WO1996036346A1 - Water-soluble extract originating in feather cockscomb - Google Patents

Water-soluble extract originating in feather cockscomb Download PDF

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Publication number
WO1996036346A1
WO1996036346A1 PCT/JP1996/001275 JP9601275W WO9636346A1 WO 1996036346 A1 WO1996036346 A1 WO 1996036346A1 JP 9601275 W JP9601275 W JP 9601275W WO 9636346 A1 WO9636346 A1 WO 9636346A1
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WIPO (PCT)
Prior art keywords
water
soluble extract
extract
soluble
liver
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PCT/JP1996/001275
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French (fr)
Japanese (ja)
Inventor
Tooru Takahashi
Koji Hase
Tsuneo Namba
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Yamanouchi Pharmaceutical Co., Ltd.
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Application filed by Yamanouchi Pharmaceutical Co., Ltd. filed Critical Yamanouchi Pharmaceutical Co., Ltd.
Priority to JP53469796A priority Critical patent/JP3984288B2/en
Priority to AU57019/96A priority patent/AU5701996A/en
Publication of WO1996036346A1 publication Critical patent/WO1996036346A1/en

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    • 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)
    • A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the present invention provides a water-soluble extract containing a polysaccharide as a main component and an active ingredient containing the water-soluble extract obtained by extraction and isolation from a crude drug, Seisho, and is particularly useful for prevention and treatment of liver injury.
  • Pharmaceutical compositions or food additives are particularly useful for prevention and treatment of liver injury.
  • the liver is also called a “silent organ” because the liver has a strong natural healing power and does not show prominent symptoms with a few disorders, and it is important for humans to metabolize, regulate blood sugar, detoxify, regulate bile circulation, store nutrients, etc. It plays an essential function in maintaining life.
  • the number of patients showing liver function abnormalities has increased with the increase in alcohol consumption.
  • the etiology and pathology of hepatic disorder are diverse, the most demanding therapeutic agents are high medical needs and chronic active hepatitis. Research and development of various therapeutic agents ranging from antiviral agents to immunomodulators are being actively conducted.
  • Kampo therapy has been practiced since ancient times, such as Sho-saiko-to (Saiko-Hanaxia ⁇ Ginseng 'Large ⁇ Licorice' ginger ⁇ Huang-gong), Furubai-to Ryo • Herbal medicines such as Kusagosha (Gomiko, Osume, Oso) are used as “cleanse the liver”, “remove the heat of the liver”, and “help the liver”.
  • the active ingredient is not clear. .
  • polysaccharide is a generic name for high molecular compounds in which a large number of monosaccharides are polymerized by glycosidic bonds, and is composed of a single polysaccharide composed of the same monosaccharide molecule and a complex composed of various monosaccharides and their derivatives.
  • polysaccharides There are polysaccharides.
  • dextran formed by linear polymerization of glucose is known as a plasma expander, and lentinan extracted from shiitake and schizophyllan extracted from shirohirotake have antitumor activity. Many have biological activity.
  • Use crude drug-derived polysaccharides for liver disease As a specific example, Japanese Patent Publication No. 6-886481 describes a polysaccharide obtained by fermenting grass fiber in a specific culture solution and having a therapeutic effect on hepatitis B. There is. _Technical issues
  • blue ginger polysaccharides extracted and isolated from seeds of blue ginger (Ce1osiaargentea L.) (hereinafter referred to as blue ginger) are obtained. It has been found that a water-soluble extract containing as a main component and further containing a protein has a remarkable inhibitory effect on liver diseases, thereby completing the present invention.
  • the present invention relates to a water-soluble extract comprising extraction and isolation from green ginger, and more particularly to a water-soluble extract obtained by the following steps (a) to (c).
  • the water-soluble extract of the present invention has a molecular weight of 1 to 300,000 and has the following physicochemical properties (1) and (2).
  • the water-soluble extract of the present invention contains 90 to 99% by weight of polysaccharide and 1 to 10% by weight of protein.
  • the present invention provides a pharmaceutical composition for humans or animals comprising the above-mentioned water-soluble extract as an active ingredient, particularly a composition useful for the prevention and treatment of liver damage in humans or animals, and the above-mentioned water-soluble extract
  • a pharmaceutical composition for humans or animals comprising the above-mentioned water-soluble extract as an active ingredient, particularly a composition useful for the prevention and treatment of liver damage in humans or animals, and the above-mentioned water-soluble extract
  • the present invention relates to a food additive containing:
  • the water-soluble extract obtained by extraction and isolation from the green ginger of the present invention contains 90 to 99% by weight of a polysaccharide and 1 to 10% by weight of a protein. It contains polysaccharide units corresponding to arabinose, rhamnose, mannose, galactose, galacturonic acid, glucose, fucose and glucuronic acid, and further contains trace amounts of sugar alcohols.
  • This water-soluble extract is acidic, has a molecular weight of 1 to 300,000, and contains 0.5 to 1.5% of protein-derived nitrogen atoms.
  • the water-soluble extract of the present invention is obtained by extracting an aqueous extract from a herbal medicine, green ginger, and then treating the aqueous extract with an organic solvent to obtain an organic solvent-insoluble precipitate. It can be obtained by subjecting the medium-insoluble precipitate to purification and isolating the water-soluble extract.
  • the method for extracting and isolating the water-soluble extract of the present invention from blue ginger is as follows.
  • the extraction is carried out by extracting a water-soluble extract with water or an aqueous solvent from a crushed product of the crude drug green ginger, the seeds of its original plant Nogatei or other homologous plants.
  • blue ginger refers to not only the dried crude herb seeds but also the crude drugs sold as green ginger, such as the mature seeds of the above-mentioned cockscomb-contaminated green ginger. Is dried.
  • Notomato which is the original plant
  • examples of such congener plants include corn beetle and japonicus.
  • the pulverized material may be roughly cut or coarsely powdered, but it is advantageous to use the powder, that is, green ginger powder, in order to increase the extraction efficiency.
  • green ginger powder which is an object to be extracted, can be directly applied to the extraction operation without being degreased in advance.
  • an organic solvent generally used for extracting lipids from crude drugs such as black form, methanol, ethanol, acetone and tetrahydrofuran, is suitably used.
  • the water used as the extraction solvent may be ordinary water, but preferably purified water is used.
  • aqueous solvent examples include various buffers commonly used for polysaccharide extraction, such as a phosphate buffer and an acetate buffer, and aqueous solutions of salts such as salt.
  • the amount of water or aqueous solvent used also depends on the method of leaching, and is not particularly limited as long as the extract of the present invention can be extracted. A suitable amount is 5 to 15 times, especially about 0.6 to 10 times.
  • the ginger extract of the present invention is a water-soluble and heat-stable extract, and the extract obtainable by hot extraction achieves the object of providing the preventive / therapeutic agent for liver injury of the present invention. Above all, the extraction is preferably performed by hot extraction.
  • the extraction is performed not only on a laboratory scale Soxhlet extractor and a heated reflux extractor, but also on an industrial scale extractor usually used for extracting physiologically active substances mainly composed of water-soluble polysaccharides from crude drugs. For example, it can be advantageously implemented by various percolators.
  • the extract thus obtained is concentrated according to a conventional method, and separated into solid and liquid by filtration or the like.
  • the extract obtained in the extraction step can be directly subjected to the separation step, but it is preferable to dry the extract in order to increase the separation efficiency by the organic solvent treatment in the separation step.
  • any drying means commonly used for drying crude drug extracts such as ventilation drying, vacuum drying, freeze drying, spray drying and the like can be applied, but freeze drying is advantageous.
  • the dried extract is then treated with an organic solvent to separate a water-soluble and organic solvent-insoluble matter from a water-soluble organic solvent-soluble matter.
  • the organic solvent may be any organic solvent that dissolves a substance that is water-soluble but soluble in the organic solvent in the organic solvent by treatment with the organic solvent.
  • organic solvent In isolating a green ginger extract, black-mouthed form and ethanol are preferred. Cloth form treatment and ethanol treatment can be applied in any order.
  • the water-soluble and organic solvent-insoluble extract of the present invention is separated as a precipitate.
  • the separated precipitate is subjected to solid-liquid separation by a conventional method such as filtration, and then dissolved in water and dialyzed to remove salts and, if necessary, low-molecular substances.
  • Dialysis is a means commonly used for desalination and purification of crude drug components such as electrodialysis, ultrafiltration membranes, and reverse osmosis, and deviations can be applied.
  • dialysis is preferably performed using an ultrafiltration membrane having a molecular sieve.
  • an ultrafiltration membrane that separates with a molecular weight of about 10,000 is suitable.
  • Dialysis with this ultrafiltration membrane usually takes 3 hours to 5 days In particular, in the separation of the green ginger extract of the present invention, about 12 hours to 3 days is preferable.
  • the dissolved and dialyzed green ginger extract is then purified from crude drugs such as chromatography by a purification means generally used for the purification of an extract containing a polysaccharide as a main component, and is isolated as a substance having a single fraction peak. Separated.
  • Chromatography is usually performed using ion exchange chromatography, gel filtration chromatography, affinity chromatography, HPLC, etc. ⁇ Ion exchange chromatography or gel Filter mouth chromatography is advantageous.
  • UV absorption spectrum Figure 1 shows the UV absorption spectrum data measured in distilled water.
  • Figure 2 shows infrared absorption spectrum data measured by the KBr method.
  • compositions for oral administration can be solid or liquid, powders, syrups, capsules, granules,? An L agent, a suspension, a drop, etc. may be used. Carriers or excipients for such compositions are well known.
  • composition may further comprise conventional materials such as binders, stabilizers, emulsifiers, suspending agents, dispersing agents, lubricants, preservatives, bulking agents and the like.
  • water-soluble extract of the present invention can be used as a food additive in health foods, functional foods, and the like.
  • the daily dose of the composition for preventing or treating hepatic injury according to the present invention may be 10 to 100 OmgZ adult in terms of polysaccharide which is a main component of the water-soluble extract. Desirable.
  • the water-soluble extract of the present invention has an inhibitory effect on toxic liver damage caused by alcohol and drugs, and immune liver damage caused by virus and virus, and is useful for prevention and treatment of liver damage.
  • the peroxidation of intracellular organ membrane lipids is caused by free radicals generated in the course of alcohol and drug metabolism, but the water-soluble extract of the present invention has an effect of inhibiting the production of lipid peroxide.
  • tumor necrosis factor (TN) has been reported to be involved in fulminant illness of viral hepatitis and exacerbation of alcoholic hepatitis. It is considered to be one of the major medias every day, but the water-soluble extract of the present invention It also showed a significant inhibitory effect on NF-induced liver injury.
  • Example 3 An aqueous extract obtained by extracting blue ginger with hot water in Example a) described below, an organic solvent-insoluble precipitate obtained by treating the aqueous extract with an organic solvent in Example 2 described below, and In Example 3, the effect of CE 1 obtained by further purifying the organic solvent-insoluble precipitate on CC 14 -induced liver injury was examined.
  • a test drug dissolved in physiological saline or suspended uniformly in a 0.5% CMC aqueous solution was administered to male Sprague-Dawley rats (6 weeks of age) p. After 12, 24 hours, 3 mgZkg of a mixed solution of CC14Z01 oil (1: 4 v / v) was injected subcutaneously into the back or intraperitoneally.
  • the test drug and CC14 were administered by different routes.
  • Condition control group saline, or after administration only 0. 5% C MC solution was injected CC 1 4 similarly.
  • the control group received 100 mg of glycyrrhizin instead of the test drug.
  • Glycyrrhizin is a compound that is used as an active ingredient in licorice such as Sho-Sho-to for liver disease.
  • CC 1 4 will cause a chemical (toxic) liver failure (T. Yokozawa et al., Pharmacognosy magazine 47, 229 (1993), N. I shi da et a to J.Hepatology 13,200 (1991)).
  • trichloromethyl radical ( ⁇ CC 1 3) is produced in the endoplasmic reticulum drug metabolism pathway, which proteins of hepatocytes, covalently bound to a lipid such as It is presumed that the function is changed and also acts on the cell membrane to produce peroxidized lipids (lipidoperoxide, LPO), altering the membrane and eventually causing cell necrosis (Paul B) McCay et al., J. Biological Chem. 259.2135 (1984)).
  • Dg a 1 actos am ine hereinafter abbreviated as D-ga
  • LPS ZL ipopo lys ac cha ri de
  • Example a) The effects of an aqueous extract obtained by extracting blue ginger with hot water in Example a) and CE 1 obtained in Example c) described below on D-ga 1ZL PS-induced liver injury were examined.
  • To the disease state control group only the solvent was administered, and then D-ga1 and LPS were similarly administered.
  • the control group received 10 OmgZ kg of glycyrrhizin instead of the test drug.
  • Blood was collected 8 hours after administration of D-ga1 and LPS, and GOT, GPT, LDH, and pyrilrubin levels were measured.
  • Dg a 1 is metabolized in cells and becomes ur idi ne 5'-di pho spha te (UDP) —galactosamine, uridine phosphide (ur idi ne 5—monophospha te (UMP), UDP, ur idi ne 5'-tri Phosphate (UTP) deficiency occurs.
  • UDP ur idi ne 5'-di pho spha te
  • UDP uridine phosphide
  • UDP ur idi ne 5'-tri Phosphate
  • mice show resistance to this liver injury.
  • Power, power, and galactosamine A remarkable liver injury model can be obtained by administering a small amount of Endotoxin (LPS) to sensitized mice.
  • LPS Endotoxin
  • L TD 4 1 euk 0 triene D 4
  • L TD 4 1 euk 0 triene D 4
  • the blood vessel temporarily becomes ischemic, and superoxide is generated when the blood flow starts flowing again with a decrease in LTD 4 concentration. by. That is, it destroys cells sensitized by Tga-Dga1 secreted from macrophages activated by super-one-year-old oxide (LPS itself is directly activated).
  • Example a) The effects of the aqueous extract obtained by extracting blue ginger with hot water in Example a) and the CE1 obtained in Example c) described below on P. acnesZLPS-induced liver injury were examined.
  • the same test was performed using CE1 and 1 OmgZkg. However, the dose of LPS was 50 mg.
  • the control group received 25 mg Zkg of glycyrrhizin instead of the test drug.
  • Each group consists of 12-14 animals, and the bioassay is K ap 1a It was performed by the n-Me iyer method / generalized Wi 1 coxon test.
  • Example c The inhibitory effect of CE 1 obtained in Example c) described later on lipid peroxide production was examined.
  • Incubated rat Kanmi closo one base Schorr completion to an arm preparative iron complex F e S_ ⁇ 4 or adenosine 5 '-di phospha te (ADP ) / F e C 1 3 and CE 1 were added 37 ° C2 0 minutes, The lipid peroxide produced was determined by the malondialdehyde method.
  • Example c The effect of CE1 obtained in Example c) described later on TNF-induced liver injury was examined.
  • Table 1 shows the levels of various serum enzymes and the magnitude of the blood protective effect when administered at 10 OmgZkg to a CC14-induced liver injury rat.
  • the blood protective action refers to the degree to which the test drug suppresses an increase in serum enzyme levels.
  • Table 2 shows the GPT level, the reduction rate, and the number of dead mice when the aqueous extract was administered to D-ga 1ZL PS-induced liver injury mice at 20 Omg / kg. Table 2 shows that the aqueous extract markedly suppressed the increase in serum GPT level and the mortality of D-gal / LPS-induced liver injury mice.
  • FIG. 3 shows the survival rate of the mice when the aqueous extract was administered to P. acnesZLPS-induced liver injury mice.
  • FIG. 3 shows that the aqueous extract significantly suppressed the mortality of P. ances / LPS-induced liver injury mice.
  • Table 3 Aqueous extract obtained from green pepper was used for black mouth form and methanol.
  • G PT glutamic- pyruvic transaminase
  • Example c The organic solvent-insoluble precipitate obtained in Example) was purified in Example c), cases administered at a dose of 2 SmgZk g of CE 1 obtained by isolated CC 1 4 induced liver failure rats Bok various serum Table 4 shows enzyme levels.
  • CE 1 is a lower dose-than glycyrrhizin, significantly suppressed won the elevated levels of serum parameters CC 1 4-induced liver injury rat, it can be seen that a much more effective liver damage inhibitory effect than glycyrrhizin.
  • Serum enzyme levels when administered at a dose of 5 mg / kg Dosage number of samples Serum GOT level Serum GPT level Serum LDH level Serum pyrilvin level Group
  • GPT glutamic-pyruvic transaminase
  • CE 1 is D—ga 1 no?
  • Table 6 shows the serum GPT levels and their reduction rates when the mice were administered at a dose of 1 OmgZkg and 50 mgZkg to mice with induced liver injury.
  • Table 6 C E1 was added to D-gajZLPS-induced liver injury mice at 10 mg gZkg.
  • G PT glutamic-pyruvic transaminase
  • FIG. 4 shows the mouse survival rate when CE1 was administered to PacnesPSS-induced liver injury mice.
  • CE 1 significantly suppressed the lethality of P. acnes / LPS-induced liver injury mice, indicating that the effect was stronger than that of glycyrrhizin.
  • Table 7 shows the results of measuring the effect of CE 1 on inhibiting lipid peroxide acidity. Table 7 shows that CE 1 suppressed the production of lipid peroxide in a dose-dependent manner.
  • ADP adenosine 5 '-di hosphate
  • Table 8 shows the serum GPT levels and their reduction rates when CE1 was administered to TNF-induced spleen-induced liver injury mice. Table 8 shows that CE 1 suppresses the increase in serum GPT levels induced by TNF-ligand.
  • Serum G ⁇ ⁇ level and its decrease rate Dose Sample No. Serum GPT level GPT decrease rate group
  • FIG. 1 is a graph showing the UV absorption spectrum data of CE1.
  • FIG. 2 is a graph showing infrared absorption spectrum data of CE1.
  • Figure 3 shows (a) a graph showing the effect of the aqueous extract on the survival rate of P. acnes s / LPS-induced liver injury mice and (b) a graph showing the effect of glycyrrhizin on the survival rate of P. acnes sZLPS-induced liver injury mice. It is a graph which shows an effect.
  • FIG. 4 is a graph showing the effect of CE1 on the survival rate of P. acnes ZLPS-induced liver injury mice.
  • FIG. 5 is a diagram showing a purification process of CE1 from blue ginger.

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Abstract

A water-soluble extract extracted and isolated from feather cockscomb (Celosia argentea, L.), which is a plant belonging to the family Amaranthaceae, and a medicinal composition or a food additive containing this extract as the active ingredient. The extract contains 90 to 99 % by weight of polysaccharides and 1 to 10 % by weight of proteins, exerts depressive effects on toxic hepatopathy caused by alcohol and drugs and immune hepatopathy caused by viruses, etc. and thus is useful as a preventive or remedy for hepatopathy.

Description

明 細 書 青しよ う子由来の水溶性抽出物 技術分野  Description Water-soluble extract derived from Ayako Aoshi Technical field
本発明は、 生薬青しょう子より抽出 '単離して得られる、 多糖類を主成分とす る水溶性抽出物及び該水溶性抽出物を有効成分とする、 特に肝障害の予防 ·治療 に有用な医薬組成物若しくは食品添加物に関する。  INDUSTRIAL APPLICABILITY The present invention provides a water-soluble extract containing a polysaccharide as a main component and an active ingredient containing the water-soluble extract obtained by extraction and isolation from a crude drug, Seisho, and is particularly useful for prevention and treatment of liver injury. Pharmaceutical compositions or food additives.
背景技術 Background art
肝臓は自然治癒力が強く少々の障害では表立った症状が表れないことから 「沈 黙の臓器」 とも呼ばれ、 物質代謝、 血糖の調節、 解毒、 胆汁循環の調節、 栄養素 の貯蔵等、 人の生命の維持に不可欠な機能を担っている。 ところが近年わが国に おいては飲酒量の増加に伴い肝機能異常を示す患者が増えている。 肝障害の病因、 病態は多種多様であるが、 治療薬の開発が最も求められているのは医療ニーズの 高レ、慢性活動性肝炎であり、 本疾患を標的として肝保護薬をはじめ原因療法とし ての抗ウィルス剤や免疫調節薬に至るまで様々な治療薬の研究開発が活発に行わ れている。  The liver is also called a “silent organ” because the liver has a strong natural healing power and does not show prominent symptoms with a few disorders, and it is important for humans to metabolize, regulate blood sugar, detoxify, regulate bile circulation, store nutrients, etc. It plays an essential function in maintaining life. However, in recent years in Japan, the number of patients showing liver function abnormalities has increased with the increase in alcohol consumption. Although the etiology and pathology of hepatic disorder are diverse, the most demanding therapeutic agents are high medical needs and chronic active hepatitis. Research and development of various therapeutic agents ranging from antiviral agents to immunomodulators are being actively conducted.
このような肝疾患に対して、 漢方療法においては、 古来より小柴胡湯 （柴胡 - 半夏 ·人参 '大そう ·甘草'生姜 ·黄ごん） 、 降ばい湯 （いん陳 ·土ぶくりょう •草河車■五味子 ·烏梅 ·大そう） 等の生薬が 「肝を清める」 「肝の熱を除く」 「肝の気を助ける」 として用いられているカ、 その有効成分は明らかではない。 一方、 多糖類は、 多数の単糖がグリコシド結合により重合した高分子化合物の 総称であり、 同一の単糖分子から構成される単一多糖類と種々の単糖類及びその 誘導体から構成される複合多糖類とがある。 多糖類のうち、 グルコースが直鎖状 に重合して成るデキストランは血漿増量剤等として知られ、 又、 シィタケから抽 出されたレンチナン及びスェヒロタケから抽出されたシゾフィラン等は抗腫瘍活 性を有する等、 生理活性を有するものも多い。 生薬由来の多糖類を、 肝疾患に適 用した具体例としては、 特公平 6— 8 6 4 8 1号に、 イネ科植物繊維を特定の培 養液中で発酵して得られ、 B型肝炎に治療効果のある多糖体についての記載があ る。 _ 技術的課題 For such liver diseases, Kampo therapy has been practiced since ancient times, such as Sho-saiko-to (Saiko-Hanaxia · Ginseng 'Large · Licorice' ginger · Huang-gong), Furubai-to Ryo • Herbal medicines such as Kusagosha (Gomiko, Osume, Oso) are used as “cleanse the liver”, “remove the heat of the liver”, and “help the liver”. The active ingredient is not clear. . On the other hand, polysaccharide is a generic name for high molecular compounds in which a large number of monosaccharides are polymerized by glycosidic bonds, and is composed of a single polysaccharide composed of the same monosaccharide molecule and a complex composed of various monosaccharides and their derivatives. There are polysaccharides. Among the polysaccharides, dextran formed by linear polymerization of glucose is known as a plasma expander, and lentinan extracted from shiitake and schizophyllan extracted from shirohirotake have antitumor activity. Many have biological activity. Use crude drug-derived polysaccharides for liver disease As a specific example, Japanese Patent Publication No. 6-886481 describes a polysaccharide obtained by fermenting grass fiber in a specific culture solution and having a therapeutic effect on hepatitis B. There is. _Technical issues
慢性活動性肝炎等の治療には、 その性質上長期間に渡る薬剤の投与が必要であ り、 応々にして副作用が問題となる。 従って、 ウィルス、 薬物中毒、 アルコール 等の肝疾患の原因を問わず高い治療効果を有し、 かつ長期間投与を続けても副作 用の無い安全な肝疾患予防 ·治療薬の開発が待たれている。 発明の開示  Treatment of chronic active hepatitis and the like requires the administration of drugs over a long period of time due to the nature of the treatment, and side effects are a problem. Therefore, development of safe and preventive and therapeutic drugs for liver disease that has a high therapeutic effect regardless of the cause of liver disease such as virus, drug poisoning, and alcohol and has no side effects even after long-term administration is awaited. ing. Disclosure of the invention
本発明者等は、 種々の生薬について検討を重ねた結果、 青しょう （C e 1 o s i a a r g e n t e a L . ) の種子（以下、 青しよう子と記載する。 ） より 抽出 ·単離して得られる、 多糖類を主成分として含有し更に蛋白質を含有する水 溶性抽出物が肝疾患に対して顕著な障害抑制効果を有することを見出し、 本発明 を完成した。  As a result of repeated studies on various crude drugs, the present inventors have found that polysaccharides extracted and isolated from seeds of blue ginger (Ce1osiaargentea L.) (hereinafter referred to as blue ginger) are obtained. It has been found that a water-soluble extract containing as a main component and further containing a protein has a remarkable inhibitory effect on liver diseases, thereby completing the present invention.
本発明に於て 「青しよう ( C e 1 0 s i a a r g e n t e a L . ) 」 とは ヒュ科の植物で和名をノゲイトウといい、 草蒿、 萋蒿、 昆侖草、 野鶏冠、 草決明 としても知られる。 その種子である 「青しょう子」 は従来漢方薬として乾燥した ものを熱水で煎じて、 そのまま服用されていたのみで、 抽出物を単離したものに ついての報告はなかった。 そして、 その適用疾病は消炎作用に基づく、 「肝臓の 熱が目にきたもの』 、 「肝火による眼部の充血 '腫脹 ·疼痛（口が苦い ·咽がか わく ·頭が脹つて痛い ·顔面紅潮 ·煩躁 ·怒りつぼいなどの "肝火" の症状をと もなう） 」 等の、 主に目に関連する疾病に使用されることが知られていた （ 「漢 方の臨床応用」 中山医学院編 医歯薬出版 （株） 第 8 3〜8 4頁 （ 1 9 8 0 ) 及び 「中薬大辞典」 上海科学技術出版社 （株） 小学館 第一巻 第 1 3 8 6 〜1 3 8 7頁 （ 1 9 8 5 ) ) 。  In the present invention, the term "blue sea (C e 10 siaargentea L.)" is a plant belonging to the family Hyu, and its Japanese name is Nogatei. Can be The seed “Seishoko”, which had been dried as a traditional Chinese medicine, was decocted in hot water and was taken as it was, and there were no reports of isolated extracts. And the applied disease is based on the anti-inflammatory effect, "Heat of the liver came to the eyes", "Hyperemia of the eye due to liver fire" swelling · Pain (bite bite · Throat swelling · Head swells and hurts · Face It has been known to be mainly used for diseases related to the eyes, such as "liver fire" symptoms such as flushing, mania, anger, etc. ("Clinical application of Chinese medicine" Nakayama Pp. 83-84 (1992) and "China Pharmaceutical Dictionary", Shanghai Academy of Science and Technology Publishing Co., Ltd. Shogakukan, Vol. 1, pp. 866-13 87 pages (19985)).
し力、しながら、 青しょう子の肝臓の疾病への使用については、 上記文献等には 「肝火を清める」 、 「肝を鎮める」 等の上記目の疾病の治療に関連する記載が認 められるのみであり、 肝臓の疾病への直接的な使用はなされてレ、なかつた。 即ち、 本発明は、 青しょう子より抽出し単離することから成る水溶性抽出物、 特に下記 （a ) 〜 （c ) の工程により得られうることを特徴とする水溶性抽出物 に関する。 The use of blue ginger in liver disease is described in the above literature. Only statements related to the treatment of the above eye diseases, such as “cleansing the liver fire” and “calming the liver”, were recognized, and direct use for liver diseases was not achieved. That is, the present invention relates to a water-soluble extract comprising extraction and isolation from green ginger, and more particularly to a water-soluble extract obtained by the following steps (a) to (c).
( a ) 青しょう子より熱水にて水性抽出液を抽出し、  (a) Extract the aqueous extract from hot spring water with hot water,
( b ) 次いで有機溶媒で処理することにより、 有機溶媒不溶性沈殿物を得、 (b) Then, by treating with an organic solvent, an organic solvent-insoluble precipitate is obtained,
( c ) 更に水溶性抽出物を単離する。 (c) further isolating the water-soluble extract.
また、 本発明の水溶性抽出物は、 分子量が 1〜3 0万であり、 且つ下記①及び ②の理化学的性質を有する。  The water-soluble extract of the present invention has a molecular weight of 1 to 300,000 and has the following physicochemical properties (1) and (2).
①ァラビノース、 ラムノース、 マンノース、 ガラク トース、 ガラクッロン酸、 グルコース、 フコース及びグルクロン酸に対応する多糖類単位を含有する。 ① Contains polysaccharide units corresponding to arabinose, rhamnose, mannose, galactose, galacturonic acid, glucose, fucose and glucuronic acid.
② 0 . 5〜1 . 5 %の、 蛋白質由来の窒素原子を含有する。 ② It contains 0.5 to 1.5% of nitrogen atoms derived from proteins.
更に、 本発明の水溶性抽出物は、 多糖類を 9 0〜9 9重量％及び蛋白質を 1〜 1 0重量％含有する。  Furthermore, the water-soluble extract of the present invention contains 90 to 99% by weight of polysaccharide and 1 to 10% by weight of protein.
また、 本発明は、 前記の水溶性抽出物を有効成分とするヒト又は動物用の医薬 組成物、 特にヒト又は動物の肝障害の予防 ·治療に有用な組成物、 及び前記の水 溶性抽出物を含有する食品添加物に関する。  Also, the present invention provides a pharmaceutical composition for humans or animals comprising the above-mentioned water-soluble extract as an active ingredient, particularly a composition useful for the prevention and treatment of liver damage in humans or animals, and the above-mentioned water-soluble extract The present invention relates to a food additive containing:
以下、 本発明の水溶性抽出物につき詳細に説明する。  Hereinafter, the water-soluble extract of the present invention will be described in detail.
本発明の青しょう子より抽出，単離して得られる水溶性抽出物は、 9 0〜9 9 重量％の多糖類及び 1 ~ 1 0重量％の蛋白質を含有し、 主成分である多糖類はァ ラビノース、 ラムノース、 マンノース、 ガラク トース、 ガラクッロン酸、 グルコ ース、 フコース、 グルクロン酸に対応する多糖類単位を含有するとともに、 微量 の糖アルコールをさらに含有する。 この水溶性抽出物は酸性を呈し、 1〜3 0万 の分子量を有するとともに、 0 . 5〜1 . 5 %の蛋白質由来の窒素原子を含有す る。  The water-soluble extract obtained by extraction and isolation from the green ginger of the present invention contains 90 to 99% by weight of a polysaccharide and 1 to 10% by weight of a protein. It contains polysaccharide units corresponding to arabinose, rhamnose, mannose, galactose, galacturonic acid, glucose, fucose and glucuronic acid, and further contains trace amounts of sugar alcohols. This water-soluble extract is acidic, has a molecular weight of 1 to 300,000, and contains 0.5 to 1.5% of protein-derived nitrogen atoms.
本発明の水溶性抽出物は、 生薬である青しょう子から水性抽出液を抽出し、 次 し、で該水性抽出液を有機溶媒で処理し有機溶媒不溶性沈殿物を得、 更に該有機溶 媒不溶性の沈殿物を精製に付して水溶性抽出物を単離することにより得られうる。 本発明の水溶性抽出物を青しょう子より抽出 ·単離する方法は以下の通りであ 。 The water-soluble extract of the present invention is obtained by extracting an aqueous extract from a herbal medicine, green ginger, and then treating the aqueous extract with an organic solvent to obtain an organic solvent-insoluble precipitate. It can be obtained by subjecting the medium-insoluble precipitate to purification and isolating the water-soluble extract. The method for extracting and isolating the water-soluble extract of the present invention from blue ginger is as follows.
( 1 ) 抽出工程  (1) Extraction process
抽出は、 生薬青しょう子、 その原植物であるノゲイトウの種子またはその他同 属植物の種子を粉砕した粉砕物から水又は水性溶媒で水溶性抽出物を抽出するこ とにより行われる。  The extraction is carried out by extracting a water-soluble extract with water or an aqueous solvent from a crushed product of the crude drug green ginger, the seeds of its original plant Nogatei or other homologous plants.
生薬青しよう子は、 市場では同属植物であるゲイトウの種子 （鶏冠子） の混入 されたものが生薬青しょう子として流通しており、 また中国北部等の地区ではァ ォゲイトウの種子が青しょう子として用いられている。 従って、 本発明にいう In the market, crude herbs mixed with the seeds of the genus Gaito, which is a congener plant, are distributed as crude herbs in the market, and in regions such as northern China, the seeds of blue ginger are used. It is used as Therefore, in the present invention,
「青しょう子」 としては、 ノゲイトウの成熟種子を乾燥した生薬青しょう子の正 品だけでなく、 青しょう子として市販されている生薬、 例えば前記の鶏冠子混入 青しょう子ゃァォゲイトウの成熟種子を乾燥したものが包含される。 また、 その 原植物であるノゲイトウの種子は勿論、 本発明青しょう子抽出物として特定され る肝障害予防 ·治療効果を発揮する水溶性抽出物を含有する同属植物の種子であ つてもよく、 そのような同属植物としてはケィトウ、 ァォゲイトウなどが挙げら れる。 The term “blue ginger” refers to not only the dried crude herb seeds but also the crude drugs sold as green ginger, such as the mature seeds of the above-mentioned cockscomb-contaminated green ginger. Is dried. In addition, not only the seeds of the wild plant, Notomato, which is the original plant, but also the seeds of the same genus plants containing a water-soluble extract which exhibits the preventive and therapeutic effects on liver damage specified as the green ginger extract of the present invention, Examples of such congener plants include corn beetle and japonicus.
粉砕物は粗切、 粗末であってもよいが、 抽出効率を高める上でその粉末、 すな わち青しょう子末を用いるのが有利である。 また、 抽出対象物である青しょう子 末等は、 予め脱脂することなく、 そのまま抽出操作に適用可能である。 脱脂を行 う場合にはクロ口ホルム、 メタノール、 エタノール、 アセトン、 テトラヒドロフ ランなど生薬から脂質を抽出する際に汎用される有機溶媒が好適に用いられる。 抽出溶媒である水は、 常水でもかまわないが、 好ましくは精製水が用いられる。 また水性溶媒としてはリン酸緩衝液や酢酸緩衝液など、 多糖類抽出に汎用される 各種の緩衝液や食塩などの塩類の水溶液が挙げられる。 なお、 水又は水性溶媒の 使用量は、 また浸出の方法によっても異なり、 本発明青しょう子抽出物を抽出可 能な量であれば特に限定はないが、 例えば青しょう子末に対して 0 . 5〜1 5倍 量、 特に 0 . 6〜1 0倍量程度が適当である。 本発明の青しょう子抽出物は水溶性でかつ熱に安定な抽出物であり、 また熱時 抽出して得られうる抽出物が本発明の肝障害予防 ·治療薬を提供する目的を達成 する上で特に好適であり、 抽出にあたっては熱時抽出が好ましい。 The pulverized material may be roughly cut or coarsely powdered, but it is advantageous to use the powder, that is, green ginger powder, in order to increase the extraction efficiency. In addition, green ginger powder, which is an object to be extracted, can be directly applied to the extraction operation without being degreased in advance. In the case of delipidation, an organic solvent generally used for extracting lipids from crude drugs, such as black form, methanol, ethanol, acetone and tetrahydrofuran, is suitably used. The water used as the extraction solvent may be ordinary water, but preferably purified water is used. Examples of the aqueous solvent include various buffers commonly used for polysaccharide extraction, such as a phosphate buffer and an acetate buffer, and aqueous solutions of salts such as salt. The amount of water or aqueous solvent used also depends on the method of leaching, and is not particularly limited as long as the extract of the present invention can be extracted. A suitable amount is 5 to 15 times, especially about 0.6 to 10 times. The ginger extract of the present invention is a water-soluble and heat-stable extract, and the extract obtainable by hot extraction achieves the object of providing the preventive / therapeutic agent for liver injury of the present invention. Above all, the extraction is preferably performed by hot extraction.
抽出は、 実験室規模のソックスレー抽出器や加熱還流抽出器だけでなく、 生薬 等から水溶性多糖類を主成分とする生理活性物質を抽出するために通常用いられ る工業用規模の抽出機、 例えば各種パーコレーターによって有利に実施できる。 このようにして得られた抽出物は、 常法に従い、 これを濃縮し、 濾過などで固 液分離する。  The extraction is performed not only on a laboratory scale Soxhlet extractor and a heated reflux extractor, but also on an industrial scale extractor usually used for extracting physiologically active substances mainly composed of water-soluble polysaccharides from crude drugs. For example, it can be advantageously implemented by various percolators. The extract thus obtained is concentrated according to a conventional method, and separated into solid and liquid by filtration or the like.
( 2 ) 分離 ·精製工程  (2) Separation and purification process
抽出工程により得られた抽出物は、 そのまま分離工程に付すこともできるが、 通常分離工程の有機溶媒処理による分離効率を高める上で、 乾燥するのが好まし い。  The extract obtained in the extraction step can be directly subjected to the separation step, but it is preferable to dry the extract in order to increase the separation efficiency by the organic solvent treatment in the separation step.
乾燥は、 通風乾燥、 真空乾燥、 凍結乾燥、 噴霧乾燥等生薬エキスを乾燥する上 で汎用される乾燥手段のいずれも適用可能であるが、 凍結乾燥が有利である。 抽出乾燥物は、 次いで、 有機溶媒で処理することにより水溶性で有機溶媒不溶 物を、 水溶性で有機溶媒可溶物から分離する。  For drying, any drying means commonly used for drying crude drug extracts such as ventilation drying, vacuum drying, freeze drying, spray drying and the like can be applied, but freeze drying is advantageous. The dried extract is then treated with an organic solvent to separate a water-soluble and organic solvent-insoluble matter from a water-soluble organic solvent-soluble matter.
有機溶媒は、 水溶性であるが有機溶媒にも可溶な物質を有機溶媒処理により有 機溶媒中に溶解させる有機溶媒であればいずれでもよいが、 特に多糖類を主成分 とする本発明の青しょう子抽出物を単離する上で、 クロ口ホルム、 エタノールが 好適である。 クロ口ホルム処理、 エタノール処理は任意の順序で適用可能である。 この有機溶媒処理によって、 本発明の水溶性で有機溶媒に不溶な抽出物は、 沈 殿物として分離される。 分離された沈殿物は濾過など常法により固液分離し、 次 いで水に溶解して透析することにより塩、 必要により低分子物質を除去する。 透析は電気透析、 限外濾過膜、 逆浸透等生薬成分の脱塩精製に汎用される手段 のし、ずれも適用可能であるが、 脱塩とともに多糖類の効用を目的として低分子物 質を除去するためには分子ふるいの限外濾過膜を用いて透析するのが好ましい。 分子量 1万以上の本発明抽出物を取得する目的においては、 分子量 1万程度で分 離する限外濾過膜が好適である。 この限外濾過膜による透析は通常 3時間〜 5日 程度、 特に本発明青しょう子抽出物分離においては、 1 2時間〜 3日程度が好適 である。 The organic solvent may be any organic solvent that dissolves a substance that is water-soluble but soluble in the organic solvent in the organic solvent by treatment with the organic solvent. In isolating a green ginger extract, black-mouthed form and ethanol are preferred. Cloth form treatment and ethanol treatment can be applied in any order. By this organic solvent treatment, the water-soluble and organic solvent-insoluble extract of the present invention is separated as a precipitate. The separated precipitate is subjected to solid-liquid separation by a conventional method such as filtration, and then dissolved in water and dialyzed to remove salts and, if necessary, low-molecular substances. Dialysis is a means commonly used for desalination and purification of crude drug components such as electrodialysis, ultrafiltration membranes, and reverse osmosis, and deviations can be applied. For removal, dialysis is preferably performed using an ultrafiltration membrane having a molecular sieve. For the purpose of obtaining the extract of the present invention having a molecular weight of 10,000 or more, an ultrafiltration membrane that separates with a molecular weight of about 10,000 is suitable. Dialysis with this ultrafiltration membrane usually takes 3 hours to 5 days In particular, in the separation of the green ginger extract of the present invention, about 12 hours to 3 days is preferable.
溶解、 透析された青しょう子抽出物は、 次いでクロマトグラフィー等生薬から 多糖類を主成分とする抽出物の精製に汎用される精製手段によって精製され、 単 一の分画ピークを有する物質として単離される。  The dissolved and dialyzed green ginger extract is then purified from crude drugs such as chromatography by a purification means generally used for the purification of an extract containing a polysaccharide as a main component, and is isolated as a substance having a single fraction peak. Separated.
クロマトグラフィーは、 通常イオン交換クロマトグラフィー、 ゲル濾過クロマ トグラフィー、 ァフィ二ティクロマトグラフィー、 HPLCなどが用いられる力 \ 多糖類を主成分とする抽出物の単離手段としてはイオン交換クロマトグラフィー やゲル濾過ク口マトグラフィ一が有利である。  Chromatography is usually performed using ion exchange chromatography, gel filtration chromatography, affinity chromatography, HPLC, etc. \ Ion exchange chromatography or gel Filter mouth chromatography is advantageous.
イオン交換クロマトグラフィーのカラム充塡剤としては、 DEAE—セファロ ース （S e p h a r o s e) CL— 6 B (フアルマシア バイオテク社製） 、 D EAE—トヨパール （Toyop e a r l ) 650 M (東ソ一社製） 、 ゲル濾過 クロマトグラフィーのカラ厶充塡剤としては、 セフアデックス （S e pha d e x) G 1 50 (フアルマシア バイオテク社製） 、 バイオゲル （B i 0— G e 1 ) P— 200 (バイオラッ ド ラボラトリ一社製） 、 Toy op e a r l HW— 55F c. c. (東ツー社製） などが有利に用いられる。 溶離液は水又は水性溶 媒が用いられ、 特に食塩などの塩水溶液が好適である。 以下に本発明の、 青しょう子から精製した水溶性抽出物 （セロシアン （C e 1 0 s i a n) 1と命名し、 本明細書中では、 以下 CE 1と記載する。 ） の理化学 的特性を記載する。  Column packings for ion exchange chromatography include DEAE-Sepharose CL-6B (Pharmacia Biotech), DEAE-Toyopearl (Toyop earl) 650 M (Tosoichi), As column packing materials for gel filtration chromatography, Sephadex G150 (manufactured by Pharmacia Biotech), biogel (Bi0—Ge1) P—200 (Biorad Laboratories) ), Toy opearl HW- 55F cc (manufactured by Tohtsu) are advantageously used. As the eluent, water or an aqueous solvent is used, and an aqueous salt solution such as common salt is particularly preferable. Hereinafter, the physicochemical properties of the water-soluble extract purified from blue ginger (named Serocian (Ce10sian) 1 and described as CE 1 in the present specification) of the present invention will be described. I do.
青しよう子由来成分 CE 1の理化学的特性  Physicochemical properties of green soybean-derived component CE1
( 1 ) 元素分析  (1) Elemental analysis
H： 6. 04%, C ： 37. 82 %, N： 0. 89%  H: 6.04%, C: 37.82%, N: 0.89%
(2) 分子量 約 1〜30万、 主として約 1 9万 （ゲルろ過法による）  (2) Molecular weight of about 1 to 300,000, mainly about 190,000 (by gel filtration method)
(3) 融点または分解点  (3) Melting point or decomposition point
融点不明確。 約 255 °Cで炭化する。  Melting point unclear. Carbonizes at about 255 ° C.
(4) 紫外線吸収スぺク トル 図 1に蒸留水中で測定した紫外線吸収スぺクトルデータを示す。 (4) UV absorption spectrum Figure 1 shows the UV absorption spectrum data measured in distilled water.
(5) 赤外線吸収スぺク トル  (5) Infrared absorption spectrum
図 2に KB r法にて測定した赤外線吸収スぺクトルデータを示す。  Figure 2 shows infrared absorption spectrum data measured by the KBr method.
(6) 溶剤に対する溶解性  (6) Solubility in solvents
水に溶解し、 水酸化カリウム、 水酸化ナトリウム、 水酸化アンモニゥ厶、 炭酸カリウム等の特にアルカリ性水溶液によく溶解する。 メタノール、 ェ タノ一ル、 プロパノール、 ブ夕ノール、 アセトン、 クロ口ホルム、 ェ一テ ルに難溶である。  It dissolves in water and dissolves well in particularly alkaline aqueous solutions such as potassium hydroxide, sodium hydroxide, ammonium hydroxide and potassium carbonate. Poorly soluble in methanol, ethanol, propanol, butanol, acetone, chloroform, and ether.
( 7 ) 呈色反応  (7) Color reaction
フ ノール Z硫酸法、 ロウリ一法に陽性  Positive for phenol Z sulfuric acid method and Lowry method
( 8 ) 性質  (8) Property
弱酸性  Mild acidity
( 9 ) 主な化学組成  (9) Main chemical composition
(a) 糖 （モル比）  (a) Sugar (molar ratio)
ァラビノース 32. 9  Arabinose 32. 9
ラムノース 1 8. 5  Rhamnose 18.5
マンノース 1 8. 3  Mannose 1 8.3
ガラク トース 1 1. 4  Galactose 1 1.4
ガラクッロン酸 8. 3  Galacturonic acid 8.3
グルコース 5. 7  Glucose 5.7
フコース 2. 1  Fucose 2.1
グルクロン酸 1. 9  Glucuronic acid1.9
了ラビトール 0. 8  Rabitol 0.8
ソルビトール 0. 1  Sorbitol 0.1
(b) 蛋白質  (b) protein
4% (ロウリ—法）  4% (Lowry method)
( 1 0) 比旋光度 〔ひ〕 D =+ 1 4 1. 1 (c = 0. 04% ;水） 本発明の水溶性抽出物を肝障害の予防 ·治療薬として用いる場合は、 製薬学的 に許容される担体または賦形剤と共存させることができ、 胃腸管からの吸収に好 適な形態で投与することが望ましい。 例えば、 経口投与用組成物は、 固体でも液 体でもよく、 粉末、 シロップ、 カプセル、 粒剤、 ？L剤、 懸濁剤、 ドロップ等でも よい。 この種の組成物のための担体または賦形剤は周知である。 錠剤用賦形剤の 例は、 ラクトース、 ポテトおよび可溶性澱粉、 ステアリン酸マグネシウム等で、 注射用担体の例は、 滅菌水、 生理的食塩水、 アーモンド油等で、 これらをアンプ ルに入れても、 または使用前に活性物質に加えてもよい。 (10) Specific rotation [H] D = + 1 41.1 (c = 0.04%; water) When the water-soluble extract of the present invention is used as a prophylactic / therapeutic agent for liver damage, it can be co-existed with a pharmaceutically acceptable carrier or excipient, and in a form suitable for absorption from the gastrointestinal tract. It is desirable to administer. For example, compositions for oral administration can be solid or liquid, powders, syrups, capsules, granules,? An L agent, a suspension, a drop, etc. may be used. Carriers or excipients for such compositions are well known. Examples of excipients for tablets are lactose, potato and soluble starch, magnesium stearate, etc.Examples of carriers for injection are sterile water, saline, almond oil, etc. Alternatively, it may be added to the active substance before use.
所望により、 組成物はさらに結合剤、 安定剤、 乳化剤、 懸濁剤、 分散剤、 潤滑 剤、 防腐剤、 増量剤等の常用の材料を含んでもよい。  If desired, the composition may further comprise conventional materials such as binders, stabilizers, emulsifiers, suspending agents, dispersing agents, lubricants, preservatives, bulking agents and the like.
また、 本発明の水溶性抽出物は食品添加物として、 健康食品、 機能性食品等に も用いることができる。  In addition, the water-soluble extract of the present invention can be used as a food additive in health foods, functional foods, and the like.
本発明に係わる肝障害の予防又は治療用組成物の 1 日投与量は、 水溶性抽出物 の主成分である多糖体に換算して 1 0〜1 0 0 0 O m gZ成人であることが望ま しい。  The daily dose of the composition for preventing or treating hepatic injury according to the present invention may be 10 to 100 OmgZ adult in terms of polysaccharide which is a main component of the water-soluble extract. Desirable.
尚、 本発明の水溶性抽出物を含有する青しょう子は、 生薬として永年種々の医 療目的に一般大衆に用いられてきたものであり、 少なくとも上記の投与量では毒 性は全く問題とならない。 産業上の利用性  Note that the blue ginger containing the water-soluble extract of the present invention has been used by the general public as a crude drug for various medical purposes for many years, and at least the toxicity is not a problem at the above dose. . Industrial applicability
本発明の水溶性抽出物は、 アルコール、 薬物を原因とする中毒性肝障害、 '及び ゥィルス等を原因とする免疫性肝障害に抑制効果を示し、 肝障害の予防 ·治療に 有用である。 特に前者では、 アルコール、 薬物代謝の過程で発生するフリーラジ カルによる細胞内器官膜脂質の過酸化が起因となるが、 本発明の水溶性抽出物は 過酸化脂質の産生抑制作用を有する。 また、 最近、 腫瘍壊死因子 （t u m o r n e c r o s i s f a c t o r—ひ， 以下 TN F—ひと略記する。 ） カ\ ウイ ルス性肝炎の劇症化やアルコール性肝炎の悪化に関与する旨の報告があり、 肝障 害の主要メデイエ一夕一の一^ 3と目されているが、 本発明の水溶性抽出物は、 T NF- 誘発の肝障害に対しても顕著な抑制作用を示した。 INDUSTRIAL APPLICABILITY The water-soluble extract of the present invention has an inhibitory effect on toxic liver damage caused by alcohol and drugs, and immune liver damage caused by virus and virus, and is useful for prevention and treatment of liver damage. In particular, in the former, the peroxidation of intracellular organ membrane lipids is caused by free radicals generated in the course of alcohol and drug metabolism, but the water-soluble extract of the present invention has an effect of inhibiting the production of lipid peroxide. Recently, tumor necrosis factor (TN) has been reported to be involved in fulminant illness of viral hepatitis and exacerbation of alcoholic hepatitis. It is considered to be one of the major medias every day, but the water-soluble extract of the present invention It also showed a significant inhibitory effect on NF-induced liver injury.
本発明の水溶性抽出物の肝障害に対する予防 ·治療効果を以下の方法により確 ！、レ ·~  The preventive and therapeutic effects of the water-soluble extract of the present invention on liver damage are confirmed by the following methods! , Les
1 ) 四塩化炭素 （CC 14 ) 誘発肝障害モデルによる評価  1) Evaluation using a carbon tetrachloride (CC14) -induced liver injury model
後記実施例 a)で青しょう子を熱水にて抽出して得た水性抽出物、 後記実施例 2で水性抽出物を有機溶媒にて処理して得た有機溶媒不溶性沈澱物、 及び後記実 施例 3にて有機溶媒不溶性沈澱物をさらに精製することにより得た C E 1の C C 14 誘発肝障害に対する効果を調べた。  An aqueous extract obtained by extracting blue ginger with hot water in Example a) described below, an organic solvent-insoluble precipitate obtained by treating the aqueous extract with an organic solvent in Example 2 described below, and In Example 3, the effect of CE 1 obtained by further purifying the organic solvent-insoluble precipitate on CC 14 -induced liver injury was examined.
生理食塩水に溶解するか、 又は 0. 5 %CMC水溶液に均一に懸濁した被験薬 を Sp r a gu e— Dawl e y系雄性ラット （6週齢） に p. 投与してか ら、 1、 12、 24時間後に、 CC l4 Z01 i ve o i l混合液 （1 : 4 v/v) 3mgZkgを背部皮下又は腹腔内へ注射した。 なお、 被検薬と CC 1 4 は異なる経路にて投与を行った。 病態対照群には生理食塩水、 又は 0. 5%C MC水溶液のみを投与した後、 同様に CC 14 を注射した。 又、 比較対照群には 被験薬の代わりにグリチルリチン 100 m gZk gを投与した。 なお、 グリチル リチンは小紫胡湯等として肝疾患に用いられる甘草の活性成分とされる化合物で ある。 A test drug dissolved in physiological saline or suspended uniformly in a 0.5% CMC aqueous solution was administered to male Sprague-Dawley rats (6 weeks of age) p. After 12, 24 hours, 3 mgZkg of a mixed solution of CC14Z01 oil (1: 4 v / v) was injected subcutaneously into the back or intraperitoneally. The test drug and CC14 were administered by different routes. Condition control group saline, or after administration only 0. 5% C MC solution was injected CC 1 4 similarly. The control group received 100 mg of glycyrrhizin instead of the test drug. Glycyrrhizin is a compound that is used as an active ingredient in licorice such as Sho-Sho-to for liver disease.
肝障害の程度が最も強く現れる CC 14 投与から 24時間経過後に採血を行う とともに、 犠殺して肝臓を摘出した。 採取した血液について g 1 υ t ami c- oxa l oac e t i c t rans ami na s e (G〇T) 、 g l u t am i c一 pyruv i c t r ans ami na s e (GPT) 、 ビリノレビン及び l ac t a t e dehydr ogena s e (LDH)値を測定した。 G 0 T、 GPT、 ピリルビン値はレフレトロンを用いて、 又、 LDH値は医療分析キッ ト を用いて測定した。 肝臓については湿重量を測定して肝重量ノ体重比を求めた後、 1 0 %リン酸緩衝ホルマリン液で固定し、 組織学的検査を行った。  Blood was collected 24 hours after CC 14 administration, when the degree of liver damage was most pronounced, and the liver was sacrificed and the liver was removed. About the collected blood g 1 υ t ami c- oxa l oac etict rans ami na se (G〇T), glut am ic pyr pyruv ic tr an s ami na se (GPT), virinolevin and l a c tate dehydr ogena se (LDH) The value was measured. GOT, GPT, and pyrilrubin values were measured using a Refretron, and LDH values were measured using a medical analysis kit. For the liver, the wet weight was measured to determine the liver weight-to-body weight ratio, and the liver was fixed with a 10% phosphate buffered formalin solution and histologically examined.
本障害モデルは簡便なため肝障害に対する効果の一次評価系として最も広範に 用いられている。 CC 14は化学的 （中毒性） 肝障害を起こす （T. Yokozawa et al.，生薬学雑誌 47, 229(1993), N. I shi da et aし J.Hepatology 13,200(1991))。 CC 14 による肝障害機序に関しては多くの研究があるが、 小胞体薬物代謝経路 でトリクロロメチルラジカル （ · CC 13 ) が生成され、 これが肝細胞のタンパ ク質、 脂質等と共有結合して機能変化を与えるとともに細胞膜にも作用して過酸 化脂質 （l i p i dper ox i de, LPO) を産生し膜を変質させ、 最終的 に細胞の壊死を起こすとの説が有力である（Paul B. McCay et al.， J. Biological Chem. 259.2135(1984))。 Because of the simplicity of this disorder model, it is most widely used as a primary evaluation system for effects on liver injury. CC 1 4 will cause a chemical (toxic) liver failure (T. Yokozawa et al., Pharmacognosy magazine 47, 229 (1993), N. I shi da et a to J.Hepatology 13,200 (1991)). Although there are many studies on liver damage mechanism by CC 14, trichloromethyl radical (· CC 1 3) is produced in the endoplasmic reticulum drug metabolism pathway, which proteins of hepatocytes, covalently bound to a lipid such as It is presumed that the function is changed and also acts on the cell membrane to produce peroxidized lipids (lipidoperoxide, LPO), altering the membrane and eventually causing cell necrosis (Paul B) McCay et al., J. Biological Chem. 259.2135 (1984)).
2) D-g a 1 a c t o s am i n e (以下、 D— ga 1と略記する。 ） ZL i popo l ys a c cha r i de (以下、 LPSと略記する。 ） 誘発肝障害モ デル U.Wang et al., Biochem. Pharm. 39,267(1990)、 A. Wendel et al.. Bioc hem. Pharm.35,2115(1986)) による評価  2) Dg a 1 actos am ine (hereinafter abbreviated as D-ga 1) ZL ipopo lys ac cha ri de (hereinafter abbreviated as LPS) Induced liver injury model U. Wang et al., Evaluation by Biochem. Pharm. 39,267 (1990), A. Wendel et al. Biochem. Pharm. 35, 2115 (1986))
後記実施例 a)で青しょう子を熱水にて抽出して得た水性抽出物、 及び後記実 施例 c) にて得た CE 1の D - ga 1ZL PS誘発肝障害に対する効果を調べた。 被験薬を ddy系雄性マウス （6週齢） に皮下投与してから 2、 18時間経過 後に、 D— ga 1 70 Omg/kg及び LPS 10〃 /¾：2を同時に p. 投与した。 病態対照群には溶媒のみを投与した後、 同様に D - ga 1及び LPS を投与した。 又、 比較対照群には被験薬の代わりにグリチルリチン 10 OmgZ kgを投与した。 D— ga 1及び LPSを投与してから 8時間経過後に採血し、 GOT, GPT、 LDH、 ピリルビン値を測定した。  The effects of an aqueous extract obtained by extracting blue ginger with hot water in Example a) and CE 1 obtained in Example c) described below on D-ga 1ZL PS-induced liver injury were examined. . Two to 18 hours after subcutaneous administration of the test drug to ddy male mice (6 weeks old), D-ga 170 Omg / kg and LPS 10〃 / ¾: 2 were simultaneously administered p. To the disease state control group, only the solvent was administered, and then D-ga1 and LPS were similarly administered. The control group received 10 OmgZ kg of glycyrrhizin instead of the test drug. Blood was collected 8 hours after administration of D-ga1 and LPS, and GOT, GPT, LDH, and pyrilrubin levels were measured.
本障害モデルにおいて誘発される肝障害は、 LTD4や TNF—ひ等のォータ コィド、 サイトカインを経由する反応であるため、 免疫学的肝障害発生モデルと して臨床成績との相関性が高いと思われる。 Liver injury induced in this disorder model, LTD 4 and TNF- unequal in Quota Koido, since a reaction that via cytokine and a high correlation with the clinical outcome as an immunological hepatic failure model Seem.
D-g a 1は細胞内で代謝され、 ur i d i ne 5' -d i pho spha t e (UDP) —ガラクトサミンとなりゥリジンリン酸化物 （ur i d i ne 5 —monophospha t e (UMP)、 UDP、 ur i d i ne 5 ' -t r i phospha t e (UTP) ) の欠乏が起きる。 このため RNA合成 能が低下する他、 UDP—グルコース、 UDP—グルクロン酸が減少しタンパク 質および脂質代謝が阻害される結果、 細胞壊死にいたる。  Dg a 1 is metabolized in cells and becomes ur idi ne 5'-di pho spha te (UDP) —galactosamine, uridine phosphide (ur idi ne 5—monophospha te (UMP), UDP, ur idi ne 5'-tri Phosphate (UTP) deficiency occurs. This leads to a decrease in RNA synthesis ability, a decrease in UDP-glucose and UDP-glucuronic acid, and inhibition of protein and lipid metabolism, resulting in cell necrosis.
一般にマウスはこの肝障害に対して抵抗性を示す。 し力、し、 ガラクトサミンに 感作されたマウスに Endo t ox i n (LPS) を微量投与すると顕著な肝障 害モデルが得られる。 これは LPSによって 1 e u k 0 t r i e n e D4 (L TD4 )が誘導され、 血管が一時的に虚血状態になり LTD 4 濃度の低下ととも に再び血流が流れ出す時スーパーォキシドが発生することによる。 即ち、 スーパ 一才キシドにより活性化されたマクロファージ （LPS自体でも直接活性化する） から分泌される TNF—ひが D— ga 1によって感作された細胞を破壊するので ある。 以上が現在考えられている D— g a 1ZLPSによる肝障害の作用機序で ある （広岡 慎悟ら、 医薬品研究 13.1046(1982) Sornmer B. G. et aし Transpla nt Pro c. 11.578(1979) G.Tiegs et al.. Biochem. Pharm. 38.627(1989) Kepp ler D. et al., Eur. J. Biochem.10.219(1969))。 Generally, mice show resistance to this liver injury. Power, power, and galactosamine A remarkable liver injury model can be obtained by administering a small amount of Endotoxin (LPS) to sensitized mice. This is because LPS induces 1 euk 0 triene D 4 (L TD 4 ), and the blood vessel temporarily becomes ischemic, and superoxide is generated when the blood flow starts flowing again with a decrease in LTD 4 concentration. by. That is, it destroys cells sensitized by Tga-Dga1 secreted from macrophages activated by super-one-year-old oxide (LPS itself is directly activated). The above is the currently considered mechanism of action of D-ga1ZLPS-induced liver injury (Shingo Hirooka et al., Pharmaceutical Research 13.1046 (1982) Sornmer BG et al., Transplant Proc. 11.578 (1979) G. Tiegs et al. .. Biochem. Pharm. 38.627 (1989) Keppler D. et al., Eur. J. Biochem.10.219 (1969)).
3) Pr op i on i bac t e r i um acne s (P._acne s) /L PS誘発肝障害モデル （T. Mimura et al., Biol. Pharm. Bull.17, 197(1994) 、 K. Yoshioka et al.. Hepatology 10,769(1989)、 A. horuts et al., Hepatology 13, 267(1991)) による評価  3) Pr i on i bac teri um acne s (P._acne s) / L PS-induced liver injury model (T. Mimura et al., Biol. Pharm. Bull. 17, 197 (1994), K. Yoshioka et. al..Hepatology 10,769 (1989), A. horuts et al., Hepatology 13, 267 (1991))
後記実施例 a)で青しょう子を熱水にて抽出して得た水性抽出物、 及び後記実 施例 c ) で得た C E 1の P. a c n e sZL P S誘発肝障害に対する効果を調べ た。  The effects of the aqueous extract obtained by extracting blue ginger with hot water in Example a) and the CE1 obtained in Example c) described below on P. acnesZLPS-induced liver injury were examined.
上記の水性抽出液を b a 1 1 bZc系雄性マウス （6週齢） に 25mg/kg で i. p. 投与してから 2、 1 8時間経過後にグラム陽性嫌気性菌である P r 0 p i on i bac t e r i um a cne s (Coryneba c t e r i um Two to eighteen hours after ip administration of the above aqueous extract to ba 11 bZc male mice (6 weeks old) at 25 mg / kg, Pr 0 pi on i bac teri, a gram-positive anaerobic bacterium um a cne s (Coryneba cteri um
P a r vum) の加熱死菌 1 m gを生理食塩水に懸濁し、 尾静脈を介して投与 した。 さらに、 7日後に 75 gの En do t ox i n (LPS) を尾静脈投与 し、 マウスの生存率を経時的に観察した。 病態対照群には溶媒のみを投与した後、 同様に P. acne sの加熱死菌及び L P Sを投与した。 なお、 各群は 1 1〜 1 3匹とした。 1 mg of heat-killed bacteria (Parvum) was suspended in physiological saline and administered via the tail vein. Seven days later, 75 g of Endoxin (LPS) was administered via tail vein, and the survival rate of the mice was observed over time. To the disease state control group, only the solvent was administered, and then heat-killed P. acnes and LPS were similarly administered. Each group consisted of 11 to 13 animals.
又、 CE 1、 1 OmgZkgを用いて同様な試験を行った。 ただし、 LPSの 投与量は 50〃 gとした。 比較対照群には被験薬の代わりにグリチルリチン 25 mgZkgを投与した。 なお、 各群は 12〜14匹とし、 生物検定は K a p 1 a n -Me i y e r法/一般化 W i 1 c o x o n検定により行った。 The same test was performed using CE1 and 1 OmgZkg. However, the dose of LPS was 50 mg. The control group received 25 mg Zkg of glycyrrhizin instead of the test drug. Each group consists of 12-14 animals, and the bioassay is K ap 1a It was performed by the n-Me iyer method / generalized Wi 1 coxon test.
ウィルス性肝炎、 胆汁性肝硬変、 アルコール性又は薬物性肝炎のようなある種 の肝障害においては免疫応答が重要な役割をしめているとの報告がある。 それ故、 作用機序から考えて本障害モデルは肝炎の発生メカニズム解明や、 新しい肝保護 薬の開発において極めて有用と思われる (K. Nakata et aL.Arch. Int. Pharmacod yn.309.170(1991))。  It has been reported that the immune response plays an important role in certain liver disorders such as viral hepatitis, biliary cirrhosis, alcoholic or drug-induced hepatitis. Therefore, considering the mechanism of action, this disorder model is considered to be extremely useful in elucidating the mechanism of hepatitis development and in developing new hepatoprotective drugs (K. Nakata et aL. Arch. Int. Pharmacod yn. 309.170 (1991)). ).
機序は P. acne s処理により肝内に集積したマクロファージが L P Sによ り活性化され活性酸素を放出し、 膜脂質の過酸化が起こることによる。 また最近 の研究では本病態モデルにおいて i n t e r l euk i n (I L) — lひ、 I L 一 6、 TNF—ひ等のサイト力インの濃度が上昇していることがわかり、 従来の 中毒性肝障害とは異なる免疫学的機序が関与することが示唆されている （S. Ιΐο et al. , Chem. Pharm. Bull.41, 1066(1993))。  The mechanism is that macrophages accumulated in the liver by P. acnes treatment are activated by LPS to release active oxygen, and peroxidation of membrane lipids occurs. In addition, recent studies have shown that the levels of interleukin (IL), IL-16, TNF, and other cytokins are elevated in this pathological model. Different immunological mechanisms have been implicated (S. Ιΐο et al., Chem. Pharm. Bull. 41, 1066 (1993)).
4 ) 過酸化脂質産生抑制効果  4) Lipid peroxide production inhibitory effect
後記実施例 c )で得た C E 1の過酸化脂質産生抑制効果を調べた。 ラット肝ミ クロソ一ムに了スコルべ一ト鉄錯体 Fe S〇4 又は a d e n o s i n e 5 ' -d i phospha t e (ADP) /F e C 13 及び CE 1を加えて 37°C2 0分間インキュベートし、 産生する過酸化脂質をマロンジアルデヒド法により定 した。 The inhibitory effect of CE 1 obtained in Example c) described later on lipid peroxide production was examined. Incubated rat Kanmi closo one base Schorr completion to an arm preparative iron complex F e S_〇 4 or adenosine 5 '-di phospha te (ADP ) / F e C 1 3 and CE 1 were added 37 ° C2 0 minutes, The lipid peroxide produced was determined by the malondialdehyde method.
5) TNF—ひ誘発肝障害モデルによる評価  5) TNF-evaluation using spleen-induced liver injury model
後記実施例 c )で得た C E 1の TNF -ひ誘発肝障害に対する効果を調べた。 CE 1を d dy系雄性マウス （6週齢） に 5 OmgZk gの用量で 2回皮下投 与してから 2、 1 8時間経過後に、 D— ga 1を 70 OmgZkgで i. p. 投 与した。 さらに、 D— ga 1投与 1時間後に TNF— を 4 g/kgで i. v. 投与した。 病態対照群には溶媒のみを投与した後、 同様に D - ga 1及び TNF 一ひを投与した。 TNF—ひを投与してから 7時間経過後に採血し、 GPT値を 測定した。  The effect of CE1 obtained in Example c) described later on TNF-induced liver injury was examined. Two to 18 hours after subcutaneous administration of CE1 to ddy male mice (6 weeks old) twice at a dose of 5 OmgZkg, D-ga1 was administered ip at 70 OmgZkg 2 to 18 hours later. One hour after D-ga 1 administration, TNF- was administered iv at 4 g / kg. To the disease state control group, only the solvent was administered, and then D-ga1 and TNF alone were similarly administered. Blood was collected 7 hours after the administration of TNF-hi and GPT value was measured.
(A)水性抽出物  (A) Aqueous extract
後記実施例 a)で青しよう子を熱水にて抽出することにより得た水性抽出物を CC 14 誘発肝障害ラッ トに 1 0 OmgZk gで投与した場合の、 各種血清酵素 レベル、 及び血液保護作用の大きさを表 1に示す。 なお、 血液保護作用とは被験 薬が血清酵素レベルの上昇を抑制する度合いをいう。 表 1より水性抽出物は CC 14 誘発肝障害ラッ トの血清 GO T、 GPT、 LDHレベルの上昇を有意に抑制 したことがわかる。 The aqueous extract obtained by extracting blue wilt with hot water in Example a) Table 1 shows the levels of various serum enzymes and the magnitude of the blood protective effect when administered at 10 OmgZkg to a CC14-induced liver injury rat. The blood protective action refers to the degree to which the test drug suppresses an increase in serum enzyme levels. Aqueous extract from Table 1 CC 1 4 induced liver failure rats serum GO T, GPT, it can be seen that significantly inhibited the increase in LDH levels.
表 1 青しょ う子より得た水性抽出物を C C 1 4 誘発肝障害 Table 1 Blue cane aqueous extract CC 1 4 induced liver damage resulting from Uco
ラ ッ トに 1 0 0 m g / k gで投与した場合の、 各種血清 酵素レベル及び血液保護作用の大きさ 検体数 血清酵素レベル ( I U/L) 肝臓保護作用 ( % ) 群  Serum enzyme levels and magnitude of blood protection when administered at 100 mg / kg to rats Number of samples Serum enzyme level (IU / L) Liver protection (%) group
n GOT G Ρ Τ L D Η GOT G P T L D H 無処置群 7 74±4·9 37±2.5 156±24  n GOT G Ρ Τ L D Η GOT G P T L D H Untreated group 7 74 ± 4.9 37 ± 2.5 156 ± 24
病態対照群 35 1689±325 553±76 601±154 Disease control group 35 1689 ± 325 553 ± 76 601 ± 154
水性抽出液投与群 6 320±133 ** 111±56* * 122±45' 81.1 79.9 79.7 Aqueous extract administration group 6 320 ± 133 ** 111 ± 56 * * 122 ± 45 '81.1 79.9 79.7
* P < 0. 0 5, ** P < 0. 0 1* P <0.05, ** P <0.01
OT ： glutamic-oxaloacetic transaminase  OT: glutamic-oxaloacetic transaminase
u P T ： glutamic-pyruvic transaminase  u PT: glutamic-pyruvic transaminase
L DH : lactate dehydrogenase L DH: lactate dehydrogenase
また、 水性抽出物を D— g a 1ZL PS誘発肝障害マウスに 20 Omg/kg で投与した場合の GPTレベル、 その減少率、 及び死亡マウス数を表 2に示す。 表 2より水性抽出物は D— ga l/LP S誘発肝障害マウスの血清 G P Tレベル の上昇、 及び致死率を顕著に抑制したことがわかる。 Table 2 shows the GPT level, the reduction rate, and the number of dead mice when the aqueous extract was administered to D-ga 1ZL PS-induced liver injury mice at 20 Omg / kg. Table 2 shows that the aqueous extract markedly suppressed the increase in serum GPT level and the mortality of D-gal / LPS-induced liver injury mice.
表 2 青しょ う子よ り得た水性抽出物を D - g a l / L P S誘発肝障害 Table 2 D-gal / LPS-induced liver injury in aqueous extract obtained from blue pepper
マウスに 2 0 O m g/k gで投与した場合の G P Tレベル、 その減少  GPT levels when administered to mice at 20 mg / kg, decrease
率、 及び死亡マウス数 投与量 血清 GPT レベル GPT 減少率 検体数 死亡ラ ッ ト数 群  Rate and number of dead mice Dose Serum GPT level GPT reduction rate Number of samples Number of dead rats Group
(mg/kg) (U/L) (%) n (12時間以内） 無処置群 66±17 7  (mg / kg) (U / L) (%) n (within 12 hours) Untreated group 66 ± 17 7
病態対照群 5867土 1289 11 6 水性抽出液投与群 200 64.6±12 98.9 10 0 " Disease control group 5867 Sat 1289 11 6 Aqueous extract administration group 200 64.6 ± 12 98.9 10 0 "
* P < 0. 0 5 , ** P < 0. 0 1 , *** P < 0. 0 0 1 * P <0.05, ** P <0.01, *** P <0.0.01
Lr P T ： glutamic - pyruvic transaminase Lr PT: glutamic-pyruvic transaminase
更に水性抽出物を P. acne sZL P S誘発肝障害マウスに投与した場合の マウスの生存率を図 3に示す。 図 3より水性抽出液は、 P. a n c e s/LPS 誘発肝障害マウスの致死率を有意に抑制したことがわかる。 Furthermore, FIG. 3 shows the survival rate of the mice when the aqueous extract was administered to P. acnesZLPS-induced liver injury mice. FIG. 3 shows that the aqueous extract significantly suppressed the mortality of P. ances / LPS-induced liver injury mice.
(B)有機溶媒不溶性沈殿物  (B) Organic solvent insoluble precipitate
実施例 a)で得た水性抽出物を実施例 b) においてクロ口ホルム （CHC 13 ) 及びメタノール （MeOH) にて処理することによって得た有機溶媒不溶性沈殿 物を CC 14 誘発肝障害ラットに 1
Figure imgf000019_0001
の用量で投与した場合の〇 Tレベル、 その減少率を表 3に示す。 表 3より上記の有機溶媒不溶性沈澱物が肝 障害抑制活性を有することがわかる。 The black hole Holm (CHC 1 3) and an organic solvent insoluble precipitates CC 14 induced liver failure rats were obtained by treatment with methanol (MeOH) in the aqueous extract of example b) obtained in Example a) 1
Figure imgf000019_0001
Table 3 shows the ΔT level and the rate of decrease when administered at the same dose. From Table 3, it can be seen that the above organic solvent-insoluble precipitate has a liver damage inhibiting activity.
表 3 青しょ う子より得た水性抽出物をク ロ口ホルム及びメ タノ ール Table 3 Aqueous extract obtained from green pepper was used for black mouth form and methanol.
にて処理するこ とによって得た有機溶媒不溶性沈澱物を C C 1 誘発肝障害ラ ッ 卜に 1 0 O m g/k gの用量で投与した場合の G P Tレベル、 その減少率 投与量 検体数 血清 G P Tレベル G P T減少率 群  GPT level when the organic solvent-insoluble precipitate obtained by the above treatment was administered to a CC1-induced liver injury rat at a dose of 10 mg / kg, and its reduction rate Dose number of samples Serum GPT level GPT reduction rate group
(m g / k g ) n (U/L) (%) 病態対照群 7 938土 124  (mg / kg) n (U / L) (%) Disease control group 7 938 Sat 124
C H C 1 可溶画分投与群 100 7 790±324 15.8 C H C 1 Soluble fraction administration group 100 7 790 ± 324 15.8
M e 0 H可溶画分投与群 100 5 782士 193 16.6 有機溶媒不溶性沈澱物投与群 100 6 77.5±26 … 91.7 Me 0 H soluble fraction administration group 100 5 782 193 16.6 Organic solvent insoluble precipitate administration group 100 6 77.5 ± 26… 91.7
*** P < 0. 0 0 1 *** P <0. 0 0 1
G PT ： glutamic- pyruvic transaminase G PT: glutamic- pyruvic transaminase
(C) CE 1 (C) CE 1
実施例 ） で得た有機溶媒不溶性沈澱物を実施例 c ) において精製し、 単離し て得た CE 1を CC 14 誘発肝障害ラッ 卜に 2 SmgZk gの用量で投与した場 合の各種血清酵素レベルを表 4に示す。 CE 1は、 グリチルリチンよりも低い用 量で、 CC 14 誘発肝障害ラットの血清中パラメーターのレベル上昇を有意に抑 制し、 グリチルリチンよりはるかに有効な肝障害抑制効果を示すことがわかる。 The organic solvent-insoluble precipitate obtained in Example) was purified in Example c), cases administered at a dose of 2 SmgZk g of CE 1 obtained by isolated CC 1 4 induced liver failure rats Bok various serum Table 4 shows enzyme levels. CE 1 is a lower dose-than glycyrrhizin, significantly suppressed won the elevated levels of serum parameters CC 1 4-induced liver injury rat, it can be seen that a much more effective liver damage inhibitory effect than glycyrrhizin.
表 4 青しょう子より単離した CE 1を CC 14 誘発肝障害ラッ トに Table 4 CE1 isolated from blue ginger was used as a CC14-induced liver injury rat
2 5mg/k gの用量で投与した場合の各種血清酵素レベル 投与量 検体数 血清 GOT レベル 血清 GPT レベル 血清 LDH レベル 血清ピリルビンレベル 群  2 Serum enzyme levels when administered at a dose of 5 mg / kg Dosage number of samples Serum GOT level Serum GPT level Serum LDH level Serum pyrilvin level Group
mg/kg) n (U/L) (U/L) (U/L) (/zg/dL) 病態対照群 7 1354 ±229 417±134 1059±219 561 ±58 グリチルリチン投与群 100 7 708±124 ' 170土 34 * 267 ± 46 " 445±28 mg / kg) n (U / L) (U / L) (U / L) (/ zg / dL) Disease control group 7 1354 ± 229 417 ± 134 1059 ± 219 561 ± 58 Glycyrrhizin administration group 100 7 708 ± 124 '170 Sat 34 * 267 ± 46 "445 ± 28
C E 1投与群 25 7 203±42.7 " 83.2土 24 " 92.9± 19 ** 255±23 " C E 1 group 25 7 203 ± 42.7 "83.2 Sat 24" 92.9 ± 19 ** 255 ± 23 "
* P < 0. 05, ** P < 0 . 0 1 * P <0. 05, ** P <0. 0 1
uOT： glutamic-oxaloacetic transaminase  uOT: glutamic-oxaloacetic transaminase
GPT： glutamic-pyruvic transaminase  GPT: glutamic-pyruvic transaminase
LDH : lactate dehydrogenase LDH: lactate dehydrogenase
また、 CE 1を CC 14 誘発肝障害マウスに投与した場合の肝臓の病理組織学 的検討結果を表 5に示す。 表 5より病態対照群については広範な細胞の壊死とそ れを取り囲む様に脂肪の蓄積した細胞が多く見受けられたのに対して、 CE 1投 与群では細胞の壊死、 脂肪の蓄積共に減少していることがわかる。 かくのごとく、 病理組織学的所見によっても CE 1の肝障害抑制効果が示された。 表 5 CE 1を CC 14 誘発肝障害マウスに投与した Table 5 shows the results of histopathological examination of the liver when CE1 was administered to CC14-induced liver injury mice. Table 5 shows that in the disease control group, a wide range of cell necrosis and a large number of cells with fat accumulation surrounding it were observed, whereas in the CE1 group, both cell necrosis and fat accumulation decreased. You can see that it is doing. As described above, the histopathological findings also showed that CE 1 was effective in suppressing liver damage. Table 5 CE 1 was administered to the CC 1 4-induced liver injury mouse
場合の肝臓の病理組織学的検討結果 ラット数 （匹）  Results of liver histopathological examination in cases
死 脂肪の蓄積  Fat accumulation
+ ++ 十 ++ + 十 + 十十十 病態対照群 0 0 3 4 0 0 5 2  + ++ 10 ++ + 10 + 10 10 Condition control group 0 0 3 4 0 0 5 2
C E 1投与群 3 4 0 0 1 3 3 0  C E 1 administration group 3 4 0 0 1 3 3 0
次に、 CE 1を D— ga 1ノ ？3誘発肝障害マゥスに1 OmgZkg及び 5 0 m gZk gの用量で投与した場合の血清 G P Tレベル及びその減少率を表 6に 示す。 CE 1は用量依存的に D— ga 1ZLPS誘発肝障害を抑制し、 その効果 はグリチルリチンよりもはるかに強レヽものであることがわかる。 Next, CE 1 is D—ga 1 no? Table 6 shows the serum GPT levels and their reduction rates when the mice were administered at a dose of 1 OmgZkg and 50 mgZkg to mice with induced liver injury. CE 1 dose-dependently inhibited DG 1 ZLPS-induced liver injury, indicating that its effect was much stronger than that of glycyrrhizin.
表 6 C E 1 を D - g a j ZL P S誘発肝障害マウスに 1 0 m gZk g Table 6 C E1 was added to D-gajZLPS-induced liver injury mice at 10 mg gZkg.
及び 5 O m gZk gの用量で投与した場合の血清 G P Tレベル  GPT levels when administered at doses of 5 and 5 OmgZkg
及びその減少率 投与量 検体数 血清 G P Tレベル G P T減少率 群  And its decrease rate Dose number of samples Serum GPT level GPT decrease rate group
(m g / k g ) n (U/L) (%) 病態対照群 15 2684±821  (mg / kg) n (U / L) (%) Disease control group 15 2684 ± 821
グリ チルリ チン投与群 100 10 328±27 ** 87.8Glycyrrhizin administration group 100 10 328 ± 27 ** 87.8
C E 1投与群 50 15 87±6.9 … 96.7 C E 1 administration group 50 15 87 ± 6.9… 96.7
10 15 134±27… 95.0  10 15 134 ± 27… 95.0
** P < 0. 0 1 , *** P < 0. 0 0 1 ** P <0.01, *** P <0.0.01
G PT ： glutamic-pyruvic transaminase G PT: glutamic-pyruvic transaminase
更に CE 1を P. a c n e s P S誘発肝障害マウスに投与した場合のマウ スの生存率を図 4に示す。 CE 1は P. a c n e s/LPS誘発肝障害マウスの 致死率を有意に抑制し、 その効果はグリチルリチンより強いことがわかる。 Furthermore, FIG. 4 shows the mouse survival rate when CE1 was administered to PacnesPSS-induced liver injury mice. CE 1 significantly suppressed the lethality of P. acnes / LPS-induced liver injury mice, indicating that the effect was stronger than that of glycyrrhizin.
C E 1の過酸化脂質酸性抑制効果を測定した結果を表 7に示す。 表 7より C E 1は用量依存的に過酸化脂質の產生を抑制することがわかる。 Table 7 shows the results of measuring the effect of CE 1 on inhibiting lipid peroxide acidity. Table 7 shows that CE 1 suppressed the production of lipid peroxide in a dose-dependent manner.
7 C E 1の過酸化脂質酸性抑制効果 マロンジアルデヒドの生成 5¾ Effect of 7 C E 1 on suppression of lipid peroxide acidity Formation of malondialdehyde 5¾
牛  Cow
濃 度 検体数 ァスコルべ一卜 ADP/F e2 + ァスコルべ一ト ADP/F e 2 + 群 鉄錯体./ Fe2 + 鉄錯体ノ Fe2 + Concentration Number of samples Ascorbate ADP / F e 2 + Ascorbate ADP / F e 2 + group Iron complex./ Fe 2 + Iron complex Fe 2 +
(nig/ml) n (nmol/ml) (nmol/ml) (%) (%) 病態対照群 5 3.05±0.156 4.67 ±0.223  (nig / ml) n (nmol / ml) (nmol / ml) (%) (%) Disease control group 5 3.05 ± 0.156 4.67 ± 0.223
0.01 5 2.36 ±0.295 4.22±0.097 22.6 9.67 0.01 5 2.36 ± 0.295 4.22 ± 0.097 22.6 9.67
C E 1投与群 0.1 4 0.637± 0.047 "* 3.99±0.115 · 79.1 14.7 C E 1 administration group 0.1 4 0.637 ± 0.047 "* 3.99 ± 0.11579.1 14.7
1.0 5 0.085±0.073 … 1.06±0.383 *** 97.2 77.4  1.0 5 0.085 ± 0.073… 1.06 ± 0.383 *** 97.2 77.4
* P < 0. 0 5, *** P < 0. 0 0 1 * P <0.05, *** P <0.0.01
ADP ： adenosine 5 ' -di hosphate ADP: adenosine 5 '-di hosphate
CE 1を TNF—ひ誘発肝障害マウスに投与した場合の血清 GPTレベル及び その減少率を表 8に示す。 表 8より CE 1は TNF—ひによって誘発される血清 GPTレベルの上昇を抑制することがわかる。 Table 8 shows the serum GPT levels and their reduction rates when CE1 was administered to TNF-induced spleen-induced liver injury mice. Table 8 shows that CE 1 suppresses the increase in serum GPT levels induced by TNF-ligand.
表 8 C E 1を TN F— α誘発肝障害マウスに投与した場合の Table 8 When CE1 was administered to TNF-α-induced liver injury mice
血清 G Ρ Τレベル及びその減少率 投与量 検体数 血清 G P Tレベル G P T減少率 群  Serum G Ρ Τ level and its decrease rate Dose Sample No. Serum GPT level GPT decrease rate group
(m g / k g ) n (U/L) (%) 病態対照群 10 603± 145  (mg / kg) n (U / L) (%) Disease control group 10 603 ± 145
C Ε 1投与群 50 11 75.9±12.7 *** 87.4  C Ε 1 group 50 11 75.9 ± 12.7 *** 87.4
*** P < 0. 0 0 1 *** P <0. 0 0 1
LJ P T ： glutamic-pyruvic transaminase LJ PT: glutamic-pyruvic transaminase
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下に実施例を掲記し本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail with reference to Examples.
a)青しよう子の乾燥粉末 300 gに水 2 を加えて加熱還流抽出器により 3 時間熱水抽出した。 次いで混合物をろ過し、 得られた水溶性ろ液を凍結乾燥して 7]性抽出物 34 gを得た。  a) Water 2 was added to 300 g of dried powder of blue syrup, and extracted with hot water for 3 hours using a heated reflux extractor. Then, the mixture was filtered, and the obtained water-soluble filtrate was lyophilized to obtain 34 g of a 7] -soluble extract.
b)実施例 a)で得た水性抽出物 13 gをソックスレー抽出機によりクロロホ ルム抽出し、 クロ口ホルム可溶画分と残渣を得た。 残渣を更にソックスレー抽出 機によりメタノール抽出し、 メタノール可溶画分と残渣を得た。 いずれも凍結乾 燥して、 クロ口ホルム可溶画分 1. 3g、 メタノール可溶画分 2. Og及び有機 溶媒不溶性沈澱物 8. 9gを得た。  b) 13 g of the aqueous extract obtained in Example a) was extracted with Chloroform using a Soxhlet extractor to obtain a solubilized form-soluble fraction and a residue. The residue was further extracted with methanol using a Soxhlet extractor to obtain a methanol-soluble fraction and a residue. All were freeze-dried to obtain 1.3 g of a form-soluble fraction in black mouth, 2.Og of a methanol-soluble fraction and 8.9 g of an organic solvent-insoluble precipitate.
c )実施例 b )で得た有機溶媒不溶性沈澱物 8. 9 gを水に溶解して分子量 1 万の透析膜にて透折を行った。 分子量 1万以上の非透析画分を D EAE-Toy opear l 650M (東ソ一社製） カラムに付した。 0. 3MNaC lで溶 出した画分を凍結乾燥して 234. 5mgの抽出物を得た。  c) 8.9 g of the organic solvent-insoluble precipitate obtained in Example b) was dissolved in water, and the solution was subjected to filtration through a dialysis membrane having a molecular weight of 10,000. The non-dialyzed fraction having a molecular weight of 10,000 or more was applied to a DEAE-Toyopearl 650M (manufactured by Tosoichi) column. The fraction eluted with 0.3 M NaCl was lyophilized to give 234.5 mg of extract.
更に、 この抽出物を Toy op ear 1 HW- 55 F c. c. (東ソ一社製） カラムに付し、 0. 2MNaC 1で溶出を行い、 ピーク画分を分取する操作を繰 り返すことにより、 乾燥重量 46. 9mgの CE 1の凍結乾燥品を得た。 以上の 精製過程を図 5に示す。 図面の簡単な説明  Furthermore, this extract was applied to a Toy opear 1 HW-55 Fcc (manufactured by Toso Ichisha) column, eluted with 0.2 M NaC1, and the procedure of collecting the peak fraction was repeated. A freeze-dried product of CE 1 having a dry weight of 46.9 mg was obtained. Figure 5 shows the above purification process. BRIEF DESCRIPTION OF THE FIGURES
図 1は CE 1の紫外線吸収スぺクトルデータを示すグラフである。  FIG. 1 is a graph showing the UV absorption spectrum data of CE1.
図 2は CE 1の赤外線吸収スぺクトルデータを示すグラフである。  FIG. 2 is a graph showing infrared absorption spectrum data of CE1.
図 3は、 (a) P. acne s/LP S誘発肝障害マウスの生存率に対する水 性抽出物の効果を示すグラフ及び（b) P. a c n e sZLPS誘発肝障害マウ スの生存率に対するグリチルリチンの効果を示すグラフである。  Figure 3 shows (a) a graph showing the effect of the aqueous extract on the survival rate of P. acnes s / LPS-induced liver injury mice and (b) a graph showing the effect of glycyrrhizin on the survival rate of P. acnes sZLPS-induced liver injury mice. It is a graph which shows an effect.
図 4は P. acne s ZL P S誘発肝障害マウスの生存率に対する C E 1の効 果を示すグラフである。  FIG. 4 is a graph showing the effect of CE1 on the survival rate of P. acnes ZLPS-induced liver injury mice.
図 5は青しょう子からの C E 1の精製過程を示す図である。  FIG. 5 is a diagram showing a purification process of CE1 from blue ginger.

Claims

請 求 の 範 囲 The scope of the claims
1. 青しょう （Ce l o s i a a r gen t e a L. ) の種子（青しょう 子） より抽出し、 単離することから成る水溶性抽出物。 1. A water-soluble extract that is extracted and isolated from the seeds of blue ginger (CelosaiargenteaL.).
2. 下記（a) 〜 （c) の工程により得られうることを特徴とする請求の範囲 第 1項記載の水溶性抽出物。 2. The water-soluble extract according to claim 1, which can be obtained by the following steps (a) to (c).
(a)青しょう子より熱水にて水性抽出液を抽出し、  (a) Extract the aqueous extract with hot water from blue ginger,
(b) 次いで有機溶媒で処理することにより、 有機溶媒不溶性沈殿物を得、 (b) Then, by treating with an organic solvent, an organic solvent-insoluble precipitate is obtained,
( c ) 更に水溶性抽出物を単離する。 (c) further isolating the water-soluble extract.
3. 1〜30万の分子量を有する請求の範囲第 1又は 2項のいずれかに記載の 水溶性抽出物。 3. The water-soluble extract according to claim 1, having a molecular weight of 10,000 to 300,000.
4. 下記①及び②の理化学的性質を有する請求の範囲第 1〜 3項のいずれかに 記載の水溶性抽出物。 4. The water-soluble extract according to any one of claims 1 to 3, having the following physicochemical properties (1) and (2).
①ァラビノース、 ラムノース、 マンノース、 ガラク トース、 ガラクッロン酸、 グ ルコース、 フコース及びグルクロン酸に対応する多糖類単位を含有する。  ① Contains polysaccharide units corresponding to arabinose, rhamnose, mannose, galactose, galacturonic acid, glucose, fucose and glucuronic acid.
② 0. 5〜1. 5 %の、 蛋白質由来の窒素原子を含有する。  ② Contains 0.5 to 1.5% of protein-derived nitrogen atoms.
5. 請求の範囲第 2項記載の工程により得られ、 多糖類を 90〜99重量％及 び蛋白質を 1〜 1 0重量 含有する水溶性抽出物。 5. A water-soluble extract obtained by the process according to claim 2 and containing 90 to 99% by weight of a polysaccharide and 1 to 10% by weight of a protein.
6. 請求の範囲第 1〜 5項のレ、ずれかに記載の水溶性抽出物を有効成分として 含有する医薬組成物。 6. A pharmaceutical composition comprising the water-soluble extract according to any one of claims 1 to 5 as an active ingredient.
7. 請求の範囲第 1〜 5項のいずれかに記載の水溶性抽出物を有効成分として 含有する、 ヒト又は動物の肝障害の予防 ·治療用組成物。 7. A composition for preventing or treating hepatic injury in humans or animals, comprising the water-soluble extract according to any one of claims 1 to 5 as an active ingredient.
8 . 請求の範囲第 1〜 5項のいずれかに記載の水溶性抽出物を有効成分として 含有する食品添加物。 8. A food additive containing the water-soluble extract according to any one of claims 1 to 5 as an active ingredient.
PCT/JP1996/001275 1995-05-17 1996-05-15 Water-soluble extract originating in feather cockscomb WO1996036346A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP53469796A JP3984288B2 (en) 1995-05-17 1996-05-15 Preventive and therapeutic composition derived from green pepper
AU57019/96A AU5701996A (en) 1995-05-17 1996-05-15 Water-soluble extract originating in feather cockscomb

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP11758495 1995-05-17
JP7/117584 1995-05-17

Publications (1)

Publication Number Publication Date
WO1996036346A1 true WO1996036346A1 (en) 1996-11-21

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JP (1) JP3984288B2 (en)
AU (1) AU5701996A (en)
WO (1) WO1996036346A1 (en)

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Publication number Priority date Publication date Assignee Title
JP2002527457A (en) * 1998-10-16 2002-08-27 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム Slowly digestible protein substances and their use
JP2005220101A (en) * 2004-02-06 2005-08-18 Maruzen Pharmaceut Co Ltd Anti-ageing agent, platelet aggregation inhibitor, antioxidant, anti-allergic agent, and food and drink
JP2005320271A (en) * 2004-05-07 2005-11-17 Maruzen Pharmaceut Co Ltd Aldose reductase activity inhibitor, diabetic complication-preventing/treating agent and diabetic complication-preventing/treating food and beverage

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JPS4891211A (en) * 1972-03-11 1973-11-28
JPH02223526A (en) * 1988-11-04 1990-09-05 Freunt Ind Co Ltd Composition containing extracts extracted with aqueous organic solvent
JPH03223217A (en) * 1989-03-03 1991-10-02 Asahi Breweries Ltd Composition having antiactive oxygen effect and antiactive oxygen-agent, food, cosmetics and drug containing the same composition as active component
JPH03255032A (en) * 1990-03-05 1991-11-13 Sanyo Kokusaku Pulp Co Ltd Antiulcer agent
JPH0899889A (en) * 1994-08-02 1996-04-16 Taisho Pharmaceut Co Ltd Therapeutic agent for atopic dermatitis

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JPS4891211A (en) * 1972-03-11 1973-11-28
JPH02223526A (en) * 1988-11-04 1990-09-05 Freunt Ind Co Ltd Composition containing extracts extracted with aqueous organic solvent
JPH03223217A (en) * 1989-03-03 1991-10-02 Asahi Breweries Ltd Composition having antiactive oxygen effect and antiactive oxygen-agent, food, cosmetics and drug containing the same composition as active component
JPH03255032A (en) * 1990-03-05 1991-11-13 Sanyo Kokusaku Pulp Co Ltd Antiulcer agent
JPH0899889A (en) * 1994-08-02 1996-04-16 Taisho Pharmaceut Co Ltd Therapeutic agent for atopic dermatitis

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* Cited by examiner, † Cited by third party
Title
KANEYOSHI AKAMATSU, (Author), "New Edit. Chinese and Japanese Medicine", 1st Edit., 5th Print, 15 October 1980, ISHIYAKU SHUPPAN K.K., (TOKYO), p. 478. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002527457A (en) * 1998-10-16 2002-08-27 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム Slowly digestible protein substances and their use
JP2005220101A (en) * 2004-02-06 2005-08-18 Maruzen Pharmaceut Co Ltd Anti-ageing agent, platelet aggregation inhibitor, antioxidant, anti-allergic agent, and food and drink
JP2005320271A (en) * 2004-05-07 2005-11-17 Maruzen Pharmaceut Co Ltd Aldose reductase activity inhibitor, diabetic complication-preventing/treating agent and diabetic complication-preventing/treating food and beverage
JP4623999B2 (en) * 2004-05-07 2011-02-02 丸善製薬株式会社 Aldose reductase activity inhibitor, preventive / therapeutic agent for diabetic complications, and food and drink for the prevention / treatment of diabetic complications

Also Published As

Publication number Publication date
AU5701996A (en) 1996-11-29
JP3984288B2 (en) 2007-10-03

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