a 0.22 μm membrane filter. The filtrate was left at room temperature for 7 days to precipitate crystals, and the mixture was filtered through a commonly used filter paper to obtain 18.7 mg of crystals of compound 5. The 9th fraction eluted from the "FS-1830" packed column was evaporated to remove the solvent and dried to obtain 500 mg of solid, which was dissolved in 0.5 ml of ethyl acetate and 40 ml of "SILICAGEL 60K650" (Japan Was added to a column packed with a mixture of ethyl acetate and chloroform (= 2: 8, 3: 7, 4: 6, 5: 5, 6: 4). , 7: 3, 8: 2 and 9: 1 (volume ratio), and 40 ml of chloroform and methanol were added in this order. 10 ml of the eluate from the column was fractionated to collect the 17th and 21st fractions, and the "SILICAGEL 60F ₂₅₄ " (thin layer for separation from Merck, USA) and toluene, ethyl acetate and acetic acid (5: 5: 1 as developer) And volume layer chromatography treatment using the liquid mixture of (volume ratio). After development, a portion of the silica gel at a position of Rf of about 0.6 was recovered and extracted with an appropriate amount of methanol to obtain the developed material. The extract was evaporated to remove the solvent and dried to give 15.4 mg of crystals of compound 6.

Of 4.6 g of solid obtained from fraction 4, 4 g was dissolved by mixing with 40 ml of methanol, but some did not dissolve but formed a precipitate. The precipitate was recovered, and 800 ml of methanol was added thereto, followed by sufficient dissolution. The mixture was left at room temperature for 2 days to precipitate a red crude crystal. This crude crystal was recovered, and the supernatant was left under the same conditions as above to precipitate another red crude crystal. The new crystals were collected and combined with the previously obtained crystals, washed with an appropriate amount of methanol, and dissolved in a sufficient amount of methanol. The obtained solution was filtered by a conventional method, the filtrate was evaporated to remove the solvent and dried to give 44.5 mg of the crystal of Compound 7.

Exact data are not available, but fraction 5 was treated in column "FS-1830" and column "SILICAGEL 60K650" in the same manner as for fraction 3, and crystallization in methanol afforded crystals of compound 8. Then, fraction 6 was treated in the column of "FS-1830" and crystallized in acetonitrile in the same manner as applied for fraction 3, thereby obtaining crystals of compound 9.

Experiment 3: Identification of Compound 1

Experiment 3-1: Mass Spectrometry

Compound 1, obtained by the method of Experiment 2, obtained peaks at m / z 249 ([M + H] ⁺ ) when the mass spectrometry was measured by high-speed atomic shock mass spectrometry (hereinafter simply referred to as "FAB-MS"). And peaks in m / z 249.0694 ([M + H] ⁺ ) as measured by high resolution mass spectrometry.

Experiment 3-2: Nuclear Magnetic Resonance Absorption Spectrum

Nuclear magnetic resonance absorption by Compound 1 obtained by the method of Experiment 2 by ¹ H-nuclear magnetic resonance spectroscopy and ¹³ C-nuclear magnetic resonance spectroscopy (hereinafter, simply referred to as ¹ H-NMR and ¹³ C-NMR, respectively) The spectrum was measured.

The chemical shifts and the assignments of hydrogen and carbon atoms observed in each spectrum are shown in Table 1.

        Chemical shift δ (ppm)     belonging ¹ H-NMR 8.48 (1H, d, J = 7.9 Hz) 8.32 (1H, d, J = 7.7 Hz) 7.95 (2H, J = 3.7 Hz, 9.2 Hz) 7.88 (1H, d, J = 7.3 Hz) 7.87 (1H, t, J = 7.9 Hz) 7.74 (1H, m, J = 4.2 Hz) 7.48 (1H, t, J = 7.5 Hz)    H-10 H-1 H-3, H-4 H-7 H-9 H-2 H-8 ¹³ C-NMR 182.4 157.6 146.4 145.9 144.9 137.7 135.1 129.8    C-6 C-12 C-5a C-4a C-10a C-9 C-3 C-4         129.8 126.9 126.8 124.7 123.2 122.2 117.0   C-2 C-1 C-8 C-7 C-12a C-6a C-10

Note: measured on DMSO-d6

Based on experimental data, Compound 1 as an ethyl acetate soluble component from the side was prepared using 6,12-dihydro-6,12-dioxoindolo [2,1-b] quinazoline [tryptanthrin, C _15. H ₈ N ₂ O ₂ , MW = 248]. The structure of compound 1 is as follows.

Experiment 4: Identification of Compound 2

Experiment 4-1: Melting Point

The melting point of Compound 2 obtained by the method of Experiment 2 was measured by a conventional method, and the melting point was 298 ° C.

Experiment 4-2: UV Absorption Spectrum

Ultraviolet absorption spectra of Compound 2 obtained by the method of Experiment 2 were measured using methanol as a solvent in a conventional manner. As a result, maximum absorption spectra were shown at wavelengths 206, 240, 273 and 353 nm.

Experiment 4-3: Infrared Absorption Spectrum

1 is an infrared absorption spectrum of Compound 2 measured by a pressure tablet method using potassium bromide powder.

Experiment 4-4: Mass Spectrometry

The mass spectra of the compound 2 obtained by the method of Experiment 2 were measured by FAB-MS, and showed a peak at m / z 314 (M ⁺ ).

Experiment 4-5: Nuclear Magnetic Resonance Absorption Spectrum

For the compound 2 obtained by the method of Experiment 2, nuclear magnetic resonance absorption spectra by ¹ H-NMR and ¹³ C-NMR were measured.

The chemical shifts of each signal observed in each spectrum and the attribution of hydrogen and carbon atoms are shown in Table 2.

     Chemical shift δ (ppm)      belonging ¹ H-NMR 8.06 (2H, d, J = 9 Hz) 6.94 (2H, d, J = 9 Hz) 6.90 (1H, s)    H-2 ', H-6' H-3 ', H-5' H-8 ¹ H-NMR 6.15 (2H, s) ¹³ C-NMR 176.2 159.3 153.7 151.4 147.4 139.8 135.8 129.4 128.7 121.4 115.4 105.8 102.6 89.3 O-CH ₂ -O C-4 C-4 'C-7 C-9 C-2 C-5 C-3 C-2', C-6 'C-6 C-1' C-3 ', C -5 'C-10 O-CH ₂ -O C-8

Note: measured on DMSO-d6

Experiment 4-6: Elemental Analysis

Compound 2 obtained by the method of Experiment 2 was subjected to conventional elemental analysis, and C = 59.2%, H = 3.4%, O = 37.4%, and N <0.3%, from which Compound 2 was represented by Equation C ₁₆ H ₁₀ O _It was found that it was ₇ · 3 / 5H ₂ O.

Based on this data, compound 2, an ethyl acetate soluble component from the side, was identified as 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone (C ₁₆ H ₁₀ O ₇ , molecular weight = 314) It became. The structure of compound 2 is shown in the formula (2) below.

Experiment 5: Identification of Compound 3

Experiment 5-1: thin layer chromatography

Compound 3 obtained by the method of Experiment 2, chlorogenic acid, quercetin, camphorol, ferulic acid, cinnamic acid, kumaric acid, galangin, pinosembrine (all of which were purchased from Sigma Chemical of the United States) Conventional thin layer chromatography treatment. "KIESELGEL 60F ₂₅₄ " (Sigma Chemical, USA) was used as a thin plate, and a mixed solution of toluene, ethyl acetate and acetic acid (= 8: 1: 1, volume ratio) was used as the developing solvent system. After development, the samples on the plates were colored by ultraviolet irradiation at a wavelength of 254 nm. As a result, Rf of Compound 3 was in good agreement with Rf of camphorol, a flavonoid.

Experiment 5-2: melting point

Melting | fusing point was measured with the conventional method about the compound 3 obtained by the method of the experiment 2, and the melting point was 277 degreeC.

Experiment 5-3: ultraviolet absorption spectrum

Ultraviolet absorption spectra of Compound 3 obtained by the method of Experiment 2 were measured using methanol as a solvent in a conventional manner, and showed maximum absorption spectra such as shoulders at wavelengths 265, 365 and 320 nm.

Experiment 5-4: Infrared Absorption Spectrum

2 is an infrared absorption spectrum of Compound 3 measured by a pressurized tablet method using potassium bromide powder.

Based on these data, Compound 3, an ethyl acetate soluble component from the side, was identified as camphorol having the formula C ₁₅ H ₁₀ O ₆ and molecular weight 286. The structure of Compound 3 is shown in Formula 3 below.

Experiment 6: Identification of Compound 4

Experiment 6-1: Melting Point

As a result of measuring the melting point of Compound 4 obtained by the method of Experiment 2 by the conventional method, the melting point of this compound was 272 ° C.

Experiment 6-2: ultraviolet absorption spectrum

Compound 4 obtained by the method of Experiment 2 was prepared according to a conventional method, in which methanol was used as a solvent 1, sodium methylate-containing methanol as solvent 2, anhydrous aluminum chloride-containing methanol as solvent 3, or methanol containing anhydrous aluminum chloride and hydrochloric acid as solvent 4. The ultraviolet absorption spectrum was measured using a solvent. The maximum absorption spectra in each solvent system are shown in Table 3.

  Solvent          Absorption (nm)   Solvent 1    255sh, 268, 355sh, 365   Solvent 2    273, 320, 404   Solvent 3    270, 305sh, 365sh, 425   Solvent 4    270, 305sh, 365sh, 425

Note: The symbol "sh" indicates maximum absorption, such as shoulders.

From the results in Table 3 it can be seen that Compound 4 belongs to a flavonol having a hydroxyl group at C-3, C-7 and C-4 'or C-3, C-5, C-7 and C-4'. have.

Experiment 6-3: Nuclear Magnetic Resonance Absorption Spectrum

Nuclear Magnetism by ¹ H-NMR and ¹³ C-NMR for Compound 4 Obtained by Experiment 2

The resonance absorption spectrum was measured. The chemical shifts observed in each spectrum and the respective attributions of hydrogen and carbon atoms are shown in Table 4.

      Chemical shift δ (ppm)      belonging ¹ H-NMR 8.08 (2H, d, J = 8.97 Hz) 6.90 (2H, d, J = 8.97 Hz) 6.49 (1H, s) 3.88 (3H, s) H-3 ', H-5' H-2 ', H-6' H-8 -O-CH ₃ ¹³ C-NMR 177.7 160.7 158.8 153.8 153.0 148.4 136.9 132.4 130.8 123.8 116.4 105.0 94.9 61.0 C-4 C-4 'C-7 C-5 C-9 C-2 C-3 C-6 C-2', C-6 'C-1' C-3 ', C-5' C-10 `` C-8 -O-CH ₃

Note: Determination in Deuterated Methanol

Experiment 6-4: Mass Spectrometry

Mass 4 spectra of Compound 4 obtained by the method of Experiment 2 were measured by electron ionization mass spectrometry (EI-MS) in a conventional manner, and the peaks were obtained at m / z 316 (M ⁺ ). The peak was shown at m / z 316.0486 (M ⁺ ) by the assay.

Experiment 6-5: infrared absorption spectrum

3 is an infrared absorption spectrum of Compound 4 measured by pressure tablet method using potassium bromide powder.

Based on this data, Compound 4, an ethyl acetate soluble component from the side, is 3,5,7,4'-tetrahydroxy-6-methoxy-flavone having the formula C ₁₆ H ₁₂ O ₇ and a molecular weight of 316. Confirmed. The structure of compound 4 is shown in Formula 4 below.

Experiment 7: Identification of Compound 5

Experiment 7-1: mass spectrometry

The mass spectrum of Compound 5 obtained by the method of Experiment 2 was measured by electron ionization mass spectrometry (EI-MS) in a conventional manner, and the peak was obtained at m / z 170 (M ⁺ ).

Experiment 7-2: nuclear magnetic resonance absorption spectrum

The nuclear magnetic resonance absorption spectrum of compound 5 obtained by the method of Experiment 2 was measured by ¹ H-NMR and ¹³ C-NMR. The chemical shifts observed in each spectrum and the attribution of hydrogen and carbon atoms are shown in Table 5.

   Chemical shift δ (ppm)       belonging ¹ H-NMR 7.06 (2H, s)     H-2, H-6 ¹³ C-NMR 170.4 146.5 139.6 122.1 110.4     C-7 C-3, C-5 C-4 C-1 C-2, C-6

Note: Determination in Deuterated Methanol

Based on this data, the compound 5 which is an ethyl acetate soluble component derived from a side was identified as gallic acid having a chemical formula of C ₇ H ₆ O ₅ and a molecular weight of 170. The structure of compound 5 is shown in Formula 5 below.

Experiment 8: Identification of Compound 6

Experiment 8-1: Mass Spectrometry

About compound 4 obtained by the method of Experiment 2, the mass spectrum was measured by the electron ionization mass spectrometry (EI-MS) by a conventional method, and the peak was shown at m / z 180 (M ⁺ ).

Experiment 8-2: infrared absorption spectrum

4 is an infrared absorption spectrum of Compound 6 measured by pressure tablet method using potassium bromide powder.

The spectrum of compound 6 was in good agreement with the spectrum of caffeic acid compared to the infrared absorption spectrum of known compounds.

Experiment 8-3: Nuclear Magnetic Resonance Absorption Spectrum

For the compound 6 obtained by the method of Experiment 2, nuclear magnetic resonance absorption spectra were measured by ¹ H-NMR and ¹³ C-NMR. The chemical shifts observed in each spectrum and the attribution of hydrogen and carbon atoms are shown in Table 6.

         Chemical shift δ (ppm)   belonging ¹ H-NMR 7.43 (1H, d, J = 15.9 Hz) 7.03 (1H, d, J = 2.0 Hz) 6.91 (1H, dd, J = 2.0 Hz, 8.3 Hz) 6.77 (1H, d, J = 8.1 Hz ) 6.27 (1H, d, J = 15.6 Hz)    H-7 H-2 H-6 H-5 H-8 ¹³ C-NMR 148.9 146.8 144.7 128.7 122.4 119.2 116.5 115.1    C-4 C-3 C-7 C-1 C-6 C-8 C-5 C-2

Note: Determination in Deuterated Methanol

Based on this data, the compound 6 which is an ethyl acetate soluble component derived from a side was confirmed to be caffeic acid having a chemical formula of C ₉ H ₈ O ₄ and a molecular weight of 180. The structure of Compound 6 is shown in Formula 6 below.

Experiment 9: Identification of Compound 7

Experiment 9-1: Mass Spectrometry

Electron ionization quality in a conventional manner with respect to compound 7 obtained by the method of Experiment 2

The mass spectrum was measured by mass spectrometry (EI-MS), and the peak was obtained at m / z 262 (M ⁺ ).

Indicated.

 Experiment 9-2: infrared absorption spectrum

5 is an infrared absorption spectrum of Compound 7 measured by pressure tablet method using potassium bromide powder.

Compared with the infrared absorption spectrum of known compounds, the spectrum of compound 7 is 3- (1,3-dihydro-3-oxo = 2H-indole-2-ylidene) -1,3-dihydro-2H-indole- Good agreement with 2-one.

Experiment 9-3: Nuclear Magnetic Resonance Absorption Spectrum

The nuclear magnetic resonance absorption spectrum of compound 7 obtained by the method of Experiment 2 was measured by ¹ H-NMR and ¹³ C-NMR. The chemical shifts observed in each spectrum and the attribution of hydrogen and carbon atoms are shown in Table 7.

            Chemical shift δ (ppm)     belonging ¹ H-NMR 11.00 (1H, s) 10.87 (1H, s) 8.77 (1H, d, J = 7.81 Hz) 7.66 (1H, d, J = 7.57 Hz) 7.58 (1H, t, J = 8.06, 8.30 Hz ) 7.42 (1H, d, J = 8.06 Hz) 7.26 (1H, t, J = 7.73 Hz, 7.57 Hz) 7.02 (2H, t, J = 7.57 Hz, 7.32 Hz) 6.91 (1H, d, J = 7.57 Hz )    H-1 H-1 'H-4' H-4 H-6 H-7 H-6 'H-5, H-5' H-7 ' ¹³ C-NMR 188.53 170.86 152.42 140.83 138.27 137.01 129.19 124.59 124.27 121.39 121.18 118.97 113.35 109.49 106.51    C-3 C-2 'C-6' C-6 C-4 'C-4 C-5, C-5' C-7 'C-7

Note: measured on DMSO-d6

Based on this data, Compound 7, an ethyl acetate soluble component from the side, has 3- (1,3-dihydro-3-oxo-2H-indole-2 having the formula C ₁₆ H ₁₀ N ₂ O ₂ and a molecular weight of 262. -Ylidene) -1,3-dihydro-2H-indol-2-one, ie indirubin. The structure of compound 7 is shown in formula 7 below.

Compound 8 was identified in the same manner as in the identification of compounds 1 to 7, and found to be [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (having a chemical formula of C ₃₅ H ₃₆ N ₄ O ₅ and a molecular weight of 592.69. Methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid), i.e., pheophorbide a.

In addition, Compound 9 was identified and found to be [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4, having the formula C ₃₆ H ₃₈ N ₄ O _5, and a molecular weight of 606. It was found to be 8,13,18-tetramethyl-20-oxo-3-phosphinepropanoic acid methyl ester, ie methylfeophoride a. The structures of compound 8, which is pheoformide a, and compound 9, which is methylfeophoride, are shown in Formulas 8 and 9 below.

Experiment 10: Antimicrobial Activity

The minimum inhibitory concentrations (MIC) were examined for the bacteria of Table 6 by agar plate dilution using a streak smear for the bioactive extracts obtained by the methods of Experiments 1 and 2 as shown in Table 8 below. Brucella culture broth containing agar 1.5 w / v%, glucose 0.1 w / v%, and bacteria-free fibrin depleted horse blood 7 v / v% was used as nutrient medium for Helicobacter pylori (NCTC 11638). For other bacteria, conventional media for sensitive discs were used. The results are shown in Table 8.

         Pathogens             Minimum Stop Concentration (μg / mL)     A     B     C    D     E  Helicobacter pylori (NCTC 11638)   156    2.5   156   39.0   39.0  Bacillus cereus (IF0 3466)   156   39.0   313     -     -  Schudomonas Eruginosa (IFO 3453)    78   39.0   313     -     -  Staphylococcus Oreus (ATCC 6538P)   313   39.0   313     -     -

Note: The sign "-" indicates no minimum stop concentration test.

      Symbols A, B, C, D and E represent the bioactive extract of Experiment 1, Compound 1 of Experiment 2, Compound 2 of Experiment 2, Compound 3 of Experiment 2 and Compound 4 of Experiment 2, respectively.

From the results shown in Table 8 it can be seen that the bioactive extract of the present invention inhibited the growth of Gram-positive bacteria and Gram-negative bacteria. These extracts, and in particular, compounds 1, 3 and 4, strongly inhibited the growth of Helicobacter pylori, a pathogen of gastritis, gastric ulcer, duodenal ulcer and gastric cancer.

Experiment 11: Antiviral Activity

According to a conventional method, FL cells (ATCC CCL62), which are chemotactic cells derived from amnion tissue of normal humans, were cultured in a single plate on a microplate. Culture supernatants were removed from the microplates and hydrophobic stomatitis virus (VSV) was adsorbed to monolayer cells at a rate of 0.1 plaque forming units (PFU) per cell, and then these cells were obtained as described below in the methods of Experiments 1 and 2. The bioactive extracts shown in Table 9 were dissolved in dimethyl sulfoxide (DMSO) at different concentrations, incubated at 37 ° C. for 24 hours, and then disrupted by repeated freezing and thawing of FL cells in a microplate. The culture supernatant containing VSV was obtained by centrifuging this microplate.

Thereafter, L929 cells derived from mouse fibroblasts (RCB0081) were used as target cells, and the antiviral activity of each sample was examined according to a conventional method using cytopathic effect by virus (CPE) as an indicator. 50% growth inhibition concentration of each bioactive extract was calculated. The results are shown in Table 9.

          sample   50% growth inhibition concentration (μl / ml)   Bioactive Extract of Experiment 1             23   Compound 1 of Experiment 2             13   Compound 2 of Experiment 2             14

As shown in Table 9, the bioactive extract of the present invention showed antiviral action against pathogenic viruses. Under virus-free conditions, FL cells were cultured by conventional methods in the presence or absence of bioactive extracts at 50% growth inhibition concentration in the irradiation described above. As a result, no significant difference was found in the growth and proliferation of the cells under each condition. The herpes simplex virus (HSV-1), influenza virus, head virus (VV) and mouse cytomegalovirus of the physiologically active extracts of the present invention by the above-mentioned method using CPE as an indicator or conventional methods using plaque formation as an indicator ( Examination of antiviral action against MCMV) confirmed that the bioactive extracts of the present invention had antiviral action against these pathogenic viruses at least at the same level as found in VSV. These data show that the bioactive extracts of the present invention exert potent antiviral action against pathogenic viruses in animals and humans.

Experiment 12: Antitumor Activity

As shown in Table 10 below, RPMI1640 medium supplemented with 10 v / v% fetal bovine serum and dissolved in DMSO to a concentration of 10 mg / ml using any one of the bioactive extracts obtained by the methods of Experiments 1 and 2 as a sample. Diluted 50 times (pH 7.2) and divided into 96-well microplate 100 μl per well. The solution in each well was serially diluted with the same fresh RPMI1640 medium (pH 7.2) prepared as above. Separately, HL-60 cells from human acute myeloid leukemia patients (ATCC CCL-240), HGC-27 cells from gastric cancer patients (RCB0500) and HLC-1 cells from lung adenocarcinoma patients (RCB0083) were used in the stomach. The same freshly prepared fresh RPMI1640 medium (pH 7.2) was suspended to have cell concentrations of 4 × 10 ⁵ / ml, 4 × 10 ⁵ / ml and 2 × 10 ⁶ / ml, respectively.

Each cell suspension was divided into 50 μl of each well in a microplate and incubated for 48 hours at 37 ° C. in a 5 v / v% CO ₂ incubator. 20 microliters of 25 v / v% glutaraldehyde aqueous solution was added to each microplate, and left for 15 minutes to fix the cells. Subsequently, the cells attached to each well were washed with water, and 100 μl of each well of 0.05 w / v% methylene blue solution was added thereto, mixed, and left for 15 minutes to stain the cells. Thereafter, excess dye solution was washed with water to remove from each well. After drying, the cells were mixed with 300 μl of 0.33 N hydrochloric acid per well, mixed well, and the absorbance was measured at a wavelength of 620 nm.

In parallel, a sample-free system was prepared as a control and treated in the same manner as in the sample. The 50% growth inhibition concentration (IC ₅₀ ) of each sample was used as an indicator of antitumor activity, and the IC ₅₀ of each sample was calculated with the control cell growth as 100%. The results are shown in Table 10.

   sample        50% growth inhibition concentration (μl / ml)   HL-60 cells   HGC-27 cells   HLC-1 cells     A    24.9     61.5    276.8     B     4.2      1.5      2.2     C   243.7     37.1     40.4

Note: Symbols A, B and C represent the bioactive extract of Experiment 1, Compound 1 of Experiment 2 and Compound 2 of Experiment 2, respectively.

From the results of Table 10, it can be seen that the bioactive extract of the present invention effectively inhibits the growth of tumor cells of leukemia, gastric cancer and lung cancer known as refractory malignancies. In particular, compound 1 exhibited at least 10-fold antitumor activity than compound 2. From these data, it can be seen that the bioactive extract of the present invention has a therapeutic / prophylactic effect on malignant tumors of mammals and humans.

Experiment 13: Radical Capture

As shown in Table 11, the radical scavenging action of the bioactive extracts obtained by the methods of Experiments 1 and 2 is described in Toshio IMANARI in Igaku-no-Ayumi (Development of Medical Science), Vol. 101, pp. 496-497 (1977), and evaluated by the nitro blue tetrazolium (NBT) method. That is, the sample coexisted in a reaction system in which xanthine oxidase was acted on xanthine to generate an excess oxide and a reaction system in which the generated excess oxide converts NBT into formazan by oxidizing power. The resulting formazan was then quantified by spectroscopic analysis.

Any one of the physiologically active extracts of the present invention was dissolved in purified water at a suitable concentration, and this solution was used as a sample. Purified water was used instead of the sample as a control. 50% inhibitory activity on formazan production in the control was defined as 1 unit activity of radical scavenging. Based on this definition, the radical scavenging action of 1 g of each sample was investigated. The results are shown in Table 11.

         sample   Radical scavenging action (unit / g)   Bioactive Extract of Experiment 1           27,800   Compound 1 of Experiment 2             -   Compound 2 of Experiment 2            1,830   Compound 3 of Experiment 2           14,000   Compound 4 of Experiment 2            4,010   Compound 5 of Experiment 2          692,000   Compound 6 of Experiment 2          418,000

Note: The sign "-" indicates that the radical trapping could not be evaluated because the sample was not sufficiently dissolved in water.

As shown in Table 11, the bioactive extract of the present invention exhibits a strong radical trapping action. In particular, the radical scavenging action of the bioactive extract of Experiment 1 and the compounds 5 and 6 of Experiment 2 was remarkable. From this, the bioactive extract of the present invention has the property of effectively capturing free radicals derived from free radicals and fat peroxide, and also malignant tumor, myocardial infarction, cerebral hemorrhage, rheumatism, various diseases related to lifestyle or geriatric disease, stress and It can be seen that the treatment / prevention effect for various diseases related to the in vivo radicals, such as aging. Compound 1 lacked in water solubility and evaluation of radical scavenging by the method of Experiment 13 was not possible.

Experiment 14: Inhibition of Cellular Necrosis

Compound 1 obtained by the method of Experiment 2 was dissolved in DMSO to a concentration of 0.8 mg / ml, and diluted with RPMI1640 medium (pH 7.2) supplemented with 10 v / v% fetal bovine serum to obtain a 20 µg / ml solution. The solution was divided into 1 ml of each well into the Michaeloplate, and the solution of each well was serially diluted with the same fresh medium prepared as that used above. HL-60 cells from acute myeloid leukemia patients (ATCC CCL-240), U-937 cells from human histocytic lymphoma patients (ATCC CRL-1593.2), HGC-27 cells from gastric cancer patients (RCB0500) HLC-1 cells from lung adenocarcinoma patients (RCB0083) and C ₆ cells from rat glial tumors (ATCC CCL-107) with cell density of 1 × 10 ⁶ / ml or 2 × 10 ⁵ / ml Each was suspended in RPMI1640 medium (pH 7.2) supplemented with 10 v / v% fetal calf serum. One well of the above-described cell suspension was added to each well of the Michaeloplate containing 1 ml of Compound 1 diluted to 20, 10 or 5.0 μg / ml, and used as a test system. All test systems were made two and incubated for 24 h and 48 h, respectively, in a 37 ° C. 5 v / v% CO ₂ incubator. A system free of compound 1 was prepared and treated in the same manner as above to serve as a control. I. Nicoletti et al. in Journal of Immunlogical Methods, Vol. 139, pp. 271-279 (1991), according to the method described in the present invention, the cells of the test and control cells induced with natural spontaneous necrosis were stained with propidium iodide so that the percentage of the stained cells relative to the total cell number was determined in flow cytometry. Obtained by That is, each culture was transferred to a polypropylene tubing and centrifuged to remove the resulting supernatant, and the cell precipitate was washed with phosphate-salt buffer containing 0.3 v / v% bovine serum albumin, followed by washing. The cells were centrifuged again to remove the supernatant. 50 µg / ml of propidium iodide (sold by Sigma Chemical, USA) and 0.1 w / v% sodium citrate and 0.1 w / v% “TRITON X-100” (Sigma Chemical, USA) Activator) and the cells were mixed with 1.5 ml each and stained with propidium iodide. The stained cells were stored overnight at 4 ° C. in the dark, and then the stained cells of each test system were analyzed with “EPICS PROFILE II”, a flow cytometer sold by Beckman Coulter of the United States. Based on the data obtained, the percentage of stained cells relative to the total cell number in each test system was determined as the cell natural necrosis expression rate. The results are shown in Table 12.

Cell line ^*  Incubation time (hours)           Expression rate in cellular necrosis (%)   contrast      Compound 1 (Concentration) in Experiment 2   2.5 μg / ml   5.0 µg / ml   10 µg / ml HL-60 cells (5 × 10 ⁵ )     24 48   0.7 1.5     0.7 1.3     1.7 4.2    11.8 22.6 U-937 cells (5 × 10 ⁵ )     24 48   3.2 4.5     3.2 5.2     5.0 11.7     6.8 66.9 HGC-27 cells (1 × 10 ⁵ )     24 48   1.5 1.4     1.9 2.2     1.7 4.1     3.5 14.1 HLC-1 cells (1 × 10 ⁵ )     24 48   2.7 1.5     3.7 5.8     4.2 7.3     8.1 21.8 C ₆ cells (1 × 10 ⁵ )     24 48   0.3 0.4    15.9 38.4    18.4 64.8    27.6 67.1

The symbol "*" indicates the initial cell density (dog / ml) in each culture

As shown in Table 12, the expression rate of spontaneous necrosis in each cell system increases with the dose of the bioactive extract of the present invention. In particular, the difference between each sample and control showed a maximum level at 10 μg / ml dose of each extract. The bioactive extract of the present invention was remarkably effective in inducing cellular necrosis in U-937 cells derived from lymphoma and C ₆ cells derived from glial tumors in the cell line. Tumorization of cells is said to occur because natural necrosis, which should occur in the cell, is not caused by several factors. In this experiment, remarkable cellular necrosis was observed in cells cultured in the presence of the physiologically active extract of the present invention, suggesting that the extract has an effect of suppressing natural necrosis of living cells in a normal state. Although the data are not shown, the same results were obtained as in the case of Compound 1 when the biologically active extracts other than Compound 1 obtained by the methods of Experiments 1 and 2 were tested by the above-described method.

Experiment 15: cytokine production regulation

Compound 7 in Experiment 2 was dissolved in DMSO to a concentration of 1 mg / ml, RPMI1640 medium (pH 7.2) supplemented with 10 v / v% fetal bovine serum (hereinafter referred to simply as "serum medium" in Experiment 15). After dilution, the concentration was 50 ng / ml, and then serially diluted with serum medium. HBL-38 cells as immune cells were cultured and grown in serum medium to a predetermined cell density. The HBL-38 cells grown after incubation were washed three times by centrifugation with RPMI1640 medium (pH 7.2) (hereinafter simply referred to as "serum-free medium" in Experiment 15), followed by cell density using serum-free medium. Adjust to 1 × 10 ⁸ / ml. To 1 ml of cell suspension, 4.5 ml of serum-free medium and 0.5 ml of a dispersion prepared by dissolving dispase (produced by Godo Shusei of Japan) in a physiological saline solution at a concentration of 10,000 units / ml were added. Incubate with shaking for 90 minutes at. Thereafter, HBL-38 cells were washed three times by centrifugation with serum medium, and serum medium was added thereto to obtain a cell suspension having a cell density of 1 × 10 ⁶ cells / ml.

50 μl of one of the above dilutions of Compound 7 was taken and 50 μl of serum-free medium containing 150 μl of the above cell suspension of dispase treated HBL-38 cells and 5 μg / ml of lipopolysaccharide (LPS). Was added to each well of the microplate and then incubated for 24 hours at 37 ° C. in a 5 v / v% CO ₂ incubator as the test group. In parallel with this, a system consisting of a serum-free medium instead of a serum-free medium containing LPS of the test group as a negative control and a serum medium instead of a dilution of Compound 7 of the test group as a positive control were prepared. The treatment was performed as in the test group. After 24 hours of incubation, 50 μl of supernatant was taken from each well. The collected supernatants were each treated with conventional enzymatic immunoassays using Gg23-901-530 (obtained from the National Institute of Health, USA) as a standard for human interferon-γ to generate interferon-γ production in each system. Quantitative analysis. 50 μl of ³ H-thymidine solution with 5 μCi / ml radiation intensity was added to the cells of each well from which the supernatant was removed, and then incubated for 8 hours at 37 ° C. in a 5 v / v% CO ₂ incubator. Bait. Subsequently, the cells of each well of the microplates were collected with a glass filter, and the radiation intensity was measured by "DIRECT β-COUNTER-MATRIX 96", a β-ray detector sold by Packard Instrument of the United States of America. ³ H-thymidine uptake was examined. The results are shown in Table 13. In Table 13 the levels of ³ H-thymidine uptake are expressed as relative values (%) relative to the positive control.

Concentration of Compound 7 in Experiment 2 (ng / ml)  Interferon-γ Production (IU / ml) Relative value of ³ H-thymidine absorption (%) ^*  Voice control         0       1.367 - ^**  Training         0       5.468           100      Test group         0.07 0.15 0.31 0.62 1.25 2.5 5.0 10.0       2.753 2.195 1.979 1.958 1.288 1.342 0.932 0.965           98.0 98.3 99.7 96.4 99.6 105.7 101.1 99.4

Note: The sign "*" indicates the percentage by measurement in the positive control.

The symbol "**" indicates that ³ H-thymidine uptake was not measured.

As shown in Table 13, LPS induced interferon- [gamma] production by HBL-38 cells, and compound 7, a bioactive extract of the present invention, inhibited interferon- [gamma] production regardless of the dose. Under the condition that the concentration of compound 7 was at least 1.25 ng / ml, interferon- [gamma] production was inhibited at a level equal to or lower than that of the negative control without LPS. No significant difference was found between the control and test groups in the rate of ³ H-thymidine uptake by HBL-38 cells. From these results, it can be seen that the bioactive extract of the present invention does not affect the proliferation of immune cells and strongly inhibits interferon-γ production of cells induced by foreign bodies such as LPS. Instead of HBL-38 cells, mouse spleen cells as immune cells were prepared in a conventional manner and treated in the same manner as above to investigate the production of interleukin 10 in addition to the production of interferon-γ. As a result, it was observed that the bioactive extract of the present invention inhibits the production of interferon-γ and increases the production of interleukin 10.

The helper T cells in the immune system in vivo are composed of cell groups including Th1 and Th2, and the balance between Th1 and Th2 greatly affects the expression of the immune response. For example, diseases such as various autoimmune diseases including autoimmune and inflammatory diseases are caused when the balance is out of the normal state of the living body and Th1 becomes dominant in the living body. As discussed above, interferon-γ and interrokin 10 inhibit equilibrium so that the latter cytokines do not tend to become Th1 dominant while the latter cytokines regulate equilibrium for Th1 dominant states. Known. According to these experimental data, the physiologically active extract of the present invention regulates the in vivo equilibrium by regulating cytokine production by cells in charge of immune cells, and effectively treats / prevents various diseases caused by the abnormalities of the equilibrium. Suggests doing. Since interferon- [gamma] is known as an inflammatory cytokine, the above data also suggest that the bioactive extract of the present invention can be used as an inhibitor for the production of such cytokines.

Although no data is presented, it was confirmed that the remaining bioactive extracts of the present invention in Experiments 1 and 2 exhibited a similar effect to Compound 7 even if their activity was changed.

From the results of Experiments 1 to 15, the bioactive extract of the present invention as an ethyl acetate soluble component obtained by the method of Experiment 1, namely 6,12-dihydro-6,12-dioxoindolo [2,1-b] quina Sleepy; 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone; Camphorol; 3,5,7,4'-tetrahydroxy-6-methoxy-flavone; Gallic acid; Caffeic acid; 3- (1,3-dihydro-3-oxo-2H-indol-2-ylidene) -1,3-dihydro-2H-indol-2-one; [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid) ; And [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid It can be seen that the methyl ester exhibits various physiological actions including antiviral action, antitumor action, radical scavenging action and cellular necrosis control, cytokine production control or inhibition.

Experiment 16: Acute Toxicity Test

According to the conventional method, the bioactive extracts obtained by the methods of Experiments 1 and 2 were administered orally, intravenously, and intraperitoneally in 5-week-old ddy mice. Irrespective of the route of administration, the LD ₅₀ was 1 g / kg or more. . From this data, it can be seen that the bioactive extract of the present invention can be safely administered to mammals and humans with almost no side effects.

Preferred embodiments of the present invention will be described by the following examples.

Example 1 Preparation of Biologically Active Crude Extracts

30 kg of the ground part of the indigo plant of Shimane Prefecture, Japan, was harvested and powdered in July, and extracted three times at room temperature with 30 to 60 liters of ethyl acetate for one extraction. The obtained extracts were collected together, filtered through a filter paper, and the filtrate was recovered, and then evaporated to remove ethyl acetate and dried to obtain 168 g of an extract containing ethyl acetate soluble components derived from the side.

This extract, which has various physiological effects, is useful as a herbal medicine used in cosmetics and medicines.

Example 2: Purified Bioactive Extract

The extract obtained by the method of Example 1 was suspended in 50 v / v% methanol aqueous solution, divided into 8 portions, and then 1700 ml of "FS-1830" (gel for adsorption chromatography sold by Japan Organo, Japan) was added. The packed columns were respectively loaded, and 60, 70, 80 and 90 v / v% aqueous methanol solution, methanol and ethyl acetate were fed in this order in the same amount as the gel volume. The eluate was collected every half volume of the gel volume. The first and later fractions eluted from the column with 80 v / v% aqueous methanol solution were referred to as fraction 2 and fraction 1, respectively; The later fraction eluted from the column with 60 v / v% methanol aqueous solution is called fraction 3; The later fractions eluted from the column with 90 v / v% methanol aqueous solution were referred to as fraction 4, and these fractions were collected and evaporated separately to remove the solvent and dried to obtain a solid.

In 5.2 g of solid obtained from fraction 1, 2.6 g was suspended in 30 ml of methanol and filtered off with suction to separate the soluble and insoluble portions (876 mg). The solution of the soluble portion was added to a column filled with 1350 ml of silica gel, added in a linear gradient of the mixed solution of methanol and chloroform with a stepwise increase from methanol concentration of 5 v / v% to 100 v / v%, followed by elution 450. ml was recovered. Fraction 4 was evaporated to remove the solvent, and the solid obtained was suspended in 2 ml of methanol and washed by filtration under suction to give 6,12-dihydro-6,12-dioxoindolo [2,1-b. ] 19.4 mg of crystals of quinazoline were obtained. 278 mg of yellow needles of 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone were obtained by dissolving insoluble fraction in an appropriate amount of methanol and standing at room temperature.

10.7 g of solid obtained from fraction 2 were suspended in 40 ml of methanol, and this solution was added to a column filled with 1,520 ml of "SEPHADEX LH-20" (gel for adsorption chromatography from Pharmacia LKB Biotechnology AB, Sweden). Methanol was added to the column to recover 190 ml of an elution fraction. Each of the 15th and 18th fractions was evaporated to remove the solvent and dried to give a solid. 400 mg of the solid composition obtained from the 18th fraction was completely dissolved in about 210 ml of a mixed solvent of methanol and water (= 5: 2 volume ratio). The solution was filtered through a 0.22 μm membrane filter and left for 3 days at room temperature to precipitate crystals. This crystal was recovered using 89.3 mg of the crystals of camphorol using conventional filter paper. 500 mg of the solid obtained in the 15th fraction was suspended in 10 ml of methanol and filtered to recover insoluble matters, and then 250 ml of methanol was added thereto to dissolve completely. The solution was filtered through a 0.22 μm membrane filter and left at room temperature for 7 days to precipitate crystals. The crystals were filtered through a conventional filter paper to obtain 252.3 mg of 3,5,7,4'-tetrahydroxy-6-methoxy-flavone.

11.8 g of solid from fraction 3 was dissolved in 40 ml of methanol, and this solution was added to a column packed with 1680 ml of "SEPHADEX LH-20" (gel for adsorption chromatography from Pharmacia LKB Biotechnology AB, Sweden). Methanol was added to the column to recover 560 ml of an elution fraction. Fourth fractions were each evaporated to remove solvent and dried to give a solid. This solid was dissolved in about 10 ml of methanol, and this solution was added to a column packed with 480 ml of "FS-1830" (gel for adsorption chromatography sold by Japan Organo, Japan), and then 0, 10, 20 480 ml of 30, 40, 50, 60 and 70 v / v% aqueous methanol solution were added, and 960 ml of methanol was added again. The eluate was fractionated to give 240 ml of fractions. The fifth fraction was evaporated to remove the solvent and dried to give 150 mg of solid. This solid was then dissolved in 300 ml of ethyl acetate, the solution was filtered through a 0.22 μm membrane filter and left at room temperature for 7 days to precipitate crystals. This crystal was recovered using a conventional filter paper to give 18.7 mg of gallic acid crystals. The 9th fraction eluted from the column of "FS-1830" was evaporated to remove the solvent and dried to obtain 500 mg of solid, which was dissolved in 0.5 ml of ethyl acetate and 40 ml of "SILICAGEL 60K650" (Japan A mixture of ethyl acetate and chloroform (= 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7 :) was added to a column packed with an adsorbing chromatograph gel sold by Katayama Chemical Industries. 3, 8: 2 and 9: 1 (volume ratio), and 40 ml of chloroform and methanol were each added. The eluate from the column was fractionated to obtain 10 ml of fractions, and the 17th and 21st fractions were put together, using "SILICAGEL 60F ₂₅₄ (Product No. 5717)" (thin layer for separation from Merck, USA) as a thin plate. Then, thin layer chromatography was performed using a mixture of toluene, ethyl acetate, and acetic acid (5: 5: 1, volume ratio) as the developing system. After development, a portion of the silica gel at a position of Rf of about 0.6 was recovered and extracted with an appropriate amount of methanol to obtain the developed material. The solvent was removed from the extract by evaporation and dried to obtain 15.4 mg of caffeic acid crystals.

Of 4.6 g of solid obtained from fraction 4, 4 g was dissolved by mixing with 40 ml of methanol, but some did not dissolve but formed a precipitate. The precipitate was recovered, dissolved sufficiently in 800 ml of methanol, and left at room temperature for 2 days to precipitate a red crude crystal. After recovering this crude crystal, the supernatant was left still as before and the red crude crystal was precipitated again. The crude crystal thus obtained was collected, combined with the previously obtained red crystals, washed with an appropriate amount of methanol, and dissolved in a sufficient amount of methanol. The resulting solution was membrane filtered and the filtrate was evaporated to remove the solvent and dried to afford 3- (1,3-dihydro-3-oxo-2H-indole-2-ylidene) -1,3-dihydro-2H 44.5 mg of crystals of indole-2-one were obtained.

These compounds with various physiological actions are useful as herbals for foods, cosmetics and pharmaceuticals that require relatively highly purified ingredients.

Example 3: Bioactive Crude Extracts

15 kg of the ground part of the indigo plant of Shimane Prefecture, Japan, was harvested and powdered in July, and extracted three times at room temperature with 30 liters of ethanol for one extraction. The obtained extracts were collected together, filtered through a filter paper, and the filtrate was recovered, and then evaporated to remove ethanol and dried to obtain 560 g of an extract containing ethyl acetate soluble components derived from the side. Using the compound obtained in the method of Example 2 as a standard, the above-mentioned bioactive extract was analyzed by conventional high performance liquid chromatography or gas chromatography, and the extract was obtained as 6,12-dihydro as an ethyl acetate soluble component. -6,12-dioxoindolo [2,1-b] quinazoline; 3,5,4'-trihydroxy-6,7-methylenedioxy-flavone; Camphorol; 3,5,7,4'-tetrahydroxy-6-methoxy-flavone; Gallic acid; Caffeic acid; 3- (1,3-dihydro-3-oxo-2H-indol-2-ylidene) -1,3-dihydro-2H-indol-2-one; [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid) And [3S- (3α, 4β, 21β)] 9-ethyl-14-ethyl-21- (methoxycarbonyl) -4,8,13,18-tetramethyl-20-oxo-3-forminpropanoic acid It has been found to contain methyl esters in amounts of 65, 780, 354, 988, 250, 230, 75, 52 and 63 mg, respectively.

This extract, which has various physiological effects, is useful as a herbal medicine used in food, cosmetics and medicines.

Example 4: Snack Foods

Potatoes self-digested with reducing sugars, stored at 20 ° C and relative humidity of 85% for two weeks, are washed with water, peeled and screened according to a conventional method, and sliced with a centrifugal slicer into slices having a thickness of about 1.5 mm. It was. These slices were washed with water to remove starch from the surface, drained, and then fried in oil at 170 ° C. for 5 minutes to remove excess oil. 6 parts by weight of salt, food grade trehalose powder (registered trademark "TREHAOSE", trehalose purity of 98% or more sold by Japan Co., Ltd.) in the fried slice using a salt In addition, seasoning powder containing 1 part by weight of the bioactive extract obtained by the method of Example 3 and the appropriate amount of spices was sprinkled. The obtained slice was transferred to a weighing machine, injected and packaged to obtain a snack food.

This product has good aroma and taste, so it can be used advantageously as a health food for maintaining / promoting health.

Example 5: Tea Bag

9 parts by weight of lyophilized black tea extract powder was dissolved in an appropriate amount of water, and the obtained solution was prepared by the method of Example 3 and mixed with 1 part by weight of a bioactive extract dissolved in ethanol. The mixture was fermented in the usual manner and sprinkled with 90 parts by weight of dried black tea leaves. The black tea leaves were then sieved, cut, dried to dryness and the foreign material was removed from the sorting machine, and then tea bags were obtained by packing 2 g each using Japanese paper.

증진하는 건강식품으로 유리하게 사용할 수 있다.The product is immersed in 180 ml of cold water for about 10 minutes or rinsed for about 2 minutes in 180 ml of hot water at 90-100 ° C. Good fragrance and taste keep this product healthy

Can be used advantageously as a health food to promote.

Example 6 Dietary Supplement

Food grade trehalose powder (registered trademark "TREHAOSE", more than 98% of trehalose purity sold by Japan Corporation, Ltd.), 52 parts by weight, corn starch 40 parts by weight, biological activity obtained by the method of Example 3 3.5 parts by weight of the extract and 2.5 parts by weight of the cellulose crystals were mixed, kneaded while spraying water according to a conventional method, granulated with a fluidized bed, and pulverized to obtain a powder for tableting. 2 parts by weight of sucrose fatty acid ester was uniformly mixed with this powder, and then tableted in a tableting machine equipped with a punch having a diameter of 11 mm to obtain a tablet (about 300 mg / tablet).

증진하기 위한 건강보조 식품으로 유용하다.Easy to eat and has excellent collapsing properties in the digestive tract

Useful as a dietary supplement to promote.

Example 7: Hair Rinse

1 part by weight of food grade trehalose powder (trade name "TREHAOSE", sold by Japan Co., Ltd., Japan), 2 parts by weight of a bioactive extract obtained by the method of Example 1, α -Glucosyl Routine (trade name "αG RUTIN", sold by Toyo Sugar Refining of Japan) 2 parts by weight, distearyl methyl ammonium chloride 2 parts by weight, cetanol 2 parts by weight, silicone oil 2 parts by weight, polyoxyethylene oleate 1 part by weight of one alcohol ether, and an appropriate amount of fragrance were dissolved by heating. 3 parts by weight of 1,3-butylene glycol, 85 parts by weight of purified water and an appropriate amount of preservative were added to the solution and mixed, and the mixture was cooled to obtain a hair rinse.

증진하는 화장품으로 유리하게 사용할 수 있다.This product has good stability and little irritation to the scalp, so it maintains the health of the scalp and hair.

It can be advantageously used as a cosmetic for promoting.

 Example 8 Milk Lotion

 0.5 parts by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, 1 part by weight of soluble glyceryl monostearate, 0.5 part by weight of pyruvic acid, 0.3 part by weight of behenyl alcohol, 0.3 part by weight of avocado oil 1 part by weight, 1 part by weight of the physiologically active extract prepared by the method of Example 1, and an appropriate amount of vitamin E and a preservative by heat dissolving in a conventional manner, 1 part by weight of sodium L- lactate, 1,3- 7 parts by weight of butylene glycol, 0.1 parts by weight of carboxyvinyl polymer and 86.3 parts by weight of purified water were added and mixed, and the mixture was emulsified in a homogenizer to prepare a milk lotion.

증진하는 화장품으로 유리하게 사용할 수 있다.This product is not sticky and has good spreadability, so it maintains the health of skin

It can be advantageously used as a cosmetic for promoting.

 Example 9: Toothpaste

 45 parts by weight of dibasic calcium phosphate, 2.9 parts by weight of pullulan, 1.5 parts by weight of sodium lauryl sulfate, 20 parts by weight of glycerin, 0.5 parts by weight of polyoxyethylene sorbitan laurate, 10 parts by weight of sorbitol, 7 parts by weight of maltitol Toothpaste was prepared by mixing 13 parts by weight of purified water and 0.1 part by weight of the bioactive extract prepared by the method of Example 1.

증진하는 화장품으로 유리하게 사용할 수 있다.This product maintains oral health

It can be advantageously used as a cosmetic for promoting.

Example 10: Ointment

1 part by weight of sodium acetate trihydrate, 4 parts by weight of DL-calcium lactate, 10 parts by weight of glycerin, 0.5 parts by weight of peppermint oil, 49 parts by weight of waselin, 10 parts by weight of Japanese wax, 10 parts by weight of lanolin, 14.5 parts by weight of sesame oil, and According to the extract percentage prepared by the method of Example 1, 2 parts by weight of the composition combined with seven compounds 1 to 7 of the bioactive extract prepared by the method of Example 2 was uniformly mixed according to a conventional method to prepare an ointment It was.

증진하는 의약품으로 유리하게 사용할 수 있다.This penetrating and expanding product maintains the health of your skin.

It can be used advantageously as an promoting medicine.

As described above, the present invention is based on the finding that ethyl acetate soluble components derived from raw indigo plants exert various physiological effects on mammals and humans. When the above-mentioned components are administered to mammals and humans, antibacterial action, antiviral action, anti-tumor action, radical scavenging action, cellular necrosis control action, cytokine production control action, cytokine production inhibition action, and nitric oxide synthase expression It exhibits various physiological activities, including inhibitory action. Therefore, the bioactive extracts of the present invention containing ethyl acetate soluble components can be widely used in the food, cosmetic and pharmaceutical fields.

The leaves and seeds of the conventional spines prepared by drying in sunlight are used after being extracted with hot water. Among these components, the indole component easily deteriorates because it is easy to cause chemical change by hydrolysis and air oxidation during drying in sunlight. The bioactive composition according to the present invention can be prepared using the ethyl acetate soluble component derived from fresh side without having to dry the side in sunlight. Therefore, according to the present invention, a physiologically effective ingredient in a living indigo plant can be used as it is, even when the place is far from the place of origin. The bioactive extract of the present invention having such utility can be produced in the amount required by the method of the present invention using raw vegetables as a raw material.

The present invention having such a useful effect can be said to be a meaningful invention that contributes to this industry.

Claims (12)

6,12-dihydro-6,12-dioxoindolo [2,1-b] quinazoline and gallic acid or caffeic acid, respectively, based on solids, 0.01% or more, and 99% or less in total, (Iii) Raw extracts having Helicobacter pylori activity, antiviral activity, radical scavenging activity or cellular spontaneous necrosis control activity. delete delete delete delete delete The raw extract of claim 1, wherein the raw is fresh ground portion. delete delete delete delete delete

Images