WO2016117705A1 - Agent for inducing production of hepatocyte growth factor - Google Patents

Agent for inducing production of hepatocyte growth factor Download PDF

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Publication number
WO2016117705A1
WO2016117705A1 PCT/JP2016/051942 JP2016051942W WO2016117705A1 WO 2016117705 A1 WO2016117705 A1 WO 2016117705A1 JP 2016051942 W JP2016051942 W JP 2016051942W WO 2016117705 A1 WO2016117705 A1 WO 2016117705A1
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Prior art keywords
growth factor
hepatocyte growth
factor production
extract
production inducer
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PCT/JP2016/051942
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French (fr)
Japanese (ja)
Inventor
敏一 中村
清正 岡
日出男 大城
樹建 孫
中島　宏
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株式会社ニューロゲン
株式会社漢方医科学研究所
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Priority to JP2016570736A priority Critical patent/JP6712056B2/en
Publication of WO2016117705A1 publication Critical patent/WO2016117705A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/254—Acanthopanax or Eleutherococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/286—Carthamus (distaff thistle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/35—Caprifoliaceae (Honeysuckle family)
    • A61K36/355—Lonicera (honeysuckle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
    • A61K36/725—Ziziphus, e.g. jujube
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger

Definitions

  • the present invention relates to a hepatocyte growth factor production inducer containing an active ingredient derived from a plant.
  • Hepatocyte growth factor is a type of cytokine involved in tissue regeneration and repair in vivo. Hepatocyte growth factor acts on various cells in addition to hepatocytes. Specifically, it is known to promote proliferation of epithelial cells and endothelial cells and to act as a neurotrophic factor for nerve cells. Further, hepatocyte growth factor exhibits anti-fibrosis, anti-inflammation, angiogenesis promotion and cell death inhibitory action, and is considered to be effective for the treatment and / or prevention of various diseases.
  • hepatocyte growth factor production inducers examples include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists (see Patent Document 1) and glycosaminoglycans such as dermatan sulfate and chondroitin sulfate (Patent Document 2). And sugar compounds such as fucoidan derived from Gagome Kombu (see Patent Document 3) and the like are known.
  • Patent Document 4 a processed product of bitter gourd is used as an active ingredient
  • Patent Document 5 a processed product of Cordyceps is processed as an active ingredient
  • Hokoei which is the rooted whole plant of various plants of the genus Taraxacum such as Taraxacum mongolicum of the family Asteraceae (see Patent Document 6), eggplant, Granular foods containing extracts such as citrus yellow and potato (see Patent Document 7), bamboo leaves, Sazabaru mugwort, potato and other extracts (see Patent Document 8), nanana ginseng, udon and serpentine (Patent Document) 9) is known.
  • Inducing agents that significantly induce the production of hepatocyte growth factor in vivo are required for use in therapeutic and preventive drugs for various diseases, beauty products, functional foods, and the like.
  • the inducer is obtained from herbal medicines such as plants that have been used for many years as traditional Chinese medicines.
  • the present inventors have studied various extracts from plants used as crude drugs, and found hepatocyte growth factor production-inducing activity as an active ingredient derived from certain plants. Moreover, it discovered that the combination of the extract of a specific crude drug had the hepatocyte growth factor production induction activity which has not existed until now.
  • the present invention is a hepatocyte growth factor production inducer containing a plant-derived active ingredient.
  • the plant in the present invention is one or more selected from safflower, camellia, coconut, sarcophagus, Masutoshi Jin, jinkin, ginseng, nanana ginseng, shinofutoto, daikon, licorice, senso and lotus wick. obtain.
  • the active ingredient contained in another hepatocyte growth factor production inducer of the present invention is an extract obtained by extraction with a polar solvent that is pharmaceutically or food hygienically acceptable.
  • a polar solvent that is pharmaceutically or food hygienically acceptable.
  • the extract is obtained by mixing a plurality of plants and extracting them.
  • hepatocyte growth factor production inducer of the present invention is a mixture of safflower, cocoon, eggplant, sarcophagus, carrot, ginseng, nanana ginseng, ninfuto, daikon, licorice, senso and lotus core. This is an extract obtained by extraction.
  • the active ingredient of the hepatocyte growth factor production inducer of the present invention includes at least two types of molecules having hepatocyte growth factor production-inducing activity, and one molecule having hepatocyte growth factor production-inducing activity is negatively charged. Another molecule having activity to induce the production of hepatocyte growth factor is neutrally charged.
  • the polar solvent can be water, ethanol, acetic acid or mixtures thereof.
  • the extraction temperature may be in the range from room temperature to the boiling point of the solvent at normal pressure or under pressure.
  • the hepatocyte growth factor production inducer of the present invention may be in the form of a powder formulation, granule, tablet, pill, capsule, or liquid formulation.
  • the present invention also provides a therapeutic and / or prophylactic agent containing a hepatocyte growth factor production inducer, a cosmetic product containing a hepatocyte growth factor production inducer, and a functional food containing a hepatocyte growth factor production inducer. To do.
  • this invention provides the manufacturing method of a hepatocyte growth factor production inducer. That is, an addition step in which the above-mentioned hepatocyte growth factor production inducer is added to an anion exchange resin together with a buffer, and elution is carried out from the anion exchange resin with a buffer containing a salt having an ionic strength of 0 to 500 ⁇ mM. And a method for producing a hepatocyte growth factor production inducer. It is preferable to elute with a buffer containing a salt having an ionic strength of 100 ⁇ m and / or 300 ⁇ m.
  • the hepatocyte growth factor production inducer of the present invention acts on hepatocyte growth factor-producing cells, promotes transcription of mRNA encoding hepatocyte growth factor, or promotes protein synthesis of hepatocyte growth factor.
  • the production of cell growth factors can be induced.
  • the plant used in the present invention is a herbal medicine that has been used for many years as a traditional Chinese medicine, and is considered to be highly safe.
  • FIG. 2 is a graph showing hepatocyte growth factor production-inducing activity of a fraction obtained by elution of a DEAE anion exchange column from the hepatocyte growth factor production inducer of the present invention.
  • the hepatocyte growth factor production inducer of the present invention contains a plant-derived active ingredient. More preferably, it is an active ingredient derived from one or more selected from safflower, persimmon, coconut, sarcophagus, Masutoshi Jin, eel gold, carrot, ginseng, ginseng, ninfuto, daegu, licorice, sensu and lotus core. including.
  • the Japanese Pharmacopoeia refers to the 16th revised Japanese Pharmacopoeia.
  • Safflower (Kouka) is an annual or perennial safflower (Carthamas tinctorius L), which has been widely known as a red dye and edible oil since ancient times. Safflower root is frequently used as a blood-purifying agent, especially as a gynecological drug, and is effective for, for example, irregular menstruation, coldness, and menopause, and is also effective in improving blood due to blood circulation disorders.
  • the dried flowers are listed in the Japanese Pharmacopoeia as herbal medicines that have a blood circulation promoting action, and are used in various Chinese medicine prescriptions.
  • each part such as safflower leaves, stems, flowers, roots and the whole plant as it is or dried.
  • a flower or whole plant on the ground is preferable, and it is more preferable to use a safflower as described in the Japanese Pharmacopoeia as it is or without removing most of the yellow pigment.
  • safflower can be preferably used by pressing it into a plate shape.
  • ⁇ (Gadju) is a kind of perennial herb (Curcuma zedoaria), a kind of ginger family turmeric genus, and is a herbal medicine with an aromatic healthy stomach action recorded in the Japanese Pharmacopoeia.
  • components extracted from sputum have nerve growth factor production enhancing action
  • the dried rhizome or boiled potato as it is or dried, and the cocoon described in the Japanese Pharmacopoeia is more preferable.
  • the coconut is a fruit of wolfberry, and wolfberry (Lycium chinense Miller or Lyciumbarbatum L.) which is a deciduous shrub of the solanaceous family native to China is preferably used.
  • Herbal medicines listed in the Japanese Pharmacopoeia known for anti-fatigue anti-aging, immunostimulatory, hepatocyte protection, anti-fatty liver, blood fat lowering, blood glucose lowering, blood pressure lowering, etc.
  • the coconut fruit as it is or dried can be preferably used, and the coconut described in the Japanese Pharmacopoeia is more preferable.
  • Sekishobu has been recognized in Chinese medicine for the benefits of fragrance opening and Ningshin Anjin, and is used in combination with other Anjin herbs to treat frenzy, dementia, amnesia, tinnitus, and hearing loss. Has also been used.
  • the stalagmite can be preferably used as it is or after drying the roots, stems, or leaves of taro (Acorus gramineus Acorus gramineus).
  • Masuthijin (yakutinin) is a herbal medicine listed in the Japanese Pharmacopoeia, and is known to have a healthy stomach, antidiuretic, and salivary secretion inhibiting action.
  • Masuthijin can be preferably used as it is or dried from the fruits of Mashiko (Yakuchi; Alpinia oxyphylla Miquel). More preferred.
  • Gold is a perennial plant belonging to the ginger family and is used as a flavoring, coloring, and herbal medicine. It is well known as the English name turmeric, and it is also used as a material for curry in Indian cuisine. As a medicinal effect as a crude drug, the promotion of bile secretion and the action of a healthy stomach are known.
  • the gold can be preferably used by drying the roots or stems of ginger perennial turmeric as it is.
  • ginger perennial turmeric there are spring turmeric (Curcuma aromtica S) that attaches red flowers in the spring and autumn turmeric (Curcuma longa L) that attaches white flowers in the fall, which is rich in curcumin, the main medicinal ingredient. Turmeric is more preferable.
  • the sens can preferably be used as it is or after drying the rhizomes of Curculigo ⁇ orchioides GAERIN.
  • Ginseng is a herbal medicine listed in the Japanese Pharmacopoeia and is blended in various Kampo prescriptions. Its effects include anti-diabetic action, anti-cancer action, heart strengthening and blood pressure adjustment, liver function strengthening, gastrointestinal function strengthening, stress relieving and tonic effect, physical strength (strength) enhancement, brain function strengthening, aging suppression, radiation irradiation protection action.
  • an anemia recovery effect and a hematopoietic effect an immune function enhancement, an anti-inflammatory effect and a weak constitution improving effect are known.
  • the ginseng can be obtained by removing the roots of the perennial perennial ginseng (Panax ginseng C.A. Meyer) or lightly boiled, and the ginseng described in the Japanese Pharmacopoeia is more preferable.
  • Red ginseng that has been peeled off ginseng and dried naturally or by hot-air drying at 60 ° C. or less to a moisture content of 15% or less, or dried steamed steamed ginseng can be preferably used.
  • Denshichinin ginseng is a medicinal plant belonging to the family Araceae, native to southern China, and cultivated in the highlands of Yunnan province and Guangxi Zhuang Autonomous Region. As an effect of the ginseng, it is known to have both hemostatic action and active blood (improvement of blood circulation). Further, antiviral action, anticholesterol action, antitumor action and the like are also known.
  • Panax ginseng Panax notoginseng roots or stems, which are also referred to as Sanchi ginseng, can be preferably used as they are or dried.
  • Ninfuto is simply referred to as Shinobifu and is a herbal medicine described in the Japanese Pharmacopoeia. It has been used for the treatment of arthritis, bronchitis, etc. for a long time as it is effective for hemorrhoids (inflammation inside and outside the body). It is also known to be effective for epidemic colds, various purulent infections, redness of joints due to wet heat, swelling, protection against hepatotoxicity and reduction of cellular immunity.
  • ninto-wisteria can be preferably used with the leaves and stems of honeysuckle (Loniceraj aponica Thunberg (Caprifoliaceae)) as they are or dried, and ninfuto described in the Japanese Pharmacopoeia is more preferable.
  • ⁇ Taisu is a jujube fruit that contains sugar, mucus, malic acid, tartaric acid, etc., and is known for its tonic and sedative effects. In addition, it has sweetness and is blended in various Chinese medicines for the purpose of alleviating side effects.
  • a jujube of the family Chromaceae Zizyphus jujube Mill. Var. Inermis Rehder (Rhamnaceae)
  • a fruit of its cultivar can be preferably used as it is or dried. preferable.
  • Licorice is a herbal medicine listed in the Japanese Pharmacopoeia and contains glycyrrhizin and glycosides, and is known to have analgesic, anti-inflammatory, gastric pain, antitussive expectorant, detoxification, and duodenal ulcer.
  • the roots and strons of leguminous larvae (Glycyrrhiza uralensis Fisch) or licorice elephant (Glycyrrhiza glabra Linne (Leguminosae)), or those excluding their pericarp (peeled licorice) are either left as is or dried.
  • a licorice described in Japanese Pharmacopoeia is more preferable.
  • Rencoshin is usually drunk as tea and is said to be effective for the heart and liver and also for mental stability.
  • it is possible to preferably use a dried seed portion of a mature seed of a lotus Nelumbo nucifera which is a water lily family.
  • Another hepatocyte growth factor production inducer of the present invention is an extract obtained by extracting an active ingredient from the above-mentioned plant with a polar solvent that is pharmaceutically or food hygienically acceptable.
  • the extract used in the present invention is a herbal medicine (preferably a dry herbal medicine) composed of the above-mentioned plants as it is or is cut, chopped, crushed (preferably cut, shredded, crushed around several mm to 1 cm), or It can be obtained by extracting a pulverized product or a powdered product with an extraction solvent.
  • the hepatocyte growth factor production inducer of the present invention can be obtained by mixing the active ingredients extracted from each plant. More preferably, it is a hepatocyte growth factor production inducer obtained by extracting from a mixture obtained by mixing each plant.
  • the hepatocyte growth factor production inducer obtained by extracting from the mixture obtained by mixing each plant is more prominent than the hepatocyte growth factor production inducer obtained by mixing the extract. This is because a high hepatocyte growth factor producing effect was confirmed.
  • the extraction solvent is preferably a polar solvent that is acceptable for pharmaceuticals or food hygiene.
  • the polar solvent include water (for example, purified water, distilled water, tap water, etc.), ethanol, a mixed solvent of water and ethanol (hydrous ethanol), acetic acid, a mixed solvent of acetic acid and water, water and ethanol, and the like. Examples thereof include a mixed solvent with acetic acid. Water or water-containing ethanol is preferred. The water content in hydrous ethanol is 1 to 99% by volume.
  • An acid may be added to water or hydrous ethanol. Examples of the acid include inorganic acids such as hydrochloric acid, and organic acids such as malic acid and citric acid.
  • the extraction method is not particularly limited, and an immersion method or a countercurrent extraction method can be employed. Moreover, it can heat-extract in the range to the boiling point of a solvent under normal temperature extraction or a normal pressure. If necessary, extraction may be performed under reduced pressure or increased pressure.
  • the pressurization may be a pressure exceeding 1 atm, and preferably about 2 to 3 atm.
  • An extraction solvent (polar solvent) is added in an amount of 10 to 100 times, preferably 10 to 40 times the amount of a plant or a mixture of plants, and usually 30 minutes or more in the range from room temperature to the boiling point of the solvent under normal pressure or pressure.
  • the extract can be obtained by immersing for 5 hours, preferably 1 to 3 hours (1, 2, 3 hours) and filtering by a known method.
  • the extract can be used as an extract as it is or by mixing with another extract.
  • what concentrated the extract liquid, what was concentrated and dried or freeze-dried can also be used as an extract.
  • the active ingredient of the hepatocyte growth factor production inducer of the present invention includes at least two types of molecules having hepatocyte growth factor production-inducing activity, and one molecule having hepatocyte growth factor production-inducing activity is negatively charged. Another molecule having activity to induce the production of hepatocyte growth factor is neutrally charged.
  • the active ingredient of the present invention may contain four types of molecules having hepatocyte growth factor production-inducing activity.
  • the hepatocyte growth factor production inducer of the present invention is orally ingested by mixing an extract and a pharmaceutically or food hygienically acceptable carrier optionally blended by a known method. It can be easily prepared as a pharmaceutical or food product.
  • the food includes health foods, functional foods and supplements. It is also possible to prepare a medicine to be taken nasally or transdermally, a medicine to be administered by injection, or a cosmetic product to be used transdermally.
  • the hepatocyte growth factor production inducer of the present invention may be formulated using a pharmaceutical or food hygiene acceptable carrier.
  • the preparation include a solid preparation and a liquid preparation.
  • a carrier acceptable for pharmaceutical or food hygiene various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations and solvents in liquid preparations. , Solubilizers, suspending agents (emulsifiers, thickeners) and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners, and fragrances can be used.
  • the hepatocyte growth factor production inducer of the present invention can be prepared as a therapeutic agent, a preventive agent, a cosmetic product, or a functional food. If the content of the hepatocyte growth factor production inducer in the therapeutic agent, preventive agent, beauty product, or functional food of the present invention is, for example, a solid preparation, the extract of the plant-derived active ingredient is extracted as the extract. About 1 to 99% by weight, preferably about 5 to 90% by weight, and more preferably about 10 to 80% by weight. In the case of a liquid preparation, it is about 0.0001 to 80% by weight, preferably about 10 to 50% by weight, more preferably about 20 to 35% by weight of the preparation as a dry extract.
  • the liquid formulation can also use the concentrate which concentrated the extract of a plant as it is, or can be used as it is.
  • the extraction rate is 1 to 3 hours (1, 2, 3 hours) extracted with 10 to 40 times the polar solvent (preferably water) to the plant from which the active ingredient is derived.
  • An extract having a recovery rate of 25% to 33% can be made into a liquid preparation.
  • the therapeutic agent, preventive agent, beauty product, or functional food of the present invention is preferably taken orally. More preferably after meal, before meal or between meals.
  • the amount of intake and the number of intakes of the present invention vary depending on age, body weight, dosage form, etc., but usually about 1 mg to 500 mg per kg body weight, preferably about 5 mg as an extract obtained by extracting an active ingredient derived from a plant per adult day. Can be taken to be ⁇ 300 mg. This amount is usually taken 1 to several times, preferably 1 to 3 times.
  • a liquid preparation it is preferable to ingest an extract obtained by extraction with a polar solvent (preferably water), usually 1 to several times, more preferably 1 to 3 times.
  • a polar solvent preferably water
  • the therapeutic agent or prophylactic agent of the present invention may be administered nasally or transdermally, and may be administered intravenously, abdominally, intradermally, subcutaneously, muscle, etc. using a syringe.
  • the cosmetic product of the present invention may be administered transdermally.
  • the hepatocyte growth factor production inducer of the present invention induces the production of hepatocyte growth factor in vivo. Therefore, it exerts an effect on various diseases that are treated and / or prevented by production of hepatocyte growth factor.
  • liver diseases hepatitis, cirrhosis, liver failure, other liver disorders
  • kidney diseases glomerulonephritis, kidney failure, renal anemia, diabetic nephropathy, other kidney disorders
  • skin diseases Eye disease (corneal ulcer, etc.)
  • lung disease prneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis, pulmonary fibrosis, other lung disorders
  • gastroduodenal disease gastritis, gastric ulcer, duodenal ulcer, other gastroduodenum
  • cancer diseases and cancer treatment disorders heart or limb ischemic or arterial diseases, blood diseases (thrombocytopenia, blood flow disorders, etc.), bone diseases (osteop
  • humans are preferable, but mammals such as mice, rats, rabbits, dogs, cats, cows, monkeys, pigs, and birds , Reptiles, amphibians, fish, pet animals, livestock / cultured species, experimental animals, etc. may be administered or ingested.
  • the present invention also provides another method for producing a hepatocyte growth factor production inducer. Specifically, an addition step of adding the above-described hepatocyte growth factor production inducer together with a buffer to an anion exchange resin, and a buffer solution containing a salt having an ionic strength of 0 to 500 ⁇ m from the anion exchange resin. It is a manufacturing method of the hepatocyte growth factor production inducer containing the elution process to elute.
  • diethylethanolamine (DEAE) or the like can be used as the anion exchange resin.
  • sodium chloride etc. can be used as a salt contained in the buffer solution to elute.
  • the elution step may be an elution step of elution with a buffer containing a salt having an ionic strength of 100 ⁇ m and / or 300 ⁇ m. This is because the fraction eluted under these conditions may contain a molecule having higher hepatocyte growth factor production-inducing activity than the fraction eluted under other conditions.
  • safflower-derived extract The dried safflower was cut into several millimeters to 1 cm. Water was added to the resulting safflower, followed by boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from safflower.
  • the hepatocyte growth factor production inducing activity of the extract of Example 1 was measured using human skin fibroblasts (SF4-1 cells) having hepatocyte growth factor production ability.
  • SF4-1 cells were seeded in a 96-well plate at a cell density of 1.5 ⁇ 10 4 cells / well.
  • the medium was replaced with a DMEM medium supplemented with 1% by weight of FCS, and the extract of Example 1 was added to the culture solution of SF4-1 cells at a plurality of concentrations.
  • 1 ⁇ g / mL heparin known to have hepatocyte growth factor producing activity was added as a positive control, and distilled water was added as a negative control.
  • SF4-1 cells were cultured for 48 hours, and the amount of hepatocyte growth factor in the culture was measured by ELISA.
  • the condition for adding the extract of Example 1 with the highest output of hepatocyte growth factor is when 3% by volume of the extract of Example 1 at 0.5 mg equivalent / mL is added to the culture medium of SF4-1 cells.
  • the production amount of hepatocyte growth factor was 1.4 times relative to the negative control, where the amount of hepatocyte growth factor was 1.
  • cocoon-derived extract The dried cocoon was cut into several millimeters to 1 cm. Water was added to the obtained koji, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from koji.
  • the addition conditions of the extract of Example 2 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 2 at 0.5 mg equivalent / mL is added to the SF4-1 cell culture solution, and the amount of hepatocyte growth factor produced is defined as 1 in the negative control. The relative amount was 1.6 times.
  • the addition conditions of the extract of Example 3 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 3 at 0.5 mg equivalent / mL is added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced is 1 as the amount of hepatocyte growth factor in the negative control The relative amount was 1.3 times.
  • sarcophagus- derived extract The dried sarcophagus was cut into a few millimeters to 1 cm. Water was added to the obtained sarcophagus, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours using a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from sarcophagus.
  • the addition conditions of the extract of Example 4 with the highest production amount of hepatocyte growth factor were: This is the case where 3% by volume of the extract of Example 4 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was 1 as the amount of hepatocyte growth factor in the negative control The relative amount was 2.8 times.
  • Preparation of plating-derived extract The dried plating was cut into several millimeters to 1 cm. Water was added to the obtained plating, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from plating.
  • the addition condition of the extract of Example 5 with the highest production amount of hepatocyte growth factor was: This is a case where the extract of Example 5 at 0.5 mg equivalent / mL was added to the SF4-1 cell culture solution at 3% by volume, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.3 times.
  • ginseng-derived extract The dried ginseng was cut into several millimeters to 1 cm. Water was added to the obtained carrot, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing ginseng-derived active ingredients.
  • the hepatocyte growth factor production-inducing activity of the extract of Example 6 was measured by the same method as in Example 1, the addition conditions of the extract of Example 6 with the highest output of hepatocyte growth factor were: This is the case where 3 vol% of the extract of Example 6 at 0.5 ⁇ mg equivalent / mL was added to the SF4-1 cell culture solution, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.5 times.
  • the addition conditions of the extract of Example 7 with the highest output of hepatocyte growth factor were: This is the case where 3% by volume of the extract of Example 7 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.1 times.
  • ninto-wisteria extract The dried ninto-wister was cut into several millimeters to 1 cm. Water was added to the obtained Shinobifuto and the mixture was boiled and extracted at 2-3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from Shinobu Toto.
  • the hepatocyte growth factor production-inducing activity of the extract of Example 8 was measured by the same method as in Example 1, the addition conditions of the extract of Example 8 with the highest production amount of hepatocyte growth factor were: This is the case where 3 vol% of the extract of Example 8 at 0.5 mg equivalent / mL was added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 1.8 times.
  • the addition conditions of the extract of Example 9 with the highest output of hepatocyte growth factor were as follows: This is the case where 1 vol% of the extract of Example 9 at 0.5 mg equivalent / mL is added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced is defined as 1 in the negative control. The relative amount was 1.6 times.
  • licorice-derived extract The dried licorice was cut into several millimeters to 1 cm. Water was added to the obtained licorice, and the mixture was boiled and extracted at 2-3 atmospheres for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to prepare an extract containing an active ingredient derived from licorice.
  • the addition conditions of the extract of Example 11 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 11 at 0.5 mg equivalent / mL was added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.4 times.
  • the addition condition of the extract of Example 12 with the highest output of hepatocyte growth factor was: This is the case where 3% by volume of the extract of Example 12 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.5 times.
  • Extracts were prepared by mixing equal amounts of the extracts of Examples 1 to 12.
  • the addition conditions of the extract of Example 13 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 13 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.1 times.
  • Hepatocyte growth factor production-inducing activity by the hepatocyte growth factor production-inducing agent of the present invention The hepatocyte growth factor production-inducing activity of the extract of Example 14 is expressed as human skin fibroblasts (SF4 -1 cells).
  • SF4-1 cells were seeded in a 96-well plate at a cell density of 1.5 ⁇ 10 4 cells / well.
  • the medium was replaced with DMEM medium supplemented with 1% by weight of FCS, and 0.5 mg equivalent / mL of the extract of Example 15 was added to the culture medium of SF4-1 cells at 1%, 3%, and 10% by volume, respectively.
  • FIG. 1 shows the amount of hepatocyte growth factor in each culture medium as a relative amount with the amount of hepatocyte growth factor in the negative control as 1. It was found that a large amount of hepatocyte growth factor was produced in SF4-1 cells to which the extract of Example 14 was added.
  • Method for producing hepatocyte growth factor production inducer of the present invention (1) Two times the amount of distilled water was added to the extract of Example 14, mixed for 1 hour at room temperature, and centrifuged. The supernatant after centrifugation is added to a Sephadex LH-20 column (manufactured by GE Healthcare Biosciences) equilibrated with a 20% ethanol / water mixture, and eluted using the same solution. After adding 5%, hepatocyte growth factor production-inducing activity was measured by ELISA. The elution curve is shown in FIG. 2, and the measurement result of HGF production inducing activity is shown in FIG. The fraction that eluted earlier was the main activity, and the weakly eluted fraction also showed weak activity.
  • Method for producing hepatocyte growth factor production inducer of the present invention Two times the amount of distilled water was added to the extract of Example 14, mixed for 1 hour at room temperature, and centrifuged. The supernatant after centrifugation was equilibrated with 50 mM Tris-HCl [pH 8.0] and added to a DEAE Sepharose FF column (GE Healthcare Bioscience). The added column was washed with 50 mM Tris-HCl [pH 8.0], and then eluted sequentially with 50 mM Tris-HCl [pH 8.0] containing 100 mM, 300 mM, and 500 mM sodium chloride.
  • hepatocyte growth factor production inducing activity was measured by ELISA.
  • the elution curve is shown in FIG. 4, and the measurement results are shown in FIG.
  • Activity was observed in the elution fraction of the buffer only and the elution fraction of the buffer containing each concentration of sodium chloride. Therefore, it was revealed that a new hepatocyte growth factor production inducer was produced by the above-described method.
  • the extract of Example 14 there are at least four types of molecules having hepatocyte growth factor production-inducing activity, of which three types of molecules having hepatocyte growth factor production-inducing activity are negatively charged, The type was considered neutral charge.
  • the fraction obtained by elution with a buffer solution containing 100 ⁇ mM sodium chloride was eluted with a buffer solution containing 300 ⁇ mM sodium chloride.
  • the fraction obtained in this way was found to have high hepatocyte growth factor production-inducing activity. Therefore, another hepatocyte growth factor production-inducing agent containing a fraction with high hepatocyte growth factor production-inducing activity can be produced by the production method described above.
  • Hepatocyte growth factor production inducing activity of hepatocyte growth factor production inducer for mice The extract of Example 14 was administered to mice and the amount of hepatocyte growth factor in plasma and cerebrum was measured. A healthy mouse (ICR, 8 weeks old, female) was orally administered 30 mg of extract once a day, and blood and cerebrum were collected 7 days later. As shown in Table 1, since the production of hepatocyte growth factor was increased by administration of the extract of Example 14, it was confirmed that it had a remarkable hepatocyte growth factor-inducing activity.
  • the present invention can be used for the treatment and / or prevention of various diseases by inducing the production of hepatocyte growth factor in the fields of medicine, beauty and food.

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Abstract

[Problem] An agent for significantly inducing production of hepatocyte growth factor in the body is desired for use in drugs for the treatment and/or prevention of various diseases, as well as in beauty products, functional foods, and the like. [Solution] The present invention provides an agent for inducing the production of hepatocyte growth factor, the agent containing an active ingredient derived from a plant. Furthermore, the present invention provides an agent for inducing the production of hepatocyte growth factor in which the plant is one or several selected from safflower, white turmeric, wolfberry, Japanese sweet flag, bitter caramon, turmeric, ginseng, Panax notoginseng, Japanese honeysuckle, jujube, licorice, Curculigo orchioides, and Nelumbo nucifera.

Description

肝細胞増殖因子産出誘導剤Hepatocyte growth factor production inducer
　本発明は、植物由来の有効成分を含む、肝細胞増殖因子産出誘導剤に関する。 The present invention relates to a hepatocyte growth factor production inducer containing an active ingredient derived from a plant.
肝細胞増殖因子（Hepatocyte Growth Factor）とは、生体内で組織再生や修復に関与するサイトカインの一種である。肝細胞増殖因子は、肝細胞以外にもさまざまな細胞に作用する。具体的には、上皮細胞や内皮細胞の増殖を促進することや、神経細胞に対し神経栄養因子として働くことが知られている。また、肝細胞増殖因子は、抗線維化、抗炎症、血管新生促進や細胞死抑制作用などを示し、さまざまな疾患の治療及び／又は予防に有効であると考えられている。 Hepatocyte growth factor is a type of cytokine involved in tissue regeneration and repair in vivo. Hepatocyte growth factor acts on various cells in addition to hepatocytes. Specifically, it is known to promote proliferation of epithelial cells and endothelial cells and to act as a neurotrophic factor for nerve cells. Further, hepatocyte growth factor exhibits anti-fibrosis, anti-inflammation, angiogenesis promotion and cell death inhibitory action, and is considered to be effective for the treatment and / or prevention of various diseases.
肝細胞増殖因子産出誘導剤としては、アンジオテンシン変換酵素阻害剤及びアンジオテンシンII受容体拮抗薬（特許文献１参照）、デルマタン硫酸やコンドロイチン硫酸等のグリコサミノグリカンを有効成分とするもの（特許文献２参照）、ガゴメコンブ由来のフコイダン等の糖化合物（特許文献３参照）等が知られている。 Examples of hepatocyte growth factor production inducers include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists (see Patent Document 1) and glycosaminoglycans such as dermatan sulfate and chondroitin sulfate (Patent Document 2). And sugar compounds such as fucoidan derived from Gagome Kombu (see Patent Document 3) and the like are known.
また、肝細胞増殖因子産出誘導活性を有する植物からの抽出物としては、ニガウリ加工処理物を有効成分とするもの（特許文献４参照）、冬虫夏草加工処理物を有効成分とするもの（特許文献５参照）、キク科のモウコタンポポ（Taraxacum mongolicum）等の各種Taraxacum属植物の根つき全草である蒲公英（ホコウエイ）から得られる抽出物を有効成分とするもの（特許文献６参照）、枸杞子、熟地黄、杜仲等の抽出物（特許文献７参照）、タケの葉、サザバルヨモギ、莪朮等の抽出物（特許文献８参照）、田七人参、鬱金、及び蛇胆を配合した顆粒状食品（特許文献９参照）等が知られている。 Moreover, as an extract from a plant having hepatocyte growth factor production-inducing activity, a processed product of bitter gourd is used as an active ingredient (see Patent Document 4), and a processed product of Cordyceps is processed as an active ingredient (Patent Document 5). ), An extract obtained from Hokoei, which is the rooted whole plant of various plants of the genus Taraxacum such as Taraxacum mongolicum of the family Asteraceae (see Patent Document 6), eggplant, Granular foods containing extracts such as citrus yellow and potato (see Patent Document 7), bamboo leaves, Sazabaru mugwort, potato and other extracts (see Patent Document 8), nanana ginseng, udon and serpentine (Patent Document) 9) is known.
特開2001-002587号公報Japanese Patent Laid-Open No. 2001-002587 特開2002-003384号公報JP 2002-003384 A 特開2001-226392号公報JP 2001-226392 A 特開2008-201748号公報JP 2008-201748 特開2008-201749号公報JP 2008-201749 特開2010-043013号公報JP 2010-043013 A 特開2003-171240号公報Japanese Patent Laid-Open No. 2003-171240 国際公開第01/076614号パンフレットInternational Publication No. 01/076614 Pamphlet 特開2005-204504号公報JP 2005-204504 A
　さまざまな疾患に対する治療薬・予防薬や、美容品、機能性食品などに利用するため、生体内で肝細胞増殖因子の産出を顕著に誘導する誘導剤が求められている。その一方で、安全性を担保するためには、誘導剤は、漢方薬として長年使用されてきた実績のある植物等の生薬から得られることが望ましい。 Inducing agents that significantly induce the production of hepatocyte growth factor in vivo are required for use in therapeutic and preventive drugs for various diseases, beauty products, functional foods, and the like. On the other hand, in order to ensure safety, it is desirable that the inducer is obtained from herbal medicines such as plants that have been used for many years as traditional Chinese medicines.
そこで本発明者らは生薬として用いられる植物からの抽出物を種々検討したところ、ある種の植物由来の有効成分に肝細胞増殖因子産出誘導活性を見出した。また、特定の生薬の抽出物の組み合わせが、これまでにない肝細胞増殖因子産出誘導活性を有することも見出した。 Thus, the present inventors have studied various extracts from plants used as crude drugs, and found hepatocyte growth factor production-inducing activity as an active ingredient derived from certain plants. Moreover, it discovered that the combination of the extract of a specific crude drug had the hepatocyte growth factor production induction activity which has not existed until now.
　すなわち、本発明は、植物由来の有効成分を含む、肝細胞増殖因子産出誘導剤である。本発明における植物は、紅花、莪朮、枸杞子、石菖蒲、益智仁、欝金、人参、田七人参、忍冬藤、大棗、甘草、仙茅及び蓮子芯から選ばれる１又は複数であり得る。 That is, the present invention is a hepatocyte growth factor production inducer containing a plant-derived active ingredient. The plant in the present invention is one or more selected from safflower, camellia, coconut, sarcophagus, Masutoshi Jin, jinkin, ginseng, nanana ginseng, shinofutoto, daikon, licorice, senso and lotus wick. obtain.
　また、別の本発明の肝細胞増殖因子産出誘導剤が含む有効成分は、製薬上又は食品衛生上許容される極性溶媒により抽出して得られる抽出物である。好ましくは、植物が複数であり、複数の植物を混合してから抽出して得られる抽出物である。 In addition, the active ingredient contained in another hepatocyte growth factor production inducer of the present invention is an extract obtained by extraction with a polar solvent that is pharmaceutically or food hygienically acceptable. Preferably, there is a plurality of plants, and the extract is obtained by mixing a plurality of plants and extracting them.
　さらに好ましい本発明の肝細胞増殖因子産出誘導剤は、紅花、莪朮、枸杞子、石菖蒲、欝金、人参、田七人参、忍冬藤、大棗、甘草、仙茅及び蓮子芯を、混合してから抽出して得られる抽出物である。 Further preferred hepatocyte growth factor production inducer of the present invention is a mixture of safflower, cocoon, eggplant, sarcophagus, carrot, ginseng, nanana ginseng, ninfuto, daikon, licorice, senso and lotus core. This is an extract obtained by extraction.
　本発明の肝細胞増殖因子産出誘導剤の有効成分には少なくとも２種類の肝細胞増殖因子産出誘導活性を有する分子が存在し、１の肝細胞増殖因子産出誘導活性を有する分子は陰性荷電であり、別の肝細胞増殖因子産出誘導活性を有する分子は中性荷電である。 The active ingredient of the hepatocyte growth factor production inducer of the present invention includes at least two types of molecules having hepatocyte growth factor production-inducing activity, and one molecule having hepatocyte growth factor production-inducing activity is negatively charged. Another molecule having activity to induce the production of hepatocyte growth factor is neutrally charged.
極性溶媒が、水、エタノール、酢酸又はそれらの混合物であり得る。また、抽出温度が、常温から常圧又は加圧下で溶媒の沸点までの範囲であり得る。 The polar solvent can be water, ethanol, acetic acid or mixtures thereof. In addition, the extraction temperature may be in the range from room temperature to the boiling point of the solvent at normal pressure or under pressure.
　本発明の肝細胞増殖因子産出誘導剤は、粉末製剤、顆粒剤、錠剤、丸剤、カプセル剤、又は液状製剤の形態であり得る。 The hepatocyte growth factor production inducer of the present invention may be in the form of a powder formulation, granule, tablet, pill, capsule, or liquid formulation.
　また、本発明は、肝細胞増殖因子産出誘導剤を含む治療薬及び／又は予防薬、肝細胞増殖因子産出誘導剤を含む美容品、さらに肝細胞増殖因子産出誘導剤を含む機能性食品を提供する。 The present invention also provides a therapeutic and / or prophylactic agent containing a hepatocyte growth factor production inducer, a cosmetic product containing a hepatocyte growth factor production inducer, and a functional food containing a hepatocyte growth factor production inducer. To do.
さらに本発明は、肝細胞増殖因子産出誘導剤の製造方法を提供する。すなわち、上述の肝細胞増殖因子産出誘導剤を緩衝液とともに陰イオン交換樹脂に添加する添加工程と、該陰イオン交換樹脂から、イオン強度0 mM～500 mMの塩を含む緩衝液で溶出する溶出工程とを含有する、肝細胞増殖因子産出誘導剤の製造方法である。イオン強度100 mM及び／又は300 mMの塩を含む緩衝液で溶出することが好ましい。 Furthermore, this invention provides the manufacturing method of a hepatocyte growth factor production inducer. That is, an addition step in which the above-mentioned hepatocyte growth factor production inducer is added to an anion exchange resin together with a buffer, and elution is carried out from the anion exchange resin with a buffer containing a salt having an ionic strength of 0 to 500 μmM. And a method for producing a hepatocyte growth factor production inducer. It is preferable to elute with a buffer containing a salt having an ionic strength of 100 μm and / or 300 μm.
　本発明の肝細胞増殖因子産出誘導剤は、肝細胞増殖因子産出細胞に作用し、肝細胞増殖因子をコードするmRNAの転写を促進、又は肝細胞増殖因子のタンパク質合成を促進することで、肝細胞増殖因子の産出を誘導することができる。また、本発明に用いられる植物は、漢方薬として長年使用されてきた生薬であり、安全性が高いものと考えられる。 The hepatocyte growth factor production inducer of the present invention acts on hepatocyte growth factor-producing cells, promotes transcription of mRNA encoding hepatocyte growth factor, or promotes protein synthesis of hepatocyte growth factor. The production of cell growth factors can be induced. The plant used in the present invention is a herbal medicine that has been used for many years as a traditional Chinese medicine, and is considered to be highly safe.
本発明の肝細胞増殖因子産出誘導剤の活性を示す図である。It is a figure which shows the activity of the hepatocyte growth factor production inducer of this invention. 本発明の肝細胞増殖因子産出誘導剤のSephadex LH-20カラムでの溶出曲線を示す図である。It is a figure which shows the elution curve in the Sephadex LH-20 column of the hepatocyte growth factor production inducer of this invention. 本発明の肝細胞増殖因子産出誘導剤のSephadex LH-20カラムの溶出で得られた画分の肝細胞増殖因子産出誘導活性を示す図である。It is a figure which shows hepatocyte growth factor production-inducing activity of the fraction obtained by elution of the Sephadex® LH-20 column of the hepatocyte growth factor production inducer of the present invention. 本発明の肝細胞増殖因子産出誘導剤のDEAE陰イオン交換カラムでの溶出曲線を示す図である。It is a figure which shows the elution curve in the DEAE anion exchange column of the hepatocyte growth factor production inducer of this invention. 本発明の肝細胞増殖因子産出誘導剤からDEAE陰イオン交換カラムの溶出で得られた画分の肝細胞増殖因子産出誘導活性を示す図である。FIG. 2 is a graph showing hepatocyte growth factor production-inducing activity of a fraction obtained by elution of a DEAE anion exchange column from the hepatocyte growth factor production inducer of the present invention.
　以下、本発明について詳細に説明する。本発明の肝細胞増殖因子産出誘導剤は、植物由来の有効成分を含む。より好ましくは、紅花、莪朮、枸杞子、石菖蒲、益智仁、欝金、人参、田七人参、忍冬藤、大棗、甘草、仙茅及び蓮子芯から選ばれる１又は複数由来の有効成分を含む。なお、本明細書において日本薬局方は、第一六改正日本薬局方をいう。 Hereinafter, the present invention will be described in detail. The hepatocyte growth factor production inducer of the present invention contains a plant-derived active ingredient. More preferably, it is an active ingredient derived from one or more selected from safflower, persimmon, coconut, sarcophagus, Masutoshi Jin, eel gold, carrot, ginseng, ginseng, ninfuto, daegu, licorice, sensu and lotus core. including. In this specification, the Japanese Pharmacopoeia refers to the 16th revised Japanese Pharmacopoeia.
紅花（コウカ）は、キク科ベニバナ属の一年草又は越年草のベニバナ（Carthamas tinctorius L）をいい、古くから紅色染料や食用油として広く一般に知られている。紅花の根は、浄血薬として、とくに婦人薬として繁用され、例えば生理不順、冷え性、更年期障害などにも効き目があり、また、血行障害によるお血の改善にも効き目がある。乾燥させた花は、血行促進作用がある生薬として日本薬局方にも収載されており、種々の漢方処方に用いられている。 Safflower (Kouka) is an annual or perennial safflower (Carthamas tinctorius L), which has been widely known as a red dye and edible oil since ancient times. Safflower root is frequently used as a blood-purifying agent, especially as a gynecological drug, and is effective for, for example, irregular menstruation, coldness, and menopause, and is also effective in improving blood due to blood circulation disorders. The dried flowers are listed in the Japanese Pharmacopoeia as herbal medicines that have a blood circulation promoting action, and are used in various Chinese medicine prescriptions.
本発明においては、紅花の葉、茎、花、根などの各部位及び全草をそのまま又は乾燥したものを用いることができる。花もしくは地上部位全草が好ましく、管状花をそのまま又は黄色色素の大部分を除いた日本薬局方に記載の紅花を用いることがより好ましい。また、紅花は、圧搾して板状としたものも好ましく用いることができる。 In the present invention, it is possible to use each part such as safflower leaves, stems, flowers, roots and the whole plant as it is or dried. A flower or whole plant on the ground is preferable, and it is more preferable to use a safflower as described in the Japanese Pharmacopoeia as it is or without removing most of the yellow pigment. Further, safflower can be preferably used by pressing it into a plate shape.
　莪朮（ガジュツ）は、ショウガ科ウコン属の1種である多年草のガジュツ（Curcuma zedoaria）をいい、日本薬局方に収録される芳香健胃作用を有する生薬である。また、莪朮から抽出される成分が神経成長因子産生増強作用を有することが知られている ガ (Gadju) is a kind of perennial herb (Curcuma zedoaria), a kind of ginger family turmeric genus, and is a herbal medicine with an aromatic healthy stomach action recorded in the Japanese Pharmacopoeia. In addition, it is known that components extracted from sputum have nerve growth factor production enhancing action
本発明においては、莪朮の根茎もしくは湯通ししたものをそのまま又は乾燥したものを好ましく用いることができ、日本薬局方に記載の莪朮がより好ましい。 In the present invention, it is possible to preferably use the dried rhizome or boiled potato as it is or dried, and the cocoon described in the Japanese Pharmacopoeia is more preferable.
枸杞子（クコシ）は、クコの実をいい、中国原産のナス科の落葉低木であるクコ（Lycium chinense Miller又はLyciumbarbatum L.）が好ましく用いられる。日本薬局方に収載されている生薬で、抗疲労抗老化作用、免疫賦活作用、肝細胞保護作用、抗脂肪肝作用、血中脂肪降下作用、血糖降下作用、血圧降下作用などが知られている。本発明においては、枸杞子の果実をそのまま又は乾燥したものを好ましく用いることができ、日本薬局方に記載の枸杞子がより好ましい。 The coconut is a fruit of wolfberry, and wolfberry (Lycium chinense Miller or Lyciumbarbatum L.) which is a deciduous shrub of the solanaceous family native to China is preferably used. Herbal medicines listed in the Japanese Pharmacopoeia, known for anti-fatigue anti-aging, immunostimulatory, hepatocyte protection, anti-fatty liver, blood fat lowering, blood glucose lowering, blood pressure lowering, etc. . In the present invention, the coconut fruit as it is or dried can be preferably used, and the coconut described in the Japanese Pharmacopoeia is more preferable.
石菖蒲（セキショウブ）は、中国医学では芳香開竅、寧心安神の効能が認められており、他の安神薬草類を配合して用いられ、癲狂、痴呆の治療、あるいは健忘、耳鳴、難聴などにも使用されている。本発明においては、石菖蒲は、サトイモ科のセキショウ（Acorus gramineusAcorus gramineus）の根又は茎、葉をそのまま又は乾燥したものを好ましく用いることができる。 Sekishobu has been recognized in Chinese medicine for the benefits of fragrance opening and Ningshin Anjin, and is used in combination with other Anjin herbs to treat frenzy, dementia, amnesia, tinnitus, and hearing loss. Has also been used. In the present invention, the stalagmite can be preferably used as it is or after drying the roots, stems, or leaves of taro (Acorus gramineus Acorus gramineus).
益智仁（ヤクチニン）は、日本薬局方に収載されている生薬であり、健胃、抗利尿、唾液分泌抑制作用が知られている。本発明においては、益智仁は、ショウガ科に属する益智（ヤクチ；Alpinia oxyphylla Miquel）の果実をそのまま又は乾燥したものを好ましく用いることができ、日本薬局方に記載の益智（仁）がより好ましい。 Masuthijin (yakutinin) is a herbal medicine listed in the Japanese Pharmacopoeia, and is known to have a healthy stomach, antidiuretic, and salivary secretion inhibiting action. In the present invention, Masuthijin can be preferably used as it is or dried from the fruits of Mashiko (Yakuchi; Alpinia oxyphylla Miquel). More preferred.
欝金は、ショウガ科の多年草で、香料、着色料、生薬として用いられ、英語名のターメリックとしてよく知られ、インド料理のカレーの材料としても使われている。生薬としての薬効には、胆汁の分泌促進や健胃作用などが知られている。 Gold is a perennial plant belonging to the ginger family and is used as a flavoring, coloring, and herbal medicine. It is well known as the English name turmeric, and it is also used as a material for curry in Indian cuisine. As a medicinal effect as a crude drug, the promotion of bile secretion and the action of a healthy stomach are known.
本発明においては、鬱金は、ショウガ科の多年草ウコンの根又は茎をそのまま乾燥したものを好ましく用いることができる。また、ウコンには春に赤色の花をつける春ウコン（Curcuma aromtica S）と、秋に白色の花をつける秋ウコン（Curcuma longa L）とがあり、主な薬効成分であるクルクミンを多く含む秋ウコンがより好ましい。 In the present invention, the gold can be preferably used by drying the roots or stems of ginger perennial turmeric as it is. In addition, there are spring turmeric (Curcuma aromtica S) that attaches red flowers in the spring and autumn turmeric (Curcuma longa L) that attaches white flowers in the fall, which is rich in curcumin, the main medicinal ingredient. Turmeric is more preferable.
仙茅は、生薬として「温腎壮陽、去寒除湿」の効果があり強壮を目的に使用されてきた。また、老人の夜間尿、婦人の更年期による諸症状にも応用されている。また、免疫力賦活や興奮性機能も知られている。本発明においては、仙茅は、ヒガンバナ科のキンバイザサ（Curculigo orchioides GAERIN.）の根茎をそのまま又は乾燥したものを好ましく用いることができる。 Sensen has been used as a herbal medicine because it has the effect of “Warm kidney soyo, de-humidification”. It has also been applied to night urine in elderly people and various symptoms caused by menopause in women. In addition, immune activation and excitability are also known. In the present invention, the sens can preferably be used as it is or after drying the rhizomes of Curculigo ナ orchioides GAERIN.
　人参は、日本薬局方に収載されている生薬で種々の漢方処方に配合されている。その効能としては、抗糖尿作用、抗癌作用、心臓強化及び血圧調整、肝機能強化、胃腸機能強化、ストレス解消及び強壮効果、体力（精力）増進、脳機能強化、老化抑制、放射線照射防御作用、貧血回復効果及び造血作用、免疫機能増進、消炎作用及び虚弱体質改善効果などが知られている。 Ginseng is a herbal medicine listed in the Japanese Pharmacopoeia and is blended in various Kampo prescriptions. Its effects include anti-diabetic action, anti-cancer action, heart strengthening and blood pressure adjustment, liver function strengthening, gastrointestinal function strengthening, stress relieving and tonic effect, physical strength (strength) enhancement, brain function strengthening, aging suppression, radiation irradiation protection action In addition, an anemia recovery effect and a hematopoietic effect, an immune function enhancement, an anti-inflammatory effect and a weak constitution improving effect are known.
　本発明においては、人参は、ウコギ科の多年草オタネニンジン（Panax ginseng C.A. Meyer）の細根を除いた根又は軽く湯通ししたものを用いることができ、日本薬局方に記載の人参がより好ましい。また人参の皮を剥ぎ、自然乾燥又は６０℃以下で熱風乾燥させて水分を１５％以下とした白参、蒸気を当てて蒸したものを乾燥した紅参も好ましく用いることができる。 In the present invention, the ginseng can be obtained by removing the roots of the perennial perennial ginseng (Panax ginseng C.A. Meyer) or lightly boiled, and the ginseng described in the Japanese Pharmacopoeia is more preferable. Red ginseng that has been peeled off ginseng and dried naturally or by hot-air drying at 60 ° C. or less to a moisture content of 15% or less, or dried steamed steamed ginseng can be preferably used.
　田七人参（デンシチニンジン）は、ウコギ科の薬用植物であり、中国南部原産で、雲南省や広西チワン族自治区の高地で栽培されている。田七人参の効能としては、止血作用と活血（血液循環の改善）作用を併せ持つことが知られている。また、抗ウイルス作用、抗コレステロール作用、抗腫瘍作用なども知られている。 Denshichinin ginseng is a medicinal plant belonging to the family Araceae, native to southern China, and cultivated in the highlands of Yunnan Province and Guangxi Zhuang Autonomous Region. As an effect of the ginseng, it is known to have both hemostatic action and active blood (improvement of blood circulation). Further, antiviral action, anticholesterol action, antitumor action and the like are also known.
本発明においては、田七人参は、三七人参（サンシチニンジン）とも呼ばれるウコギ科の多年草（Panax notoginseng）の根又は茎をそのまま又は乾燥したものを好ましく用いることができる。 In the present invention, Panax ginseng (Panax notoginseng) roots or stems, which are also referred to as Sanchi ginseng, can be preferably used as they are or dried.
　忍冬藤は、単に忍冬ともいわれ、日本薬局方にも記載された漢方生薬である。昔から忍冬を癰疽（体内外の炎症）に有効であるとして関節炎、気管支炎などの治療に使用されてきた。また、流行性風邪、各種化膿性感染症、湿熱による関節の発赤、腫脹、肝毒性保護及び細胞免疫能力低下に有効であることも知られている。 Ninfuto is simply referred to as Shinobifu and is a herbal medicine described in the Japanese Pharmacopoeia. It has been used for the treatment of arthritis, bronchitis, etc. for a long time as it is effective for hemorrhoids (inflammation inside and outside the body). It is also known to be effective for epidemic colds, various purulent infections, redness of joints due to wet heat, swelling, protection against hepatotoxicity and reduction of cellular immunity.
本発明においては、忍冬藤は、スイカズラ（Loniceraj aponica Thunberg （Caprifoliaceae））の葉及び茎をそのまま又は乾燥したものを好ましく用いることができ、日本薬局方に記載の忍冬がより好ましい。 In the present invention, ninto-wisteria can be preferably used with the leaves and stems of honeysuckle (Loniceraj aponica Thunberg (Caprifoliaceae)) as they are or dried, and ninfuto described in the Japanese Pharmacopoeia is more preferable.
　大棗（タイソウ）は、クロウメモドキ科のナツメの果実で糖、粘液質、リンゴ酸、酒石酸などを含み、強壮作用・鎮静作用などが知られている。また、甘味があり、また副作用の緩和などを目的に種々の漢方薬に配合されている。本発明においては、クロウメモドキ科のナツメ（Zizyphus jujube Mill. var. inermis Rehder (Rhamnaceae)）又はその品種の果実をそのまま又は乾燥したものを好ましく用いることができ、日本薬局方に記載の大棗がより好ましい。 タ Taisu is a jujube fruit that contains sugar, mucus, malic acid, tartaric acid, etc., and is known for its tonic and sedative effects. In addition, it has sweetness and is blended in various Chinese medicines for the purpose of alleviating side effects. In the present invention, a jujube of the family Chromaceae (Zizyphus jujube Mill. Var. Inermis Rehder (Rhamnaceae)) or a fruit of its cultivar can be preferably used as it is or dried. preferable.
　甘草（カンゾウ）は、日本薬局方に収載されている生薬で、グリチルリチンや配糖体などを含有し、鎮痛、抗炎症、胃痛、鎮咳去痰、解毒、十二指腸潰瘍などの効果が知られている。本発明においては、マメ科のウラルカンゾウ（Glycyrrhiza uralensis Fisch）又はナンキンカンゾウ（Glycyrrhiza glabra Linne (Leguminosae）)の根及びストロン、又はそれらの周皮を除いたもの（皮去リカンゾウ）をそのままあるいは乾燥したものを好ましく用いることができ、日本薬局方に記載の甘草がより好ましい。 Licorice is a herbal medicine listed in the Japanese Pharmacopoeia and contains glycyrrhizin and glycosides, and is known to have analgesic, anti-inflammatory, gastric pain, antitussive expectorant, detoxification, and duodenal ulcer. In the present invention, the roots and strons of leguminous larvae (Glycyrrhiza uralensis Fisch) or licorice elephant (Glycyrrhiza glabra Linne (Leguminosae)), or those excluding their pericarp (peeled licorice) are either left as is or dried. A licorice described in Japanese Pharmacopoeia is more preferable.
　蓮子芯（レンシシン）は、通常お茶として飲まれ、心臓や肝臓に有効で精神安定にも効果があるといわれている。本発明においては、スイレン科の植物である蓮Nelumbo nuciferaの成熟した種子の胚芽部分をそのまま又は乾燥したものを好ましく用いることができる。 Rencoshin is usually drunk as tea and is said to be effective for the heart and liver and also for mental stability. In the present invention, it is possible to preferably use a dried seed portion of a mature seed of a lotus Nelumbo nucifera which is a water lily family.
　また、別の本発明の肝細胞増殖因子産出誘導剤は、前述の植物から製薬上又は食品衛生上許容される極性溶媒により有効成分を抽出して得られる抽出物である。本発明で用いられる抽出物は、前述の植物からなる生薬（好ましくは、乾燥生薬）をそのまま又は裁断、細切、破砕（好ましくは、数ｍｍ～１ｃｍ前後に裁断、細切、破砕）、もしくは粉砕したもの、あるいは粉末にしたものを抽出溶媒にて抽出することによって得ることができる。 Another hepatocyte growth factor production inducer of the present invention is an extract obtained by extracting an active ingredient from the above-mentioned plant with a polar solvent that is pharmaceutically or food hygienically acceptable. The extract used in the present invention is a herbal medicine (preferably a dry herbal medicine) composed of the above-mentioned plants as it is or is cut, chopped, crushed (preferably cut, shredded, crushed around several mm to 1 cm), or It can be obtained by extracting a pulverized product or a powdered product with an extraction solvent.
　本発明が２種以上の植物から抽出された有効成分を含む場合は、各植物からそれぞれ抽出した有効成分を混合することで本発明の肝細胞増殖因子産出誘導剤を得ることができる。より好ましくは、各植物を混合して得られた混合物から抽出することで得られた肝細胞増殖因子産出誘導剤である。抽出物を混合して得られた肝細胞増殖因子産出誘導剤よりも、各植物を混合して得られた混合物から抽出することで得られた肝細胞増殖因子産出誘導剤の方が、顕著に高い肝細胞増殖因子産出効果が確認されたためである。 When the present invention contains active ingredients extracted from two or more kinds of plants, the hepatocyte growth factor production inducer of the present invention can be obtained by mixing the active ingredients extracted from each plant. More preferably, it is a hepatocyte growth factor production inducer obtained by extracting from a mixture obtained by mixing each plant. The hepatocyte growth factor production inducer obtained by extracting from the mixture obtained by mixing each plant is more prominent than the hepatocyte growth factor production inducer obtained by mixing the extract. This is because a high hepatocyte growth factor producing effect was confirmed.
　抽出溶媒は、製薬上又は食品衛生上許容される極性溶媒が好ましい。極性溶媒としては、例えば、水（例えば、精製水、蒸留水、水道水等）、エタノール、水とエタノールとの混合溶媒（含水エタノール）、酢酸、酢酸と水との混合溶媒、水とエタノールと酢酸との混合溶媒等が挙げられる。好ましくは水、又は含水エタノールである。含水エタノールにおける含水率は１～９９容量％である。水又は含水エタノールには、酸を添加してもよい。酸としては、例えば、塩酸等の無機酸、リンゴ酸又はクエン酸等の有機酸などが挙げられる。 The extraction solvent is preferably a polar solvent that is acceptable for pharmaceuticals or food hygiene. Examples of the polar solvent include water (for example, purified water, distilled water, tap water, etc.), ethanol, a mixed solvent of water and ethanol (hydrous ethanol), acetic acid, a mixed solvent of acetic acid and water, water and ethanol, and the like. Examples thereof include a mixed solvent with acetic acid. Water or water-containing ethanol is preferred. The water content in hydrous ethanol is 1 to 99% by volume. An acid may be added to water or hydrous ethanol. Examples of the acid include inorganic acids such as hydrochloric acid, and organic acids such as malic acid and citric acid.
　抽出方法は特に限定されず、浸漬法や向流抽出法を採用することができる。また、常温抽出又は常圧下で溶媒の沸点までの範囲で加熱抽出できる。必要により、減圧又は加圧下で抽出してもよい。加圧は、１気圧を超える圧力であればよく、約２～３気圧が好ましい。 The extraction method is not particularly limited, and an immersion method or a countercurrent extraction method can be employed. Moreover, it can heat-extract in the range to the boiling point of a solvent under normal temperature extraction or a normal pressure. If necessary, extraction may be performed under reduced pressure or increased pressure. The pressurization may be a pressure exceeding 1 atm, and preferably about 2 to 3 atm.
抽出溶媒（極性溶媒）を植物又は植物の混合物に対し１０～１００倍量、好ましくは１０～４０倍量を加え、通常、常温から常圧下又は加圧下で溶媒の沸点までの範囲で３０分～５時間、好ましくは１～３時間（１、２、３時間）浸漬し、公知の方法でろ過することにより抽出液を得ることができる。抽出液は、そのまま又は他の抽出液と混合することで、抽出物として使用することができる。また、抽出液を濃縮したもの又は、濃縮乾燥もしくは凍結乾燥したものを抽出物として使用することもできる。 An extraction solvent (polar solvent) is added in an amount of 10 to 100 times, preferably 10 to 40 times the amount of a plant or a mixture of plants, and usually 30 minutes or more in the range from room temperature to the boiling point of the solvent under normal pressure or pressure. The extract can be obtained by immersing for 5 hours, preferably 1 to 3 hours (1, 2, 3 hours) and filtering by a known method. The extract can be used as an extract as it is or by mixing with another extract. Moreover, what concentrated the extract liquid, what was concentrated and dried or freeze-dried can also be used as an extract.
　本発明の肝細胞増殖因子産出誘導剤の有効成分には少なくとも２種類の肝細胞増殖因子産出誘導活性を有する分子が存在し、１の肝細胞増殖因子産出誘導活性を有する分子は陰性荷電であり、別の肝細胞増殖因子産出誘導活性を有する分子は中性荷電である。本発明の有効成分には、４種類の肝細胞増殖因子産出誘導活性を有する分子が存在する場合もある。 The active ingredient of the hepatocyte growth factor production inducer of the present invention includes at least two types of molecules having hepatocyte growth factor production-inducing activity, and one molecule having hepatocyte growth factor production-inducing activity is negatively charged. Another molecule having activity to induce the production of hepatocyte growth factor is neutrally charged. The active ingredient of the present invention may contain four types of molecules having hepatocyte growth factor production-inducing activity.
　本発明の肝細胞増殖因子産出誘導剤は、抽出物と所望により配合される医薬上又は食品衛生上許容される担体とを、公知の方法により混合して製剤化することにより、経口的に摂取できる医薬又は食品として容易に調製できる。食品とは、健康食品、機能性食品及びサプリメントを含む。また、経鼻的又は経皮的に摂取する医薬や、注射により投与する医薬、経皮的に使用する美容品としての調整も可能である。 The hepatocyte growth factor production inducer of the present invention is orally ingested by mixing an extract and a pharmaceutically or food hygienically acceptable carrier optionally blended by a known method. It can be easily prepared as a pharmaceutical or food product. The food includes health foods, functional foods and supplements. It is also possible to prepare a medicine to be taken nasally or transdermally, a medicine to be administered by injection, or a cosmetic product to be used transdermally.
　本発明の肝細胞増殖因子産出誘導剤は、医薬上又は食品衛生上許容される担体を用いて製剤化してもよい。製剤としては、例えば固形製剤又は液状製剤が挙げられる。医薬上又は食品衛生上許容される担体としては、製剤素材として慣用の各種有機又は無機担体物質が用いられ、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤や、液状製剤における溶剤、溶解補助剤、懸濁化剤（乳化剤、増粘剤）等が挙げられる。また必要に応じて、保存剤、抗酸化剤、着色剤、甘味剤、香料等の製剤添加物を用いることもできる。 The hepatocyte growth factor production inducer of the present invention may be formulated using a pharmaceutical or food hygiene acceptable carrier. Examples of the preparation include a solid preparation and a liquid preparation. As a carrier acceptable for pharmaceutical or food hygiene, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations and solvents in liquid preparations. , Solubilizers, suspending agents (emulsifiers, thickeners) and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners, and fragrances can be used.
　本発明の肝細胞増殖因子産出誘導剤は、治療薬、予防薬、美容品、又は機能性食品として調製することができる。本発明の治療薬、予防薬、美容品、又は機能性食品における肝細胞増殖因子産出誘導剤の含有量は、例えば固形製剤であれば、植物由来の有効成分を抽出した抽出物として、製剤の約１～９９重量％であり、好ましくは約５～９０重量％であり、より好ましくは、約１０～８０重量％である。液状製剤であれば、抽出乾燥物として製剤の約０．０００１～８０重量％であり、好ましくは約１０～５０重量％であり、より好ましくは、約２０～３５重量％である。 The hepatocyte growth factor production inducer of the present invention can be prepared as a therapeutic agent, a preventive agent, a cosmetic product, or a functional food. If the content of the hepatocyte growth factor production inducer in the therapeutic agent, preventive agent, beauty product, or functional food of the present invention is, for example, a solid preparation, the extract of the plant-derived active ingredient is extracted as the extract. About 1 to 99% by weight, preferably about 5 to 90% by weight, and more preferably about 10 to 80% by weight. In the case of a liquid preparation, it is about 0.0001 to 80% by weight, preferably about 10 to 50% by weight, more preferably about 20 to 35% by weight of the preparation as a dry extract.
また液状製剤は、植物の抽出液をそのまま又は濃縮した濃縮物をそのまま使用することもできる。好ましくは、有効成分の由来となる植物に対し、１０～４０倍の極性溶媒（好ましくは、水）を用い、１～３時間（１、２、３時間）抽出した場合の抽出率（エキスの回収率）が２５％～３３％である抽出液を液状製剤とすることができる。 Moreover, the liquid formulation can also use the concentrate which concentrated the extract of a plant as it is, or can be used as it is. Preferably, the extraction rate (extract of extract) is 1 to 3 hours (1, 2, 3 hours) extracted with 10 to 40 times the polar solvent (preferably water) to the plant from which the active ingredient is derived. An extract having a recovery rate of 25% to 33% can be made into a liquid preparation.
　本発明の治療薬、予防薬、美容品、又は機能性食品は、経口的に摂取されることが好ましい。食後、食前又は食間に摂取することがより好ましい。本発明の摂取量及び摂取回数は、年齢、体重、投与形態などにより異なるが、通常成人一日あたり植物由来の有効成分を抽出した抽出物として、体重１ｋｇあたり約１ｍｇ～５００ｍｇ、好ましくは約５ｍｇ～３００ｍｇとなるように服用できる。この量を、通常１～数回、好ましくは１～３回に分けて服用することが好ましい。 The therapeutic agent, preventive agent, beauty product, or functional food of the present invention is preferably taken orally. More preferably after meal, before meal or between meals. The amount of intake and the number of intakes of the present invention vary depending on age, body weight, dosage form, etc., but usually about 1 mg to 500 mg per kg body weight, preferably about 5 mg as an extract obtained by extracting an active ingredient derived from a plant per adult day. Can be taken to be ~ 300 mg. This amount is usually taken 1 to several times, preferably 1 to 3 times.
液状製剤の場合、極性溶媒（好ましくは、水）で抽出して得られる抽出液を摂取することが好ましく、通常１～数回、好ましくは１～３回に分けて摂取することがさらに好ましい。 In the case of a liquid preparation, it is preferable to ingest an extract obtained by extraction with a polar solvent (preferably water), usually 1 to several times, more preferably 1 to 3 times.
さらに、本発明の治療薬又は予防薬は、経鼻的、経皮的に投与されるものでもよく、注射器を用いて、静脈、腹腔、皮内、皮下、筋肉等に投与されてもよい。本発明の美容品は、経皮的に投与されるものであってもよい。 Furthermore, the therapeutic agent or prophylactic agent of the present invention may be administered nasally or transdermally, and may be administered intravenously, abdominally, intradermally, subcutaneously, muscle, etc. using a syringe. The cosmetic product of the present invention may be administered transdermally.
　本発明の肝細胞増殖因子産出誘導剤は、生体内での肝細胞増殖因子の産出を誘導する。したがって、肝細胞増殖因子の産出によって治療及び／又は予防されるさまざまな疾患に効果を発揮する。具体的には、肝疾患（肝炎、肝硬変、肝不全、その他の肝障害）、腎疾患（糸球体腎炎、腎不全、腎性貧血症、糖尿病性腎症、その他の腎障害）、皮膚疾患、眼疾患（角膜潰瘍等）、肺疾患（肺炎、肺気腫、肺結核、慢性閉塞性肺疾患、塵肺、肺線維症、その他の肺障害）、胃十二指腸疾患（胃炎、胃潰瘍、十二指腸潰瘍、その他の胃十二指腸障害）、癌疾患や癌治療における障害、心臓又は四肢の虚血性疾患又は動脈疾患、血液疾患（血小板減少症、血流障害等）、骨疾患（骨粗鬆症、骨異形成症、変形性関節炎、その他の骨障害）、又は中枢疾患（神経分化異常症等）等が挙げられる。本発明の治療薬、予防薬、美容品、又は機能性食品は、これらの疾患の治療及び／又は予防に用いることができる。 The hepatocyte growth factor production inducer of the present invention induces the production of hepatocyte growth factor in vivo. Therefore, it exerts an effect on various diseases that are treated and / or prevented by production of hepatocyte growth factor. Specifically, liver diseases (hepatitis, cirrhosis, liver failure, other liver disorders), kidney diseases (glomerulonephritis, kidney failure, renal anemia, diabetic nephropathy, other kidney disorders), skin diseases, Eye disease (corneal ulcer, etc.), lung disease (pneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis, pulmonary fibrosis, other lung disorders), gastroduodenal disease (gastritis, gastric ulcer, duodenal ulcer, other gastroduodenum) Disorders), cancer diseases and cancer treatment disorders, heart or limb ischemic or arterial diseases, blood diseases (thrombocytopenia, blood flow disorders, etc.), bone diseases (osteoporosis, osteodysplasia, osteoarthritis, etc.) Bone disorders), or central diseases (such as abnormal neurodifferentiation). The therapeutic agent, preventive agent, beauty product, or functional food of the present invention can be used for the treatment and / or prevention of these diseases.
　本発明の治療薬、予防薬、美容品、又は機能性食品の投与又は摂取対象としては、ヒトが好ましいが、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、サル、ブタ等の哺乳類や、鳥類、爬虫類、両生類、魚類に属する愛玩動物や畜産・養殖種、実験用動物等に投与又は摂取させてもよい。 As a subject for administration or ingestion of the therapeutic agent, preventive agent, beauty product, or functional food of the present invention, humans are preferable, but mammals such as mice, rats, rabbits, dogs, cats, cows, monkeys, pigs, and birds , Reptiles, amphibians, fish, pet animals, livestock / cultured species, experimental animals, etc. may be administered or ingested.
　また、本発明は別の肝細胞増殖因子産出誘導剤の製造方法を提供する。具体的には、上述の肝細胞増殖因子産出誘導剤を緩衝液とともに陰イオン交換樹脂に添加する添加工程と、該陰イオン交換樹脂から、イオン強度0 mM～500 mMの塩を含む緩衝液で溶出する溶出工程を含有する、肝細胞増殖因子産出誘導剤の製造方法である。 The present invention also provides another method for producing a hepatocyte growth factor production inducer. Specifically, an addition step of adding the above-described hepatocyte growth factor production inducer together with a buffer to an anion exchange resin, and a buffer solution containing a salt having an ionic strength of 0 to 500 μm from the anion exchange resin. It is a manufacturing method of the hepatocyte growth factor production inducer containing the elution process to elute.
　本発明において、陰イオン交換樹脂としては、ジエチルエタノールアミン（DEAE）等を用いることができる。また、溶出する緩衝液に含まれる塩としては、塩化ナトリウム等を用いることができる。 In the present invention, diethylethanolamine (DEAE) or the like can be used as the anion exchange resin. Moreover, sodium chloride etc. can be used as a salt contained in the buffer solution to elute.
本発明において、より好ましくは、溶出工程は、イオン強度100 mM及び／又は300 mMの塩を含む緩衝液で溶出する溶出工程であり得る。本条件で溶出された分画は、他の条件で溶出された分画よりも、高い肝細胞増殖因子産出誘導活性を有する分子を含み得るためである。 In the present invention, more preferably, the elution step may be an elution step of elution with a buffer containing a salt having an ionic strength of 100 μm and / or 300 μm. This is because the fraction eluted under these conditions may contain a molecule having higher hepatocyte growth factor production-inducing activity than the fraction eluted under other conditions.
　以下、実施例を通じて本発明をさらに詳細に説明する。しかし、本発明の範囲がこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail through examples. However, the scope of the present invention is not limited to these examples.
紅花由来抽出物の作製
乾燥された紅花を数ミリ～１ｃｍ前後に裁断加工した。得られた紅花に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、紅花由来の有効成分を含む抽出物を作製した。 Preparation of safflower-derived extract The dried safflower was cut into several millimeters to 1 cm. Water was added to the resulting safflower, followed by boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from safflower.
実施例１の抽出物が有する肝細胞増殖因子産出誘導活性を、肝細胞増殖因子産出能を有するヒト皮膚線維芽細胞（SF4-1細胞）を用いて測定した。SF4-1細胞を、1.5 x104 cells/well の細胞密度で、96 穴プレートに播種した。1重量％ のFCSを添加したDMEM培地に交換し、実施例１の抽出物をSF4-1細胞の培養液に複数の濃度となるよう添加した。また、陽性対照として肝細胞増殖因子産出活性を有することが知られているヘパリンを1μg/mL添加し、陰性対照として蒸留水を添加した。SF4-1細胞を48時間培養し、培養液中の肝細胞増殖因子量をELISA法で測定した。肝細胞増殖因子の産出量が最も高い実施例１の抽出物の添加条件は、0.5 mg当量/mLの実施例１の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で1.4倍であった。 The hepatocyte growth factor production inducing activity of the extract of Example 1 was measured using human skin fibroblasts (SF4-1 cells) having hepatocyte growth factor production ability. SF4-1 cells were seeded in a 96-well plate at a cell density of 1.5 × 10 4 cells / well. The medium was replaced with a DMEM medium supplemented with 1% by weight of FCS, and the extract of Example 1 was added to the culture solution of SF4-1 cells at a plurality of concentrations. In addition, 1 μg / mL heparin known to have hepatocyte growth factor producing activity was added as a positive control, and distilled water was added as a negative control. SF4-1 cells were cultured for 48 hours, and the amount of hepatocyte growth factor in the culture was measured by ELISA. The condition for adding the extract of Example 1 with the highest output of hepatocyte growth factor is when 3% by volume of the extract of Example 1 at 0.5 mg equivalent / mL is added to the culture medium of SF4-1 cells. The production amount of hepatocyte growth factor was 1.4 times relative to the negative control, where the amount of hepatocyte growth factor was 1.
莪朮由来抽出物の作製
乾燥された莪朮を数ミリ～１ｃｍ前後に裁断加工した。得られた莪朮に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、莪朮由来の有効成分を含む抽出物を作製した。実施例２の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例２の抽出物の添加条件は、0.5 mg当量/mLの実施例２の抽出物をSF4-1細胞の培養液に1容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で1.6倍であった。 Preparation of cocoon-derived extract The dried cocoon was cut into several millimeters to 1 cm. Water was added to the obtained koji, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from koji. When the hepatocyte growth factor production-inducing activity of the extract of Example 2 was measured by the same method as in Example 1, the addition conditions of the extract of Example 2 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 2 at 0.5 mg equivalent / mL is added to the SF4-1 cell culture solution, and the amount of hepatocyte growth factor produced is defined as 1 in the negative control. The relative amount was 1.6 times.
枸杞子由来抽出物の作製
乾燥された枸杞子を数ミリ～１ｃｍ前後に裁断加工した。得られた枸杞子に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、枸杞子由来の有効成分を含む抽出物を作製した。実施例３の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例３の抽出物の添加条件は、0.5 mg当量/mLの実施例３の抽出物をSF4-1細胞の培養液に1容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で1.3倍であった。 Preparation of extract derived from eggplant The dried eggplant was cut into several millimeters to about 1 cm. Water was added to the resulting insulator, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from eggplant. When the hepatocyte growth factor production-inducing activity of the extract of Example 3 was measured by the same method as in Example 1, the addition conditions of the extract of Example 3 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 3 at 0.5 mg equivalent / mL is added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced is 1 as the amount of hepatocyte growth factor in the negative control The relative amount was 1.3 times.
石菖蒲由来抽出物の作製
乾燥された石菖蒲を数ミリ～１ｃｍ前後に裁断加工した。得られた石菖蒲に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、石菖蒲由来の有効成分を含む抽出物を作製した。実施例４の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例４の抽出物の添加条件は、0.5 mg当量/mLの実施例４の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で2.8倍であった。 Preparation of sarcophagus- derived extract The dried sarcophagus was cut into a few millimeters to 1 cm. Water was added to the obtained sarcophagus, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours using a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from sarcophagus. When the hepatocyte growth factor production-inducing activity of the extract of Example 4 was measured by the same method as in Example 1, the addition conditions of the extract of Example 4 with the highest production amount of hepatocyte growth factor were: This is the case where 3% by volume of the extract of Example 4 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was 1 as the amount of hepatocyte growth factor in the negative control The relative amount was 2.8 times.
欝金由来抽出物の作製
乾燥された欝金を数ミリ～１ｃｍ前後に裁断加工した。得られた欝金に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、欝金由来の有効成分を含む抽出物を作製した。実施例５の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例５の抽出物の添加条件は、0.5 mg当量/mLの実施例５の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で2.3倍であった。 Preparation of plating-derived extract The dried plating was cut into several millimeters to 1 cm. Water was added to the obtained plating, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from plating. When the hepatocyte growth factor production-inducing activity of the extract of Example 5 was measured by the same method as in Example 1, the addition condition of the extract of Example 5 with the highest production amount of hepatocyte growth factor was: This is a case where the extract of Example 5 at 0.5 mg equivalent / mL was added to the SF4-1 cell culture solution at 3% by volume, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.3 times.
人参由来抽出物の作製
乾燥された人参を数ミリ～１ｃｍ前後に裁断加工した。得られた人参に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、人参由来の有効成分を含む抽出物を作製した。 Preparation of ginseng-derived extract The dried ginseng was cut into several millimeters to 1 cm. Water was added to the obtained carrot, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing ginseng-derived active ingredients.
実施例６の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例６の抽出物の添加条件は、0.5 mg当量/mLの実施例６の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で2.5倍であった。 When the hepatocyte growth factor production-inducing activity of the extract of Example 6 was measured by the same method as in Example 1, the addition conditions of the extract of Example 6 with the highest output of hepatocyte growth factor were: This is the case where 3 vol% of the extract of Example 6 at 0.5 μmg equivalent / mL was added to the SF4-1 cell culture solution, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.5 times.
田七人参由来抽出物の作製
乾燥された田七人参を数ミリ～１ｃｍ前後に裁断加工した。得られた田七人参に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、田七人参由来の有効成分を含む抽出物を作製した。実施例７の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例７の抽出物の添加条件は、0.5 mg当量/mLの実施例７の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で3.1倍であった。 Production of extract derived from ginseng ginseng The dried ginseng was cut into several millimeters to 1 cm. Water was added to the obtained ginseng, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to prepare an extract containing an active ingredient derived from the ginseng. When the hepatocyte growth factor production-inducing activity of the extract of Example 7 was measured by the same method as in Example 1, the addition conditions of the extract of Example 7 with the highest output of hepatocyte growth factor were: This is the case where 3% by volume of the extract of Example 7 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.1 times.
忍冬藤由来抽出物の作製
乾燥された忍冬藤を数ミリ～１ｃｍ前後に裁断加工した。得られた忍冬藤に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、忍冬藤由来の有効成分を含む抽出物を作製した。実施例８の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例８の抽出物の添加条件は、0.5 mg当量/mLの実施例８の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で1.8倍であった。 Preparation of ninto-wisteria extract The dried ninto-wister was cut into several millimeters to 1 cm. Water was added to the obtained Shinobifuto and the mixture was boiled and extracted at 2-3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from Shinobu Toto. When the hepatocyte growth factor production-inducing activity of the extract of Example 8 was measured by the same method as in Example 1, the addition conditions of the extract of Example 8 with the highest production amount of hepatocyte growth factor were: This is the case where 3 vol% of the extract of Example 8 at 0.5 mg equivalent / mL was added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 1.8 times.
大棗由来抽出物の作製
乾燥された大棗を数ミリ～１ｃｍ前後に裁断加工した。得られた大棗に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、大棗由来の有効成分を含む抽出物を作製した。実施例９の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例９の抽出物の添加条件は、0.5 mg当量/mLの実施例９の抽出物をSF4-1細胞の培養液に1容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で1.6倍であった。 Preparation of extracts from potatoes The dried potatoes were cut into several millimeters to 1 cm. Water was added to the obtained large koji, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours with a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to prepare an extract containing an active ingredient derived from Taiho. When the hepatocyte growth factor production-inducing activity of the extract of Example 9 was measured by the same method as in Example 1, the addition conditions of the extract of Example 9 with the highest output of hepatocyte growth factor were as follows: This is the case where 1 vol% of the extract of Example 9 at 0.5 mg equivalent / mL is added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced is defined as 1 in the negative control. The relative amount was 1.6 times.
甘草由来抽出物の作製
乾燥された甘草を数ミリ～１ｃｍ前後に裁断加工した。得られた甘草に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、甘草由来の有効成分を含む抽出物を作製した。 Preparation of licorice-derived extract The dried licorice was cut into several millimeters to 1 cm. Water was added to the obtained licorice, and the mixture was boiled and extracted at 2-3 atmospheres for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to prepare an extract containing an active ingredient derived from licorice.
仙茅由来抽出物の作製
乾燥された仙茅を数ミリ～１ｃｍ前後に裁断加工した。得られた仙茅に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、仙茅由来の有効成分を含む抽出物を作製した。実施例１１の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例１１の抽出物の添加条件は、0.5 mg当量/mLの実施例１１の抽出物をSF4-1細胞の培養液に1容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で3.4倍であった。 Preparation of extract derived from senpo The dried senpo was cut into a few millimeters to 1 cm. Water was added to the obtained Sensen, and it was filtered by boiling at 2 to 3 atm for 2 hours using a pressure extractor. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from Sengan. When the hepatocyte growth factor production-inducing activity of the extract of Example 11 was measured by the same method as in Example 1, the addition conditions of the extract of Example 11 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 11 at 0.5 mg equivalent / mL was added to the culture solution of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.4 times.
蓮子芯由来抽出物の作製
乾燥された蓮子心を数ミリ～１ｃｍ前後に裁断加工した。得られた蓮子心に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、蓮子心由来の有効成分を含む抽出物を作製した。実施例１２の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例１２の抽出物の添加条件は、0.5 mg当量/mLの実施例１２の抽出物をSF4-1細胞の培養液に3容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で2.5倍であった。 Preparation of an extract derived from a lotus core The dried lotus core was cut into a few millimeters to about 1 cm. Water was added to the obtained lotus core, and the mixture was boiled and extracted at 2 to 3 atm for 2 hours with a pressure extractor and then filtered. The obtained filtrate was packaged in an aluminum pack to produce an extract containing an active ingredient derived from Renko heart. When the hepatocyte growth factor production-inducing activity of the extract of Example 12 was measured by the same method as in Example 1, the addition condition of the extract of Example 12 with the highest output of hepatocyte growth factor was: This is the case where 3% by volume of the extract of Example 12 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 2.5 times.
複数種の植物を用いた抽出物の作製（１）
実施例１～実施例１２の抽出物を等量ずつ混合した抽出物を作製した。実施例１３の抽出物が有する肝細胞増殖因子産出誘導活性を、実施例１と同様の方法で測定したところ、肝細胞増殖因子の産出量が最も高い実施例１３の抽出物の添加条件は、0.5 mg当量/mLの実施例１３の抽出物をSF4-1細胞の培養液に1容量％添加した場合であり、肝細胞増殖因子産出量は、陰性対照における肝細胞増殖因子量を1とした相対量で3.1倍であった。 Preparation of extracts using multiple types of plants (1)
Extracts were prepared by mixing equal amounts of the extracts of Examples 1 to 12. When the hepatocyte growth factor production-inducing activity of the extract of Example 13 was measured by the same method as in Example 1, the addition conditions of the extract of Example 13 with the highest production amount of hepatocyte growth factor were: This is the case where 1 vol% of the extract of Example 13 at 0.5 mg equivalent / mL was added to the culture medium of SF4-1 cells, and the amount of hepatocyte growth factor produced was defined as 1 in the negative control. The relative amount was 3.1 times.
複数種の植物を用いた抽出物の作製（２）
乾燥された紅花、莪朮、枸杞子、石菖蒲、欝金、人参、田七人参、忍冬藤、大棗、甘草、仙茅及び蓮子芯を、それぞれ数ミリ～１ｃｍ前後に裁断加工し、混合した。得られた混合物に水を加え、加圧抽出機にて、２～３気圧で２時間煮沸抽出後濾過した。得られた濾液をアルミパックに分包し、これら１２種類の植物由来の有効成分を含む抽出物を作製した。 Preparation of extracts using multiple types of plants (2)
Dried safflower, persimmon, eggplant, stone carp, shinkin, carrot, nanana ginseng, ninfuto, daikon, licorice, sensu and lotus core are each cut into several millimeters to 1 cm and mixed. . Water was added to the obtained mixture, and the mixture was subjected to boiling extraction at 2 to 3 atm for 2 hours using a pressure extractor, followed by filtration. The obtained filtrate was packaged in an aluminum pack to produce an extract containing these 12 kinds of plant-derived active ingredients.
本発明の肝細胞増殖因子産出誘導剤による肝細胞増殖因子産出誘導活性
　実施例１４の抽出物が有する肝細胞増殖因子産出誘導活性を、肝細胞増殖因子産出能を有するヒト皮膚線維芽細胞（SF4-1細胞）を用いて測定した。SF4-1細胞を、1.5 x104 cells/well の細胞密度で、96 穴プレートに播種した。1重量％ のFCSを添加したDMEM培地に交換し、0.5 mg当量/mLの実施例１５の抽出物をSF4-1細胞の培養液にそれぞれ1容量％、3容量％、10容量％添加した。また、陽性対照として肝細胞増殖因子産出活性を有することが知られているヘパリンを1μg/mL添加し、陰性対照として蒸留水を添加した。SF4-1細胞を48時間培養し、培養液中の肝細胞増殖因子量をELISA法で測定した。図１に、各培養液中における肝細胞増殖因子の量を、陰性対照における肝細胞増殖因子量を1とした相対量で示す。実施例１４の抽出物を添加したSF4-1細胞で、大量の肝細胞増殖因子が産出されていることがわかった。 Hepatocyte growth factor production-inducing activity by the hepatocyte growth factor production-inducing agent of the present invention The hepatocyte growth factor production-inducing activity of the extract of Example 14 is expressed as human skin fibroblasts (SF4 -1 cells). SF4-1 cells were seeded in a 96-well plate at a cell density of 1.5 × 10 4 cells / well. The medium was replaced with DMEM medium supplemented with 1% by weight of FCS, and 0.5 mg equivalent / mL of the extract of Example 15 was added to the culture medium of SF4-1 cells at 1%, 3%, and 10% by volume, respectively. In addition, 1 μg / mL heparin known to have hepatocyte growth factor producing activity was added as a positive control, and distilled water was added as a negative control. SF4-1 cells were cultured for 48 hours, and the amount of hepatocyte growth factor in the culture was measured by ELISA. FIG. 1 shows the amount of hepatocyte growth factor in each culture medium as a relative amount with the amount of hepatocyte growth factor in the negative control as 1. It was found that a large amount of hepatocyte growth factor was produced in SF4-1 cells to which the extract of Example 14 was added.
本発明の肝細胞増殖因子産出誘導剤の製造方法（１）
実施例１４の抽出物に２倍量の蒸留水を加え、室温で１時間混和し、遠心した。遠心後の上清を20% エタノール/水混合液により平衡化したSephadex LH-20カラム（GEヘルスケアバイオサイエンス社製）に添加し、同溶液を用いて溶出し、各画分を培養液に5%添加して肝細胞増殖因子産出誘導活性をELISA法で測定した。溶出曲線を図２に、HGF産出誘導活性測定結果を図３に示す。早く溶出される画分がメインの活性であり、遅く溶出される画分にも弱い活性が認められた。 Method for producing hepatocyte growth factor production inducer of the present invention (1)
Two times the amount of distilled water was added to the extract of Example 14, mixed for 1 hour at room temperature, and centrifuged. The supernatant after centrifugation is added to a Sephadex LH-20 column (manufactured by GE Healthcare Biosciences) equilibrated with a 20% ethanol / water mixture, and eluted using the same solution. After adding 5%, hepatocyte growth factor production-inducing activity was measured by ELISA. The elution curve is shown in FIG. 2, and the measurement result of HGF production inducing activity is shown in FIG. The fraction that eluted earlier was the main activity, and the weakly eluted fraction also showed weak activity.
本発明の肝細胞増殖因子産出誘導剤の製造方法（２）
　実施例１４の抽出物に２倍量の蒸留水を加え、室温で１時間混和し、遠心した。遠心後の上清を50 mM Tris-HCl[pH 8.0]で平衡化し、DEAE Sepharose FFカラム（GEヘルスケアバイオサイエンス社製）に添加した。添加したカラムを50 mM Tris-HCl[pH 8.0]で洗浄後、100 mM、300 mM、及び500 mMの塩化ナトリウムを含む50 mM Tris-HCl[pH 8.0]で順に溶出した。溶出された各画分を培養液に3%添加して肝細胞増殖因子産出誘導活性をELISA法で測定した。溶出曲線を図４に、測定結果を図５に示す。緩衝液のみの溶出画分及び各濃度の塩化ナトリウムを含む緩衝液の溶出画分で活性が認められた。したがって、上述の方法により、新たな肝細胞増殖因子産出誘導剤が製造されることが明らかとなった。また、実施例１４の抽出物には、少なくとも４種類の肝細胞増殖因子産出誘導活性を有する分子が存在し、うち肝細胞増殖因子産出誘導活性を有する分子の３種類は陰性荷電であり、１種類は中性荷電であると考えられた。 Method for producing hepatocyte growth factor production inducer of the present invention (2)
Two times the amount of distilled water was added to the extract of Example 14, mixed for 1 hour at room temperature, and centrifuged. The supernatant after centrifugation was equilibrated with 50 mM Tris-HCl [pH 8.0] and added to a DEAE Sepharose FF column (GE Healthcare Bioscience). The added column was washed with 50 mM Tris-HCl [pH 8.0], and then eluted sequentially with 50 mM Tris-HCl [pH 8.0] containing 100 mM, 300 mM, and 500 mM sodium chloride. Each eluted fraction was added to the culture solution at 3%, and hepatocyte growth factor production inducing activity was measured by ELISA. The elution curve is shown in FIG. 4, and the measurement results are shown in FIG. Activity was observed in the elution fraction of the buffer only and the elution fraction of the buffer containing each concentration of sodium chloride. Therefore, it was revealed that a new hepatocyte growth factor production inducer was produced by the above-described method. Further, in the extract of Example 14, there are at least four types of molecules having hepatocyte growth factor production-inducing activity, of which three types of molecules having hepatocyte growth factor production-inducing activity are negatively charged, The type was considered neutral charge.
　また、本実施例で得られた各画分の活性を比較すると、100 mMの塩化ナトリウムを含む緩衝液で溶出して得られた分画と、300 mMの塩化ナトリウムを含む緩衝液で溶出して得られた分画の肝細胞増殖因子産出誘導活性が高いことがわかった。したがって、上述の製造方法により、肝細胞増殖因子産出誘導活性の高い分画を含む、別の肝細胞増殖因子産出誘導剤を製造することができる。 In addition, comparing the activities of the fractions obtained in this Example, the fraction obtained by elution with a buffer solution containing 100 μmM sodium chloride was eluted with a buffer solution containing 300 μmM sodium chloride. The fraction obtained in this way was found to have high hepatocyte growth factor production-inducing activity. Therefore, another hepatocyte growth factor production-inducing agent containing a fraction with high hepatocyte growth factor production-inducing activity can be produced by the production method described above.
マウスに対する肝細胞増殖因子産出誘導剤の肝細胞増殖因子産出誘導活性
実施例１４の抽出物をマウスに投与して血漿および大脳中の肝細胞増殖因子量の測定を行った。健常マウス（ICR、8週齢、雌）に１日１回 30 mg量の抽出物を経口投与し、７日後に血液および大脳を回収した。表１に示すように、実施例１４の抽出物の投与によって肝細胞増殖因子の産出量の増加が認められたことから、顕著な肝細胞増殖因子誘導活性を有することが確認された。 Hepatocyte growth factor production inducing activity of hepatocyte growth factor production inducer for mice The extract of Example 14 was administered to mice and the amount of hepatocyte growth factor in plasma and cerebrum was measured. A healthy mouse (ICR, 8 weeks old, female) was orally administered 30 mg of extract once a day, and blood and cerebrum were collected 7 days later. As shown in Table 1, since the production of hepatocyte growth factor was increased by administration of the extract of Example 14, it was confirmed that it had a remarkable hepatocyte growth factor-inducing activity.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　本発明は、医薬、美容、及び食品分野において、肝細胞増殖因子の産出を誘導することによるさまざまな疾患の治療及び／又は予防に用いることができる。 The present invention can be used for the treatment and / or prevention of various diseases by inducing the production of hepatocyte growth factor in the fields of medicine, beauty and food.

Claims (13)

  1. 　植物由来の有効成分を含む、肝細胞増殖因子産出誘導剤 Hepatocyte growth factor production inducer containing active ingredients derived from plants
  2. 　前記植物が、紅花、莪朮、枸杞子、石菖蒲、益智仁、欝金、人参、田七人参、忍冬藤、大棗、甘草、仙茅及び蓮子芯から選ばれる１又は複数である、請求項１に記載の肝細胞増殖因子産出誘導剤 The plant is one or more selected from safflower, camellia, coconut, sarcophagus, masochito, jinkin, ginseng, ginseng, ginseng, ninfuto, daiso, licorice, sensu and lotus wick. Item 1. The hepatocyte growth factor production inducer according to Item 1.
  3. 　前記有効成分には少なくとも２種類の肝細胞増殖因子産出誘導活性を有する分子が存在し、１の肝細胞増殖因子産出誘導活性を有する分子は陰性荷電であり、別の肝細胞増殖因子産出誘導活性を有する分子は中性荷電である、請求項１又は２に記載の肝細胞増殖因子産出誘導剤 The active ingredient includes at least two kinds of molecules having hepatocyte growth factor production-inducing activity, one molecule having hepatocyte growth factor production-inducing activity is negatively charged, and another hepatocyte growth factor production-inducing activity. 3. The hepatocyte growth factor production inducer according to claim 1 or 2, wherein the molecule having a neutral charge is neutrally charged.
  4. 前記有効成分は、製薬上又は食品衛生上許容される極性溶媒により抽出して得られる抽出物である、請求項１～３いずれか一項に記載の肝細胞増殖因子産出誘導剤 The hepatocyte growth factor production inducer according to any one of claims 1 to 3, wherein the active ingredient is an extract obtained by extraction with a pharmaceutically or food hygienically acceptable polar solvent.
  5. 前記植物が複数であり、前記複数の植物を混合してから抽出して抽出物を得ることを特徴とする、請求項４に記載の肝細胞増殖因子産出誘導剤 5. The hepatocyte growth factor production inducer according to claim 4, wherein the plant is plural, and the plurality of plants are mixed and extracted to obtain an extract.
  6. 　極性溶媒が、水、エタノール、酢酸又はそれらの混合物である、請求項４又は請求項５記載の肝細胞増殖因子産出誘導剤 The hepatocyte growth factor production inducer according to claim 4 or 5, wherein the polar solvent is water, ethanol, acetic acid or a mixture thereof.
  7. 　抽出温度が、常温から常圧又は加圧下で溶媒の沸点までの範囲である、請求項４～請求項６いずれか一項に記載の肝細胞増殖因子産出誘導剤 The hepatocyte growth factor production inducer according to any one of claims 4 to 6, wherein the extraction temperature is in a range from room temperature to normal pressure or the boiling point of the solvent under pressure.
  8. 　粉末製剤、顆粒剤、錠剤、丸剤、カプセル剤、又は液状製剤の形態である、請求項１～７いずれか一項に記載の肝細胞増殖因子産出誘導剤 The hepatocyte growth factor production inducer according to any one of claims 1 to 7, which is in the form of a powder preparation, granule, tablet, pill, capsule, or liquid preparation.
  9. 　請求項１～７いずれか一項に記載の肝細胞増殖因子産出誘導剤を含む治療薬及び／又は予防薬 A therapeutic and / or prophylactic agent comprising the hepatocyte growth factor production inducer according to any one of claims 1 to 7.
  10. 　請求項１～７いずれか一項に記載の肝細胞増殖因子産出誘導剤を含む美容品 Cosmetics comprising the hepatocyte growth factor production inducer according to any one of claims 1 to 7.
  11. 　請求項１～７いずれか一項に記載の肝細胞増殖因子産出誘導剤を含む機能性食品 A functional food comprising the hepatocyte growth factor production inducer according to any one of claims 1 to 7.
  12. 請求項１～７いずれか一項に記載の肝細胞増殖因子産出誘導剤を緩衝液とともに陰イオン交換樹脂に添加する添加工程と、
    該陰イオン交換樹脂から、イオン強度0 mM～500 mMの塩を含む緩衝液で溶出する溶出工程を含有する、肝細胞増殖因子産出誘導剤の製造方法     An addition step of adding the hepatocyte growth factor production inducer according to any one of claims 1 to 7 together with a buffer to an anion exchange resin;
    A method for producing a hepatocyte growth factor production inducer, comprising an elution step of elution from the anion exchange resin with a buffer containing a salt having an ionic strength of 0 mM to 500 mM.
  13. 前記溶出工程が、イオン強度100 mM及び／又は300 mMの塩を含む緩衝液で溶出する溶出工程である、請求項１２に記載の肝細胞増殖因子産出誘導剤の製造方法 The method for producing an agent for inducing hepatocyte growth factor production according to claim 12, wherein the elution step is an elution step of elution with a buffer containing a salt having an ionic strength of 100 mM and / or 300 mM.
PCT/JP2016/051942 2015-01-23 2016-01-23 Agent for inducing production of hepatocyte growth factor WO2016117705A1 (en)

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