WO2009147702A1 - Cerebroprotective agent - Google Patents

Cerebroprotective agent Download PDF

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Publication number
WO2009147702A1
WO2009147702A1 PCT/JP2008/001521 JP2008001521W WO2009147702A1 WO 2009147702 A1 WO2009147702 A1 WO 2009147702A1 JP 2008001521 W JP2008001521 W JP 2008001521W WO 2009147702 A1 WO2009147702 A1 WO 2009147702A1
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Prior art keywords
purple
koji
citric acid
sweet potato
purple sweet
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PCT/JP2008/001521
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French (fr)
Japanese (ja)
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鎌田照男
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トーシン株式会社
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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)
    • A61K36/39—Convolvulaceae (Morning-glory family), e.g. bindweed
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the present invention relates to a brain protective agent, and more particularly to a brain protective agent that is naturally derived and excellent in safety.
  • Edaravone (trade name Rajcut), aspirin, warfarin, etc. are currently used as brain protective agents such as cerebral infarction preventives and thrombus preventives. These are chemical substances and are concerned about long-term side effects.
  • Natural food-derived brain protective agents include ginkgo biloba extract (Japanese Patent Laid-Open No. 2007-277183), Yamabushitake (2006-129743), safflower extract extract (Japanese Patent Laid-Open No. 2001-346555), hot water extract of Cordyceps sinensis (Japanese Patent Laid-Open No. 2003-292451) and the like are known.
  • An object of the present invention is to provide a food-derived and safe brain protective agent similar to the above-mentioned natural food-derived brain protective agent.
  • this invention provides the brain protective agent which has a purple koji fermentation liquid as a main component.
  • JP-A-2001-69957 citric acid-containing beverage and method for producing the same
  • JP-A-2003-33163 sweet potato juice
  • Japanese Patent Application Laid-Open Publication No. 2004-121221 health food materials and health drinks using sweet potato shochu distilled spirit as raw materials and methods for producing them.
  • purple koji fermented liquid has a brain protective action such as prevention of cerebral infarction.
  • the purple koji fermented liquid is, for example, (1) a step of peeling the purple koji and chilling the surface of the peeled koji koji, followed by freezing. (2) thawing frozen purple koji, (3) steaming the thawed purple persimmon, (4) charging steamed purple rice bran and rice bran into a tank and fermenting purple rice cake at a temperature of 50 to 70 ° C. for 18 to 28 hours; (5) It is obtained by pressing the fermented purple koji and separating it into juice and solid residue, and (6) adding citric acid to the juice and adjusting the acidity. .
  • the purple koji fermentation broth is also (1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing (2) thawing frozen purple koji, (3) squeezing the thawed purple koji and separating it into squeezed juice and solid residue; and (4) charging the squeezed liquor and rice koji into a tank at a temperature of 50 to 70 ° C. for 18 to 28 hours. Fermenting the koji, (5) It is obtained by adding citric acid to the juice and adjusting the acidity.
  • the brain protective agent of the present invention may be one obtained by freeze-drying the purple koji fermented liquid and processing it into a powder or a tablet.
  • the brain protective agent of the present invention is particularly useful for prevention of cerebral infarction.
  • Purple grape contains abundant minerals such as polyphenols, citric acid, iron and potassium, but is low in protein. Moreover, purple koji fermented liquor has a higher circulatory activity than other vinegars.
  • the cerebral protective agent containing the purple koji fermented liquid of the present invention as a main component is expected to prevent cerebral infarction caused by circulatory disturbance.
  • the main component of the brain protective agent of the present invention is purple koji fermented liquid.
  • the purple koji fermentation broth can be prepared by the following steps.
  • Citric acid treatment / freezing process The purple candy is peeled off, and the surface of the peeled purple candy is treated with citric acid and then frozen.
  • the raw material purple koji is not particularly limited as long as it is purple koji, and examples thereof include Ayamurasaki, Nakamura Saki, Tanegashima purple, Yamakawa purple, Purple Sweet Road, Miyano 36 and Kyushu 109.
  • the citric acid used may be in any form such as citrus-fermented sweet potato or other starchy raw materials or crystalline citric acid, but citric acid derived from sweet potato is preferred in terms of imparting a strawberry flavor.
  • a particularly preferable raw material is citric acid contained in the purple koji solid residue obtained in the squeezing step (5) described later, or a residue obtained by subjecting the residue to citric acid fermentation.
  • the means of citric acid treatment is usually application or immersion of a citric acid aqueous solution.
  • concentration of the citric acid aqueous solution is usually 10 to 50%, preferably 20 to 30%.
  • the purple koji coated with citric acid is usually stored frozen at a temperature of ⁇ 80 to ⁇ 10 ° C., preferably ⁇ 25 to ⁇ 15 ° C. Freezing is effective in destroying the purple cell membrane.
  • the appropriately cut purple koji is usually steamed at a temperature of 80 to 100 ° C., preferably 90 to 100 ° C., for 20 to 60 minutes, preferably 20 to 40 minutes. Steaming kills germs and promotes the subsequent growth of koji molds.
  • the rice bran to be used is usually a koji mold belonging to the genus Aspergius and having a high starch resolving power.
  • Examples include Aspergillus oryzae, Aspergillus awamori, Aspergillus niger, Aspergillus kawachi, and the like. Of these, jaundice is preferred.
  • the spore of the koji mold is in contact with steamed rice and left at a temperature of about 35 ° C. for about 3 days. Thereafter, spores germinate, and rice bran is obtained in which steamed rice is covered with mycelia of koji mold.
  • rice bran commercially available ones may be used, for example, “Koji mold” (manufactured by Tokushima Seiko Co., Ltd.).
  • Steamed purple rice bran and rice bran into a tank and add water The amount of rice bran used is usually 100 to 230 parts by weight, preferably 130 to 200 parts by weight, per 100 parts by weight of steamed purple rice bran.
  • the sugar content is usually in the range of 5 to 35%, preferably 10 to 35%, particularly preferably 20 to 35%.
  • the fermented purple koji is put in a hemp bag and compressed with a press or the like.
  • the degree of compression is usually from 6 to 14 MPa, preferably from 8 to 12 MPa.
  • the obtained juice is appropriately filtered and filtered through a hollow fiber membrane or centrifuged to remove fine suspended matters.
  • the juice may be concentrated or diluted as appropriate.
  • the acidity is adjusted by adding citric acid to the clarified purple koji juice obtained in step (5) as appropriate.
  • the aqueous citric acid solution is preferably derived from a purple koji solid residue in order to maintain the flavor of koji.
  • the citric acid concentration suitable as a purple koji processed beverage product is usually 500 to 4000 mg / 100 ml, preferably 800 to 3000 mg / 100 ml, particularly preferably 1000 to 2800 mg / 100 ml.
  • citric acid powder is added to the purple koji juice to adjust the citric acid concentration to the above range.
  • the obtained purple koji fermentation broth is preferably sterilized by heating at a temperature of 80 ° C. or higher, preferably 90 to 98 ° C. for 10 to 15 minutes.
  • the purple koji fermented liquid has the following steps: (1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing (2) thawing frozen purple koji, (3) squeezing the thawed purple koji and separating it into squeezed juice and solid residue; and (4) charging the squeezed liquor and rice koji into a tank at a temperature of 50 to 70 ° C. for 18 to 28 hours. Fermenting the koji, (5) It can also be produced by a step of adjusting the acidity by adding citric acid to the fermentation broth.
  • This production method is the same as the production method described above, except that ginger is squeezed and then fermented with rice bran.
  • a step of sterilizing the juice may be added between step (3) and step (4). Sterilization is usually performed at 70 to 80 ° C. for 1 to 10 minutes.
  • the concentration (dilution degree) of the purple koji fermented solution contained in the brain protective agent of the present invention varies depending on the amount of brain protective agent ingested, but it may usually be 1 to 100% by weight, preferably 10 to 10%. 100% by weight, more preferably 100% by weight (purple fermented broth stock solution).
  • concentration of the purple koji fermented liquid is 1% by weight or less, it may not be possible to take in an amount necessary for obtaining a brain protective action.
  • diluting distilled water is preferable.
  • the cerebral protective agent of the present invention may be a syrup composed of an extract obtained by concentrating the above solution. Syrups are formulated in the form of bottles or capsules. Furthermore, the brain protective agent of the present invention may be processed into a powder, granule, tablet, capsule or the like in addition to the liquid or syrup. The purple koji fermented liquid is lyophilized to a powder and then tableted or encapsulated as appropriate.
  • the brain protective agent of the present invention includes pharmacologically usable carriers / excipients, binders, lubricants, lubricants, disintegrants, surfactants,
  • the effects of the present invention include emulsifiers, solubilizers, absorption enhancers, pH adjusters, brighteners, stabilizers, antioxidants, preservatives, wetting agents, colorants, fragrance excipients, and other auxiliaries. Can be added in a range that does not inhibit the above.
  • the usage of the brain protective agent of the present invention is oral administration.
  • the dose of the cerebral protective agent of the present invention can be determined in consideration of various factors that affect the patient's symptoms, body weight, administration interval, administration method, and other clinical effects.
  • the intake amount of the purple koji fermentation broth per day for an adult male may normally be 10 to 40 ml, preferably 20 to 30 ml, more preferably 25 to 30 ml.
  • Example 1 (Preparation of brain protective agent) The purple candy (variety: Ayamurasaki) was washed and peeled. The peeled purple koji was dipped in a citric acid aqueous solution having a citric acid concentration of 25% (derived from a purple koji solid residue) and stored frozen at ⁇ 25 ° C. for 10 days.
  • the purple koji preserved in a frozen state was thawed to room temperature, and then cut into a size that was easy to cook (about 5 cm square).
  • the cut purple koji was steamed at a temperature of 100 ° C. for 40 minutes.
  • the fermented purple koji was put in a hemp sack and squeezed by applying a load of 10 MPa with a squeezing machine to separate into squeezed juice and solid residue.
  • the resulting juice was further filtered to remove impurities.
  • aqueous citric acid solution was added to the filtered juice to adjust the acidity to 1400 mg / 100 ml.
  • the pH of the obtained juice was 3.0.
  • the squeezed juice was sterilized at a temperature of 90 ° C. for 10 minutes to obtain a purple koji fermentation broth having a liquid volume of 230 L.
  • LPS neuroprotection against endotoxin
  • NO production by LPS can be used as an indicator that LPS causes tissue damage by some mechanism.
  • Rat cerebellar granule cells were prepared by the following procedure. First, using 10 Wistar rats (obtained from the Charles River strain) of the same litter of 8 days of age without discrimination between males and females, each cerebellum was extracted. The cerebellum was minced to about 400 um 3 and treated in Krebs-HEPES buffer containing bovine serum albumin, trypsin Mg 2+ , Ca 2+ and the like, and granule cells were collected. Fractionated cells, fetal bovine serum, KCI, Basal Medium Eagle medium containing glutamine (product name GL-1, Kohjin Bio Co.) were seeded into, 5% CO 2 incubator maintained at 37 ° C. (ASTEC Ltd. The culture was started in).
  • glia cells 6: 4
  • the cultured cells were washed with Krebs-HEPES buffer (136 mM NaCl, 10 mM glucose, 20 mM HEPES, 5 mM KCI, 1.3 mM MgCl 2 , 1.2 mM CaCl 2 pH 7.4). Experiments were performed with liquid.
  • the purple koji fermentation broth obtained above is diluted to final concentrations of 0.0626%, 0.125%, 0.25% and 0.5%, respectively, and applied to cultured brain neurons simultaneously with the addition of LPS. did. The condition was 20 hours at 37 ° C.
  • black vinegar product name: product name: Sakamoto brewing vinegar
  • rice vinegar product name: Fukuyama brewing rice vinegar
  • NO 2 and NO 3 detected measurement peaks at about 4 minutes and 6 minutes, respectively, after the start of measurement.
  • the measurement results were compared by NO 2 and NO 3 area (AUC).
  • LPS E. coli 026: B6, DIFCOLABORATORIES, MI, USA
  • Measurement of NO 2 / NO 3 was started immediately after LPS administration. As the measurement result, the sum of NO 2 and NO 3 of each value was obtained and displayed as NOx.
  • the normal extracellular NOx value of primary cultured brain neurons was about 50 pmol (43.1 ⁇ 8.5), but when LPS (10 ug / mI) was applied to cultured cells for 20 hours, NOx production was about 1. The amount was increased 7 times (75.0 ⁇ 4.5).
  • Fig. 1 shows the effect of adding purple koji fermented liquid on LPS-induced NOx production.
  • the vertical axis (inhibition rate) in FIG. 1 is obtained by calculating the percentage of the results with each concentration of purple koji fermented liquid when the measured value of LPS alone is 100%. Groups with significant differences in LPS-induced NOx production values were marked with * (*: P ⁇ 0.05). As FIG. 1 shows, a significant inhibitory effect was observed at 0.25 and 0.5% purple koji fermentation broth concentrations.
  • Fig. 2 shows the effect of black vinegar on LPS-induced NOx production.
  • the display method is the same as that of the purple koji fermentation broth.
  • black vinegar was observed to be suppressed at 0.25 and 0.5% concentrations.
  • inhibition of weight gain at 0.5% concentration was not a statistically significant result.
  • Fig. 3 shows the effect of rice vinegar on LPS-induced NOx production.
  • the display method is the same as that of the purple koji fermentation broth.
  • rice vinegar was observed to be inhibited at concentrations of 0.25 and 0.5%. However, these were not statistically significant differences.
  • the embolus used was an 11 mm long 8-0 nylon monofilament (Ethylon: Ethicon. NJ. USA) coated 4 mm from the tip with a silicone resin (Xantopren: Bayer Dental. Osaka. Japan). The diameter of the coating portion was selected as appropriate depending on the body weight. The occlusion time was 4 hours.
  • mice were decapitated by intraperitoneal administration of an excessive amount of pentobarbital 24 hours after middle cerebral artery occlusion.
  • the removed brain was frozen with dry ice, and pre-slice slices of the brain containing the cerebral cortex were prepared at 2 mm intervals. Sections were incubated in a 2% 2.3.5-triphenyltetrazolium chloride (TTC, purchased from Sigma) solution at 37 ° C. for 30 minutes. They were arranged on a slide glass and photographed with a digital still camera (MVC-FD91, manufactured by Sony Corporation).
  • TTC 2.3.5-triphenyltetrazolium chloride
  • FIG. 4 shows the influence of purple koji fermented liquid, black vinegar, and rice vinegar on the infarct volume that occurs after middle cerebral artery occlusion.
  • Neurological symptoms In order to visually judge the movement disorder due to middle cerebral artery occlusion / reperfusion, it was scored in 5 stages. The criteria for the score are as follows. In addition, it measured immediately after restarting. 0: normal posture 1: flexion of torso and of contralateral forI im uplifting of the animal by the tail 2: Circulating to the contralateral side but normal post at rest 3: Continuous circling to the contralateral side 4: rolling to the contralateral side Table 1 shows the average score and standard error of neurological symptoms (immediately after recommencement).
  • FIG. 4 shows that administration of purple koji fermented liquid suppressed the size of the infarction and was a clear dose-dependent suppression.
  • the stock solution ( ⁇ 1) was continuously administered, a statistically significant change was observed for infarct suppression.
  • the neurological symptom average values shown in Table 1 also in the purple koji fermented liquid, a dose-dependent improvement was observed, and a statistically significant change (*: p ⁇ 0.05) was observed in the stock solution administration group. It was.
  • Black vinegar showed a clear inhibitory effect in continuous administration in the stock solution administration group (FIG. 4).
  • the neurological symptoms in Table 1 although the overall score decreased, no significant improvement was observed. Rather, a behavior that rubs the belly was observed.
  • Rice vinegar had no effect on the infarct in any of the administration groups with continuous administration (FIG. 4). No improvement effect was observed for neurological symptoms (Table 1).

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Abstract

It is intended to provide a safe cerebroprotective agent originating in a food. Namely, a cerebroprotective agent which comprises fermented purple sweet potato liquor as the main component. The fermented purple sweet potato liquor is the one obtained by, for example, the following steps: (1) the step of peeling the purple sweet potato, treating the surface of the peeled purple sweet potato with citric acid and then freezing; (2) the step of thawing the frozen purple sweet potato; (3) the step of cooking the thawed purple sweet potato; (4) the step of feeding the cooked purple sweet potato and rice koji in a tank and fermenting the purple sweet potato at a temperature of 50 to 70oC for 18 to 28 hours; (5) the step of pressing the fermented purple sweet potato followed by separation into the press juice and the solid residue; and (6) the step of further adding citric acid to the press juice as described above to thereby control the acidity. This cerebroprotective agent is particularly efficacious in preventing cerebral infarction.

Description

脳保護作用剤Brain protective agent
　本発明は、脳保護作用剤に関し、より詳細には天然由来で安全性に優れた脳保護作用剤に関する。 The present invention relates to a brain protective agent, and more particularly to a brain protective agent that is naturally derived and excellent in safety.
　高齢化社会がますます進展する中、多くの高齢者が高血圧、動脈硬化や心疾患が発端となって、脳内血流が停滞し、脳梗塞になり、また脳神経細胞死を招く危険にさらされている。これらの症状が過激であると、身体麻痺、痴呆症等に進行することもある。脳梗塞等で損傷した脳は、現状では再生が困難であるため、日常から動脈硬化、心疾患、脳梗塞等を予防するような生活を送ることが大事となる。 As the aging society progresses more and more, many elderly people are exposed to the risk of hypertension, arteriosclerosis and heart disease, cerebral blood flow stagnation, cerebral infarction, and neuronal cell death. Has been. If these symptoms are extreme, they may progress to body paralysis, dementia and the like. Since the brain damaged by cerebral infarction or the like is difficult to regenerate at present, it is important to live a life that prevents arteriosclerosis, heart disease, cerebral infarction and the like from the daily routine.
　脳梗塞予防剤、血栓予防剤などの脳保護作用剤として、現在、エダラボン（商品名ラジカット）、アスピリン、ワーファリンなどが使用されている。これらは、化学物質であり、長期にわたる服用での副作用が心配である。 Edaravone (trade name Rajcut), aspirin, warfarin, etc. are currently used as brain protective agents such as cerebral infarction preventives and thrombus preventives. These are chemical substances and are worried about long-term side effects.
　天然の食物由来の脳保護作用剤としては、イチョウ葉エキス（特開２００７－２７７１８３）、ヤマブシタケ（２００６－１２９７４３）、紅花の搾汁エキス混合物（特開２００１－３４６５５５）、冬虫夏草の熱水抽出物（特開２００３－２９２４５２）などが公知である。 Natural food-derived brain protective agents include ginkgo biloba extract (Japanese Patent Laid-Open No. 2007-277183), Yamabushitake (2006-129743), safflower extract extract (Japanese Patent Laid-Open No. 2001-346555), hot water extract of Cordyceps sinensis (Japanese Patent Laid-Open No. 2003-292451) and the like are known.
　本発明の目的は、上記の天然食物由来の脳保護作用剤と同様に、食物由来で安全な脳保護作用剤を提供することにある。 An object of the present invention is to provide a food-derived and safe brain protective agent similar to the above-mentioned natural food-derived brain protective agent.
　本発明者は、上記課題に対して鋭意研究開発を進めた結果、紫芋発酵液の脳保護作用が顕著であることを発見し、本発明に至った。すなわち、本発明は、紫芋発酵液を主成分とする脳保護作用剤を提供する。 As a result of intensive research and development on the above problems, the present inventor discovered that the brain-protecting action of the purple koji fermented liquid is remarkable, leading to the present invention. That is, this invention provides the brain protective agent which has a purple koji fermentation liquid as a main component.
　甘藷をクエン酸発酵して得られるクエン酸を含有する飲料とする従来技術として、特開２００１－６９９５７号公報（クエン酸含有飲料及びその製造方法）、特開２００３－３３１６３号公報（サツマイモ搾汁液の濃縮方法、健康飲料の製造方法及び健康飲料）、特開２００４－１２１２２１号公報（甘しょ焼酎蒸留粕を原料とする健康食品素材と健康飲料及びそれらの製造方法）などがある。しかし、これらの従来技術からは、紫芋発酵液に脳梗塞予防等の脳保護作用があることは全く知られていなかった。 JP-A-2001-69957 (citric acid-containing beverage and method for producing the same), JP-A-2003-33163 (sweet potato juice) as conventional techniques for producing a sweet potato-containing beverage obtained by fermenting sweet potato with citric acid Concentration method, health drink manufacturing method and health drink), and Japanese Patent Application Laid-Open Publication No. 2004-121221 (health food materials and health drinks using sweet potato shochu distilled spirit as raw materials and methods for producing them). However, from these prior arts, it has not been known at all that purple koji fermented liquid has a brain protective action such as prevention of cerebral infarction.
　上記紫芋発酵液は、例えば
（１）紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する工程、
（２）冷凍された紫芋を解凍する工程、
（３）解凍された紫芋を蒸煮する工程、
（４）蒸された紫芋と米麹とをタンクに仕込み、５０～７０℃の温度で１８～２８時間、紫芋を発酵させる工程、
（５）発酵された紫芋を圧搾し、搾汁と固形残渣とに分離する工程、および
（６）前記搾汁にさらにクエン酸を添加して酸度を調整する工程
により得られたものである。 The purple koji fermented liquid is, for example, (1) a step of peeling the purple koji and chilling the surface of the peeled koji koji, followed by freezing.
(2) thawing frozen purple koji,
(3) steaming the thawed purple persimmon,
(4) charging steamed purple rice bran and rice bran into a tank and fermenting purple rice cake at a temperature of 50 to 70 ° C. for 18 to 28 hours;
(5) It is obtained by pressing the fermented purple koji and separating it into juice and solid residue, and (6) adding citric acid to the juice and adjusting the acidity. .
　前記紫芋発酵液は、また、
（１）紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する工程、
（２）冷凍された紫芋を解凍する工程、
（３）解凍した紫芋を圧搾し、搾汁と固形残渣とに分離する工程、および
（４）搾汁液と米麹とをタンクに仕込み、５０～７０℃の温度で１８～２８時間、紫芋を発酵させる工程、
（５）前記搾汁にさらにクエン酸を添加して酸度を調整する工程
により得られたものである。 The purple koji fermentation broth is also
(1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing
(2) thawing frozen purple koji,
(3) squeezing the thawed purple koji and separating it into squeezed juice and solid residue; and (4) charging the squeezed liquor and rice koji into a tank at a temperature of 50 to 70 ° C. for 18 to 28 hours. Fermenting the koji,
(5) It is obtained by adding citric acid to the juice and adjusting the acidity.
　本発明の脳保護作用剤は、前記紫芋発酵液を凍結乾燥し、粉末または錠剤状に加工したものでもよい。 The brain protective agent of the present invention may be one obtained by freeze-drying the purple koji fermented liquid and processing it into a powder or a tablet.
　本発明の脳保護作用剤は、特に脳梗塞の予防に有用である。 The brain protective agent of the present invention is particularly useful for prevention of cerebral infarction.
　紫芋にはポリフェノール類、クエン酸、鉄分やカリウム分等のミネラルが豊富に含まれるが、低タンパク質である。また、紫芋発酵液は、他の酢よりも循環系の亢進作用が高い。本発明の紫芋発酵液を主成分とする脳保護作用剤には、循環障害によって惹起される脳梗塞の予防作用が期待される。 Purple grape contains abundant minerals such as polyphenols, citric acid, iron and potassium, but is low in protein. Moreover, purple koji fermented liquor has a higher circulatory activity than other vinegars. The cerebral protective agent containing the purple koji fermented liquid of the present invention as a main component is expected to prevent cerebral infarction caused by circulatory disturbance.
ＬＰＳ誘発のＮＯｘ産生に対する紫芋発酵液添加による抑制効果を示す図である。It is a figure which shows the inhibitory effect by the purple koji fermentation liquid addition with respect to LPS induction NOx production. ＬＰＳ誘発のＮＯｘ産生に対する黒酢添加による抑制効果を示す図である。It is a figure which shows the inhibitory effect by black vinegar addition with respect to LPS induction NOx production. ＬＰＳ誘発のＮＯｘ産生に対する米酢添加による抑制効果を示す図である。It is a figure which shows the inhibitory effect by the rice vinegar addition with respect to NOS production induced by LPS. 紫芋発酵液、黒酢および米酢の脳梗塞に及ぼす影響を梗塞巣の体積での比較した図である。It is the figure which compared the influence which it has on the cerebral infarction of purple koji fermented liquid, black vinegar, and rice vinegar in the volume of the infarct.
　以下、本発明の脳保護作用剤をより詳細に説明する。本発明の脳保護作用剤の主成分は、紫芋発酵液である。紫芋発酵液は、例えば以下の工程により調製することができる。 Hereinafter, the brain protective agent of the present invention will be described in more detail. The main component of the brain protective agent of the present invention is purple koji fermented liquid. For example, the purple koji fermentation broth can be prepared by the following steps.
（１）クエン酸処理・冷凍工程
　紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する。原料の紫芋は、紫芋であれば特に制限が無く、例えばアヤムラサキ、ナカムラサキ、種子島紫、山川紫、パープルスイートロード、宮農３６号および九州１０９号が挙げられる。 (1) Citric acid treatment / freezing process The purple candy is peeled off, and the surface of the peeled purple candy is treated with citric acid and then frozen. The raw material purple koji is not particularly limited as long as it is purple koji, and examples thereof include Ayamurasaki, Nakamura Saki, Tanegashima purple, Yamakawa purple, Purple Sweet Road, Miyano 36 and Kyushu 109.
　紫芋の表皮近くには、不良部分やアク成分が多いため、１～２ｍｍ剥皮して取り除く。次いで、剥いた紫芋の表面をクエン酸処理する。このクエン酸処理は、主に保存状態の向上や酸化防止のために行われる。 ¡There are many defective parts and aqua components near the surface of purple coral, so remove it by 1-2mm. Next, the peeled purple koji surface is treated with citric acid. This citric acid treatment is mainly performed for improving the storage state and preventing oxidation.
　使用するクエン酸は、甘藷その他のでんぷん質原料をクエン酸発酵させたものや結晶クエン酸などのいかなる形態のものでよいが、芋風味を付与する点で甘藷由来のクエン酸が好ましい。特に好ましい原料は、後述する搾汁工程（５）で得られる紫芋固形残渣に含有されるクエン酸、または残渣をクエン酸発酵させたものである。 The citric acid used may be in any form such as citrus-fermented sweet potato or other starchy raw materials or crystalline citric acid, but citric acid derived from sweet potato is preferred in terms of imparting a strawberry flavor. A particularly preferable raw material is citric acid contained in the purple koji solid residue obtained in the squeezing step (5) described later, or a residue obtained by subjecting the residue to citric acid fermentation.
　クエン酸処理の手段は、通常、クエン酸水溶液の塗布や浸漬である。クエン酸水溶液の濃度は、通常１０～５０％、好ましくは２０～３０％である。 The means of citric acid treatment is usually application or immersion of a citric acid aqueous solution. The concentration of the citric acid aqueous solution is usually 10 to 50%, preferably 20 to 30%.
　クエン酸の塗布された紫芋は、通常、－８０～－１０℃、好ましくは－２５～－１５℃の温度にて冷凍保管される。冷凍は、紫芋の細胞膜を破壊するのに有効である。 The purple koji coated with citric acid is usually stored frozen at a temperature of −80 to −10 ° C., preferably −25 to −15 ° C. Freezing is effective in destroying the purple cell membrane.
（２）解凍工程
　冷凍された紫芋を、室温まで解凍する。冬場は常温、夏場は冷蔵庫で解凍することが好ましい。解凍した芋は、蒸煮しやすいように、例えば１～２ｃｍ四方に切断するとよいが、切断しなくてもよい。 (2) Thawing process Thaw frozen purple persimmon to room temperature. It is preferable to thaw in a refrigerator in winter and at room temperature in summer. The thawed potato is cut into, for example, 1 to 2 cm square so that it can be easily cooked, but it does not need to be cut.
（３）蒸煮工程
　適宜切断した紫芋を、通常、８０～１００℃、好ましくは９０～１００℃の温度で、２０～６０分間、好ましくは２０～４０分間、蒸煮する。蒸煮は、雑菌を死滅させ、その後の麹菌の増殖を促す。 (3) Steaming step The appropriately cut purple koji is usually steamed at a temperature of 80 to 100 ° C., preferably 90 to 100 ° C., for 20 to 60 minutes, preferably 20 to 40 minutes. Steaming kills germs and promotes the subsequent growth of koji molds.
（４）発酵工程
　蒸された紫芋と米麹とをタンクに仕込み、紫芋を高温で発酵させる。米麹は、蒸した米に麹菌を繁殖させたものである。米麹には、でんぷんをブドウ糖に分解するアミラーゼが多く含まれ、これが紫芋のでんぷん質を分解し、さらに発酵させる。また、米麹に含まれる糖分が、紫芋発酵液の甘みの増強に寄与する。 (4) Fermentation process Steamed purple koji and rice koji are charged into a tank, and the koji is fermented at a high temperature. Rice bran is obtained by breeding koji mold on steamed rice. Rice bran contains a lot of amylase that breaks starch into glucose, which breaks down the starch quality of purple rice bran and ferments it further. Moreover, the sugar contained in rice bran contributes to the enhancement of sweetness of the purple koji fermented liquid.
　使用する米麹は、通常、アスペルギウス属に属する麹菌であってデンプン分解能の大きいものであればよい。例えば黄麹菌（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）、黒麹菌（Ａｓｐｅｒｇｉｌｌｕｓ　ａｗａｍｏｒｉ，　Ａｓｐｅｒｇｉｌｌｕｓ　ｎｉｇｅｒ）、白麹菌（Ａｓｐｅｒｇｉｌｌｕｓ　ｋａｗａｃｈｉ）などが挙げられる。中でも、黄麹菌が好ましい。 The rice bran to be used is usually a koji mold belonging to the genus Aspergius and having a high starch resolving power. Examples include Aspergillus oryzae, Aspergillus awamori, Aspergillus niger, Aspergillus kawachi, and the like. Of these, jaundice is preferred.
　前記米麹を作る（製麹）するには、上記麹菌の胞子を蒸米に接菌し、約３５℃の温度で約３日間放置する。その後、胞子が発芽し、蒸米が麹菌の菌糸で覆われた米麹が得られる。米麹は、市販のものを使用してもよく、例えば「黄麹菌」（徳島精工株式会社製）などがある。 To make the rice bran (i.e., koji making), the spore of the koji mold is in contact with steamed rice and left at a temperature of about 35 ° C. for about 3 days. Thereafter, spores germinate, and rice bran is obtained in which steamed rice is covered with mycelia of koji mold. As the rice bran, commercially available ones may be used, for example, “Koji mold” (manufactured by Tokushima Seiko Co., Ltd.).
　蒸した紫芋と米麹とを、タンクに仕込み、加水する。米麹の使用量は、蒸した紫芋１００重量部に対して、通常、１００～２３０重量部、好ましくは１３０～２００重量部である。 Steamed purple rice bran and rice bran into a tank and add water. The amount of rice bran used is usually 100 to 230 parts by weight, preferably 130 to 200 parts by weight, per 100 parts by weight of steamed purple rice bran.
　次いで、５０～７０℃、好ましくは５５～６５℃の温度で、通常、１８～２８時間、好ましくは２０～２４時間放置して、紫芋のでんぷん質を分解させ、発酵させる。その間、適宜、攪拌してもよい。 Then, it is left at a temperature of 50 to 70 ° C., preferably 55 to 65 ° C., usually for 18 to 28 hours, preferably 20 to 24 hours to decompose and ferment purple starch. In the meantime, you may stir suitably.
　糖度は、通常、５～３５％の範囲にあり、好ましくは１０～３５％、特に好ましくは２０～３５％である。 The sugar content is usually in the range of 5 to 35%, preferably 10 to 35%, particularly preferably 20 to 35%.
（５）搾汁工程
　発酵された紫芋を麻袋に入れて、圧搾機などで圧縮する。圧縮の程度は、通常、６～１４ＭＰａでよく、好ましくは８～１２ＭＰａである。得られた搾汁液は、適宜、フィルター、中空糸膜でのろ過や遠心分離機にかけて、微細な浮遊物を除去する。搾汁液は、適宜、濃縮または希釈してもよい。 (5) Juice process The fermented purple koji is put in a hemp bag and compressed with a press or the like. The degree of compression is usually from 6 to 14 MPa, preferably from 8 to 12 MPa. The obtained juice is appropriately filtered and filtered through a hollow fiber membrane or centrifuged to remove fine suspended matters. The juice may be concentrated or diluted as appropriate.
（６）酸度調整工程
　工程（５）で得られた適宜、清澄済みの紫芋搾汁液へ、クエン酸を添加して酸度を調整する。クエン酸水溶液は、紫芋固形残渣由来のものが芋の風味を保つ上で好ましい。紫芋加工飲料製品として適するクエン酸濃度は、通常、５００～４０００ｍｇ／１００ｍｌであり、好ましくは８００～３０００ｍｇ／１００ｍｌ、特に好ましくは１０００～２８００ｍｇ／１００ｍｌである。具体的には、紫芋搾汁液へ、クエン酸の粉末を添加して、クエン酸濃度を上記範囲へ調整する。 (6) Acidity adjustment process The acidity is adjusted by adding citric acid to the clarified purple koji juice obtained in step (5) as appropriate. The aqueous citric acid solution is preferably derived from a purple koji solid residue in order to maintain the flavor of koji. The citric acid concentration suitable as a purple koji processed beverage product is usually 500 to 4000 mg / 100 ml, preferably 800 to 3000 mg / 100 ml, particularly preferably 1000 to 2800 mg / 100 ml. Specifically, citric acid powder is added to the purple koji juice to adjust the citric acid concentration to the above range.
　得られた紫芋発酵液は、８０℃以上、好ましくは９０～９８℃の温度で、１０～１５分の加熱殺菌することが好ましい。 The obtained purple koji fermentation broth is preferably sterilized by heating at a temperature of 80 ° C. or higher, preferably 90 to 98 ° C. for 10 to 15 minutes.
　紫芋発酵液は、以下に示す工程：
（１）紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する工程、
（２）冷凍された紫芋を解凍する工程、
（３）解凍した紫芋を圧搾し、搾汁と固形残渣とに分離する工程、および
（４）搾汁液と米麹とをタンクに仕込み、５０～７０℃の温度で１８～２８時間、紫芋を発酵させる工程、
（５）前記発酵液にさらにクエン酸を添加して酸度を調整する工程
によっても製造可能である。 The purple koji fermented liquid has the following steps:
(1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing
(2) thawing frozen purple koji,
(3) squeezing the thawed purple koji and separating it into squeezed juice and solid residue; and (4) charging the squeezed liquor and rice koji into a tank at a temperature of 50 to 70 ° C. for 18 to 28 hours. Fermenting the koji,
(5) It can also be produced by a step of adjusting the acidity by adding citric acid to the fermentation broth.
　この製造方法は、生芋を搾汁した後に米麹とともに発酵させる以外は、前述の製造方法と同様である。 This production method is the same as the production method described above, except that ginger is squeezed and then fermented with rice bran.
　工程（３）と工程（４）との間に、搾汁液を殺菌する工程を付加してもよい。殺菌は、通常、７０～８０℃で１～１０分間行われる。 A step of sterilizing the juice may be added between step (3) and step (4). Sterilization is usually performed at 70 to 80 ° C. for 1 to 10 minutes.
　本発明の脳保護作用剤に含有される紫芋発酵液の濃度（希釈度）は、脳保護作用剤の摂取量によって変わってくるが、通常、１～１００重量％でよく、好ましくは１０～１００重量％、より好ましくは１００重量％（紫芋発酵液原液）である。紫芋発酵液の濃度が１重量％以下であると、脳保護作用を得るのに必要な量を摂取できない場合がある。なお、希釈する場合は、蒸留水が好ましい。 The concentration (dilution degree) of the purple koji fermented solution contained in the brain protective agent of the present invention varies depending on the amount of brain protective agent ingested, but it may usually be 1 to 100% by weight, preferably 10 to 10%. 100% by weight, more preferably 100% by weight (purple fermented broth stock solution). When the concentration of the purple koji fermented liquid is 1% by weight or less, it may not be possible to take in an amount necessary for obtaining a brain protective action. In addition, when diluting, distilled water is preferable.
　本発明の脳保護作用剤は、上記液剤を濃縮したエキスからなるシロップ剤でもよい。シロップ剤は、瓶詰めやカプセルの形態で処方される。さらに、本発明の脳保護作用剤は、液剤やシロップ剤のほかに、粉末、顆粒、錠剤、カプセルのような形態に加工されてもよい。紫芋発酵液を凍結乾燥して、粉末にした後、適宜、打錠やカプセル化する。 The cerebral protective agent of the present invention may be a syrup composed of an extract obtained by concentrating the above solution. Syrups are formulated in the form of bottles or capsules. Furthermore, the brain protective agent of the present invention may be processed into a powder, granule, tablet, capsule or the like in addition to the liquid or syrup. The purple koji fermented liquid is lyophilized to a powder and then tableted or encapsulated as appropriate.
　本発明の脳保護作用剤には、必須成分の紫芋発酵液のほかに、薬理学上使用可能な担体・賦形剤、結合剤、滑沢剤、潤滑剤、崩壊剤、界面活性剤、乳化剤、溶解補助剤、吸収促進剤、ｐＨ調整剤、光沢剤、安定化剤、酸化防止剤、保存剤、湿潤剤、着色剤、芳香剤賦形剤、その他の助剤を、本発明の効果を阻害しない範囲で添加することができる。 In addition to the essential component purple koji fermented liquid, the brain protective agent of the present invention includes pharmacologically usable carriers / excipients, binders, lubricants, lubricants, disintegrants, surfactants, The effects of the present invention include emulsifiers, solubilizers, absorption enhancers, pH adjusters, brighteners, stabilizers, antioxidants, preservatives, wetting agents, colorants, fragrance excipients, and other auxiliaries. Can be added in a range that does not inhibit the above.
　本発明の脳保護作用剤の用法は、経口投与である。本発明の脳保護作用剤の用量は、患者の症状、体重、投与間隔、投与方法、他の臨床的作用を左右する種々の因子を考慮して決定され得る。典型的には、成人男性一日あたりの紫芋発酵液原液の摂取量として、通常、１０～４０ｍｌでよく、好ましくは、２０～３０ｍｌであり、さらに好ましくは２５～３０ｍｌである。 The usage of the brain protective agent of the present invention is oral administration. The dose of the cerebral protective agent of the present invention can be determined in consideration of various factors that affect the patient's symptoms, body weight, administration interval, administration method, and other clinical effects. Typically, the intake amount of the purple koji fermentation broth per day for an adult male may normally be 10 to 40 ml, preferably 20 to 30 ml, more preferably 25 to 30 ml.
〔実施例１〕
（脳保護作用剤の調製）
　紫芋（品種：アヤムラサキ）を洗浄後、剥皮した。剥いた紫芋を、用意しておいたクエン酸濃度２５％のクエン酸水溶液（紫芋固形残渣由来のもの）へ浸した後、－２５℃で１０日間、冷凍保存した。 [Example 1]
(Preparation of brain protective agent)
The purple candy (variety: Ayamurasaki) was washed and peeled. The peeled purple koji was dipped in a citric acid aqueous solution having a citric acid concentration of 25% (derived from a purple koji solid residue) and stored frozen at −25 ° C. for 10 days.
　冷凍保存された紫芋を室温まで解凍し、次いで蒸煮しやすい大きさ（約５ｃｍ四方）に切断した。切断した紫芋を、１００℃の温度で４０分間蒸煮した。 The purple koji preserved in a frozen state was thawed to room temperature, and then cut into a size that was easy to cook (about 5 cm square). The cut purple koji was steamed at a temperature of 100 ° C. for 40 minutes.
　次いで、蒸した紫芋６０ｋｇと、市販の乾燥米麹（製品名ＫＬ－３、徳島精工株式会社製）１００ｋｇとを容積１５００Ｌのタンクに仕込み、約６０℃で２０時間放置し、紫芋を発酵させた。 Next, 60 kg of steamed purple koji and 100 kg of commercially available dried rice koji (product name KL-3, manufactured by Tokushima Seiko Co., Ltd.) are placed in a 1500 L tank and left at about 60 ° C. for 20 hours to ferment purple koji. I let you.
　発酵された紫芋を麻袋に入れて、圧搾機にて１０ＭＰａの荷重をかけて圧搾し、搾汁と固形残渣とに分離した。得られた搾汁をさらにフィルターろ過して、不純物を除去した。 The fermented purple koji was put in a hemp sack and squeezed by applying a load of 10 MPa with a squeezing machine to separate into squeezed juice and solid residue. The resulting juice was further filtered to remove impurities.
　ろ過した搾汁にクエン酸水溶液を添加し、酸度を１４００ｍｇ／１００ｍｌに調整した。得られた搾汁のｐＨは、３．０であった。搾汁を温度９０℃で１０分間殺菌して、液量２３０Ｌの紫芋発酵液を得た。 An aqueous citric acid solution was added to the filtered juice to adjust the acidity to 1400 mg / 100 ml. The pH of the obtained juice was 3.0. The squeezed juice was sterilized at a temperature of 90 ° C. for 10 minutes to obtain a purple koji fermentation broth having a liquid volume of 230 L.
　本発明の紫芋発酵液の脳保護作用を、黒酢および米酢と比較するために、初代培養脳神経細胞を用いた内毒素（Ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ、以下、単に「ＬＰＳ」という）誘発細胞障害に対する神経保護作用、ならびに、中大脳動脈閉塞マウスモデルの梗塞体積に対する阻害作用を調査した。 In order to compare the brain protective effect of the purple koji fermented liquid of the present invention with black vinegar and rice vinegar, neuroprotection against endotoxin (Lipopolysaccharide, hereinafter simply referred to as “LPS”)-induced cell damage using primary cultured brain neurons. The effect and the inhibitory effect on the infarct volume of the middle cerebral artery occlusion mouse model were investigated.
１．内毒素誘発細胞障害に対する神経保護作用
（各酢の内毒素誘発細胞障害に及ぼす影響）
　細胞組織におけるＮＯ発生をＬＰＳによる組織障害の程度の指標とした。その理由を、以下に説明する。一般的に、組織障害の生じる際、細胞内へのＣａ２＋が異常流入することが知られている。この異常の流入は、種々の細胞内情報系を活性させるが、その中の一つにＰｈｏｓｐｈｏｌｉｐａｓｅ　Ａ２を主体としたアラキドン酸カスケードの活性化が挙げられる。アラキドン酸の過剰産生は、その代謝酵素であるシクロオキシゲナーゼ（殊に、組織障害時や炎症発現時に誘発されるＩＩ型）の活性上昇を誘発させ、プロスタグランジン類の産生を促進する。この産生が炎症性反応を増長させ、組織内ではＮＯ合成酵素の活性化に繋がる。ＮＯは、さらに、活性酸素をはじめとするフリーラジカルの余剰産生を誘導し、組織障害へと発展する。これらの根拠から、ＬＰＳによるＮＯ産生は、何らかの機序でＬＰＳが組織障害を惹起することの指標として使用可能である。 1. Neuroprotective action against endotoxin-induced cytotoxicity (Effect of each vinegar on endotoxin-induced cytotoxicity)
NO generation in the cell tissue was used as an index of the degree of tissue damage caused by LPS. The reason will be described below. In general, it is known that when tissue damage occurs, Ca 2+ abnormally flows into cells. This abnormal inflow activates various intracellular information systems, one of which is activation of the arachidonic acid cascade mainly composed of phospholipase A2. Overproduction of arachidonic acid induces an increase in the activity of its metabolic enzyme, cyclooxygenase (particularly type II induced during tissue damage or inflammation), and promotes the production of prostaglandins. This production increases the inflammatory response and leads to activation of NO synthase in the tissue. NO further induces excessive production of free radicals including active oxygen, and develops into tissue damage. For these reasons, NO production by LPS can be used as an indicator that LPS causes tissue damage by some mechanism.
（初代培養神経細胞の作製）
　以下の手順で、ラット小脳顆粒細胞を作成した。まず、Ｗｉｓｔａｒ系ラット（チャールスリバー株より入手）で同腹の生後８日齢を雌雄の区別なく１０匹用い、それぞれの小脳を摘出した。小脳は、約４００ｕｍ３に細切後、牛血清アルブミン、トリプシンＭｇ２＋、Ｃａ２＋等を含有するＫｒｅｂｓ－ＨＥＰＥＳ緩衝液中で処理し顆粒細胞を分取した。分取した細胞は、牛胎児血清、ＫＣＩ、グルタミンを含有するＢａｓａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ液（製品名ＧＬ－１、コージンバイオ株製）中に播種し、３７℃に保温した５％ＣＯ２インキュベーター（ＡＳＴＥＣ製）内で培養を開始した。 (Preparation of primary cultured neurons)
Rat cerebellar granule cells were prepared by the following procedure. First, using 10 Wistar rats (obtained from the Charles River strain) of the same litter of 8 days of age without discrimination between males and females, each cerebellum was extracted. The cerebellum was minced to about 400 um 3 and treated in Krebs-HEPES buffer containing bovine serum albumin, trypsin Mg 2+ , Ca 2+ and the like, and granule cells were collected. Fractionated cells, fetal bovine serum, KCI, Basal Medium Eagle medium containing glutamine (product name GL-1, Kohjin Bio Co.) were seeded into, 5% CO 2 incubator maintained at 37 ° C. (ASTEC Ltd. The culture was started in).
　小脳顆粒細胞を播種後８～１０日間培養することにより、神経細胞とグリア細胞が共存（神経細胞：グリア細胞＝６：４）する初代培養細胞を作製し、以下の実験に用いた。なお、培養細胞は、Ｋｒｅｂｓ－ＨＥＰＥＳ緩衝液（１３６ｍＭ　ＮａＣｌ、１０　ｍＭ　ｇｌｕｃｏｓｅ、２０　ｍＭ　ＨＥＰＥＳ、５　ｍＭ　ＫＣＩ、１．３　ｍＭ　ＭｇＣｌ２、　１．２　ｍＭ　ＣａＣｌ２　ｐＨ７．４）で洗浄し、同緩衝液で実験を行った。 By culturing cerebellar granule cells for 8 to 10 days after seeding, primary cultured cells in which nerve cells and glial cells coexist (neuron cells: glia cells = 6: 4) were prepared and used in the following experiments. The cultured cells were washed with Krebs-HEPES buffer (136 mM NaCl, 10 mM glucose, 20 mM HEPES, 5 mM KCI, 1.3 mM MgCl 2 , 1.2 mM CaCl 2 pH 7.4). Experiments were performed with liquid.
（紫芋発酵液の適用）
　上記で得られた紫芋発酵液を最終濃度が０．０６２６％、０．１２５％、０．２５％および０．５％となるようにそれぞれ希釈し、ＬＰＳの添加と同時に培養脳神経細胞に適用した。条件は、３７℃で２０時間とした。また、比較例として、黒酢（製品名：製品名：坂元醸造のくろ酢）または米酢（製品名：福山醸造の米酢）を上記と同じ濃度で希釈し、前記細胞に適用した。 (Application of purple koji fermentation broth)
The purple koji fermentation broth obtained above is diluted to final concentrations of 0.0626%, 0.125%, 0.25% and 0.5%, respectively, and applied to cultured brain neurons simultaneously with the addition of LPS. did. The condition was 20 hours at 37 ° C. Moreover, as a comparative example, black vinegar (product name: product name: Sakamoto brewing vinegar) or rice vinegar (product name: Fukuyama brewing rice vinegar) was diluted at the same concentration as above and applied to the cells.
（ＮＯｘ（ＮＯ２／ＮＯ３）の測定）
　細胞外のＮＯｘは、ＨＰＬＣ－ＵＶ　ｓｙｓｔｅｍ（製品名ＥＮＯ－１０、ＥｌＣＯＭ製）を用いてＮＯ２およびＮＯ３に分類し、Ｇｒｉｅｓｓ法により定量測定を行った。ＮＯ２／ＮＯ３の分離および還元カラム、ならびに反応コイルは、３５℃に設置したカラムオーブン（製品名ＡＴＣ－１０、ＥＩＣＯＭ製）中で使用した。 (Measurement of NOx (NO 2 / NO 3 ))
Extracellular NOx was classified into NO 2 and NO 3 using HPLC-UV system (product name ENO-10, manufactured by ElCOM), and quantitative measurement was performed by Griess method. The NO 2 / NO 3 separation and reduction column and the reaction coil were used in a column oven (product name ATC-10, manufactured by EICOM) installed at 35 ° C.
　ＮＯ２およびＮＯ３は、測定開始後、それぞれ、約４分および６分で測定ピークを検出した。測定結果は、ＮＯ２およびＮＯ３の面積（ＡＵＣ）で比較した。ＬＰＳ（Ｅ．ｃｏｌｉ０２６：Ｂ６、ＤＩＦＣＯＬＡＢＯＲＡＴＯＲＩＥＳ、ＭＩ、ＵＳＡ）は、１０および２０ｕｇ／ｍｌの各濃度で用いた。ＬＰＳを投与した直後からＮＯ２／ＮＯ３の測定を開始した。測定結果は、各値のＮＯ２とＮＯ３の和を求めてＮＯｘとして表示した。 NO 2 and NO 3 detected measurement peaks at about 4 minutes and 6 minutes, respectively, after the start of measurement. The measurement results were compared by NO 2 and NO 3 area (AUC). LPS (E. coli 026: B6, DIFCOLABORATORIES, MI, USA) was used at concentrations of 10 and 20 ug / ml. Measurement of NO 2 / NO 3 was started immediately after LPS administration. As the measurement result, the sum of NO 2 and NO 3 of each value was obtained and displayed as NOx.
　初代培養脳神経細胞の正常細胞外におけるＮＯｘ値は約５０ｐｍｏｌ（４３．１±８．５）であったが、ＬＰＳ（１０ｕｇ／ｍＩ）を２０時間培養細胞に適用すると、ＮＯｘ産生は、約１．７倍（７５．０±４．５）に増量した。 The normal extracellular NOx value of primary cultured brain neurons was about 50 pmol (43.1 ± 8.5), but when LPS (10 ug / mI) was applied to cultured cells for 20 hours, NOx production was about 1. The amount was increased 7 times (75.0 ± 4.5).
　ＬＰＳ誘発のＮＯｘ生産に対する紫芋発酵液の添加効果を図１に示す。図１の縦軸（阻害率）は、ＬＰＳ単独での実測値を１００％とした場合の各濃度の紫芋発酵液での成績を百分率で計算したものである。ＬＰＳ誘発のＮＯｘ産生値に対して、その違いが統計的に有意な差を有する群には＊印を付けた（＊：Ｐ＜０．０５）。図１が示すように、０．２５および０．５％の紫芋発酵液濃度で有意な抑制効果が観察された。 Fig. 1 shows the effect of adding purple koji fermented liquid on LPS-induced NOx production. The vertical axis (inhibition rate) in FIG. 1 is obtained by calculating the percentage of the results with each concentration of purple koji fermented liquid when the measured value of LPS alone is 100%. Groups with significant differences in LPS-induced NOx production values were marked with * (*: P <0.05). As FIG. 1 shows, a significant inhibitory effect was observed at 0.25 and 0.5% purple koji fermentation broth concentrations.
　ＬＰＳ誘発のＮＯｘ産生に対する黒酢の影響を図２に示す。表示の仕方は紫芋発酵液と同じである。ＬＰＳ誘発のＮＯｘ量の増量に対して、黒酢は、０．２５および０．５％濃度で抑制が観察された。しかし、０．５％濃度での増量抑制は統計学的に有意な結果ではなかった。 Fig. 2 shows the effect of black vinegar on LPS-induced NOx production. The display method is the same as that of the purple koji fermentation broth. In contrast to the increase in LPS-induced NOx, black vinegar was observed to be suppressed at 0.25 and 0.5% concentrations. However, inhibition of weight gain at 0.5% concentration was not a statistically significant result.
　ＬＰＳ誘発のＮＯｘ産生に対する米酢の影響を図３に示す。表示の仕方は紫芋発酵液と同じである。ＬＰＳ誘発のＮＯｘ量の増量に対して、米酢は、０．２５および０．５％の濃度で抑制が観察された。しかし、これらは統計的に有意な差ではなかった。 Fig. 3 shows the effect of rice vinegar on LPS-induced NOx production. The display method is the same as that of the purple koji fermentation broth. In contrast to the LPS-induced increase in NOx, rice vinegar was observed to be inhibited at concentrations of 0.25 and 0.5%. However, these were not statistically significant differences.
　以上の結果をまとめると、紫芋発酵液や黒酢は、ＬＰＳ誘発のＮＯ産生を明らかに抑制したことから、細胞保護作用を有することが示唆される。 Summarizing the above results, it is suggested that purple persimmon fermentation broth and black vinegar have a cytoprotective effect because they clearly suppressed LPS-induced NO production.
２．中大脳動脈閉塞マウスの脳梗塞に及ぼす影響
（紫芋発酵液、黒酢または米酢の投与）
　原液（×１）、１０倍希釈液（×１０）または１００倍希釈液（×１００）を、マウスに２１日間経口投与した。なお、希釈には、蒸留水を用いた。 2. Effects on middle cerebral artery occlusion mice cerebral infarction (administration of purple koji fermented liquid, black vinegar or rice vinegar)
Stock solutions (× 1), 10-fold dilution (× 10) or 100-fold dilution (× 100) were orally administered to mice for 21 days. Dilution water was used for dilution.
（中大脳動脈閉塞マウスモデルの作製）
　マウスを２％　ｈａｌｏｔｈａｎｅ吸収により麻酔を導入させ、続いて１％　ｈａｌｏｔｈａｎｅ吸入によって麻酔を維持し、手術台上に固定した。手術用顕微鏡（オリンパス　ＯＭＫ－１Ｆ）下で、頸部の中央を切開し、左側総頸動脈と外頸動脈を結紮した。総頸動脈を切開し、塞栓子が中大脳動脈（ＭＣＡ）の起始部に到達するように、内頸動脈と外頸動脈の分岐部から内頸動脈を経由して９ｍｍ挿入した。再灌流は、塞栓子を手前に引くことによって行った。塞栓子は、長さ１１ｍｍの８－０ナイロンモノフィラメント（Ｅｔｈｉｌｏｎ：Ｅｔｈｉｃｏｎ．　ＮＪ．　ＵＳＡ）の先端から４ｍｍをシリコン樹脂（Ｘａｎｔｏｐｒｅｎ：Ｂａｙｅｒ　Ｄｅｎｔａｌ．　Ｏｓａｋａ．　Ｊａｐａｎ）でコーティングしたものを用いた。コーティングの部分の直径は、体重により適切な太さを選択した。閉塞時間は４時間とした。 (Preparation of middle cerebral artery occlusion mouse model)
Mice were anesthetized by 2% halothane absorption, followed by 1% halothane inhalation to maintain anesthesia and fixation on the operating table. Under the surgical microscope (Olympus OMK-1F), the center of the neck was incised, and the left common carotid artery and external carotid artery were ligated. The common carotid artery was incised, and 9 mm was inserted through the internal carotid artery from the bifurcation of the internal carotid artery and external carotid artery so that the embolus reached the origin of the middle cerebral artery (MCA). Reperfusion was performed by pulling the embolus forward. The embolus used was an 11 mm long 8-0 nylon monofilament (Ethylon: Ethicon. NJ. USA) coated 4 mm from the tip with a silicone resin (Xantopren: Bayer Dental. Osaka. Japan). The diameter of the coating portion was selected as appropriate depending on the body weight. The occlusion time was 4 hours.
（ＴＴＣ染色による梗塞巣体積の測定）
　中大脳動脈閉塞２４時間後に、マウスに過量のペントバルビタールを腹腔内投与し、断頭した。取り出した脳をドライアイスで凍結し、大脳皮質を含む脳の前額断スライスを２ｍｍ間隔で作製した。切片を、２％　２．３．５－ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ　ｃｈｌｏｒｉｄｅ（ＴＴＣ、Ｓｉｇｍａ社より購入）溶液中で、３７℃、３０分間インキュベートした。スライドガラス上に並べ、デジタルスチルカメラ（ＭＶＣ－ＦＤ９１、ＳＯＮＹ（株）製）で撮影した。梗塞巣体積は、割面の写真から梗塞巣面積を画像解析ソフト（ＮＩＨ　ｌｍａｇｅ　１．６２）で測定した。中大脳動脈閉塞後に生じる梗塞体積に及ぼす紫芋発酵液、黒酢および米酢の影響を、図４に示す。 (Measurement of infarct volume by TTC staining)
Mice were decapitated by intraperitoneal administration of an excessive amount of pentobarbital 24 hours after middle cerebral artery occlusion. The removed brain was frozen with dry ice, and pre-slice slices of the brain containing the cerebral cortex were prepared at 2 mm intervals. Sections were incubated in a 2% 2.3.5-triphenyltetrazolium chloride (TTC, purchased from Sigma) solution at 37 ° C. for 30 minutes. They were arranged on a slide glass and photographed with a digital still camera (MVC-FD91, manufactured by Sony Corporation). For the infarct volume, the infarct area was measured from the photograph of the cleavage plane with image analysis software (NIH image 1.62). FIG. 4 shows the influence of purple koji fermented liquid, black vinegar, and rice vinegar on the infarct volume that occurs after middle cerebral artery occlusion.
（神経症状）
　中大脳動脈閉塞・再灌流による運動障害を肉眼的に判断するために、５段階にスコア化した。スコアの基準は、以下の通りである。なお、再開通直後に測定した。
０：ｎｏｒｍａｌ　ｐｏｓｔｕｒｅ
１：ｆｌｅｘｉｏｎ　ｏｆ　ｔｏｒｓｏ　ａｎｄ　ｏｆ　ｃｏｎｔｒａｌａｔｅｒａｌ　ｆｏｒｅＩ　ｉｍｂ　ｕｐｏｎ　ｌｉｆｔｉｎｇ　ｏｆ　ｔｈｅ　ａｎｉｍａｌ　ｂｙ　ｔｈｅ　ｔａｉｌ
２：ｃｉｒｃｌｉｎｇ　ｔｏ　ｔｈｅ　ｃｏｎｔｒａｌａｔｅｒａｌ　ｓｉｄｅ　ｂｕｔ　ｎｏｒｍａｌ　ｐｏｓｔｕｒｅ　ａｔ　ｒｅｓｔ
３：ｃｏｎｔｉｎｕｏｕｓ　ｃｉｒｃｌｉｎｇ　ｔｏ　ｔｈｅ　ｃｏｎｔｒａｌａｔｅｒａｌ　ｓｉｄｅ
４：ｒｏｌｌｉｎｇ　ｔｏ　ｔｈｅ　ｃｏｎｔｒａｌａｔｅｒａｌ　ｓｉｄｅ
　神経症状（再開通直後）のスコア平均値ならびに標準誤差を表１に示す。 (Neurological symptoms)
In order to visually judge the movement disorder due to middle cerebral artery occlusion / reperfusion, it was scored in 5 stages. The criteria for the score are as follows. In addition, it measured immediately after restarting.
0: normal posture
1: flexion of torso and of contralateral forI im uplifting of the animal by the tail
2: Circulating to the contralateral side but normal post at rest
3: Continuous circling to the contralateral side
4: rolling to the contralateral side
Table 1 shows the average score and standard error of neurological symptoms (immediately after recommencement).
Figure JPOXMLDOC01-appb-T000001
 ＊：ｐ＜０．０５
Figure JPOXMLDOC01-appb-T000001
 *: P <0.05
　図４から、紫芋発酵液の投与は、梗塞の大きさを抑制し、しかも、明らかな用量依存的な抑制であったことがわかる。特に、原液（×１）を連続投与した群では、梗塞抑制に対して統計学的に有意な変化が認められた。表１に示す神経症状のスコア平均値についても、紫芋発酵液には用量依存的に改善が認められ、原液投与群で統計学的に有意な変化（＊：ｐ＜０．０５）が認められた。 FIG. 4 shows that administration of purple koji fermented liquid suppressed the size of the infarction and was a clear dose-dependent suppression. In particular, in the group to which the stock solution (× 1) was continuously administered, a statistically significant change was observed for infarct suppression. As for the neurological symptom average values shown in Table 1, also in the purple koji fermented liquid, a dose-dependent improvement was observed, and a statistically significant change (*: p <0.05) was observed in the stock solution administration group. It was.
　黒酢には、原液投与群で連続投与での明らかな抑制効果が認められた（図４）。表１の神経症状については、全体的なスコアが低下したものの、顕著な改善が認められなかった。むしろ、腹を擦るような行動が観察された。 Black vinegar showed a clear inhibitory effect in continuous administration in the stock solution administration group (FIG. 4). Regarding the neurological symptoms in Table 1, although the overall score decreased, no significant improvement was observed. Rather, a behavior that rubs the belly was observed.
　米酢には、連続投与でいずれの投与群も梗塞巣に及ぼす影響が無かった（図４）。神経症状に関しても、改善効果は認められなかった（表１）。また、米酢の原液で連続投与した群では、強酸による胃腸障害、殊に漬瘍が剖検で認められた。このため、実験遂行途中で死亡した例があった。 Rice vinegar had no effect on the infarct in any of the administration groups with continuous administration (FIG. 4). No improvement effect was observed for neurological symptoms (Table 1). In addition, gastrointestinal disturbances caused by strong acids, especially pickles, were observed at autopsy in the group continuously administered with the stock solution of rice vinegar. For this reason, there were cases of death during the course of the experiment.
　以上の結果は、紫芋発酵液や黒酢の連続投与が中大脳動脈閉塞によって生じる脳梗塞の抑制効果を有し、脳梗塞の予防に効果的であることを示唆する。しかし、黒酢の原液による連続投与は、漬瘍形成を促進することもあり、原液使用の場合においては紫芋発酵液の方が安全であると思われる。 These results suggest that continuous administration of purple koji fermented liquid or black vinegar has an effect of suppressing cerebral infarction caused by middle cerebral artery occlusion, and is effective in preventing cerebral infarction. However, continuous administration with a black vinegar stock solution may promote the formation of sores, and in the case of using the stock solution, the purple koji fermented solution seems to be safer.
　以上のとおり、Ｉｎ　Ｖｉｔｒｏ系とＩｎ　Ｖｉｖｏ系での脳機能障害モデルに対して紫芋発酵液原液の連続的な投与は、脳梗塞の予防となり得ることが示唆された。 As described above, it was suggested that continuous administration of the purple koji fermented liquid stock solution can prevent cerebral infarction against brain function disorder models in the In Vitro system and the In Vivo system.

Claims (5)

  1. 　紫芋発酵液を主成分とする脳保護作用剤。 A brain-protecting agent whose main ingredient is purple persimmon fermentation broth.
  2. 　前記紫芋発酵液が、
    （１）紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する工程、
    （２）冷凍された紫芋を解凍する工程、
    （３）解凍された紫芋を蒸煮する工程、
    （４）蒸された紫芋と米麹とをタンクに仕込み、５０～７０℃の温度で１８～２８時間、紫芋を発酵させる工程、
    （５）発酵された紫芋を圧搾し、搾汁と固形残渣とに分離する工程、および
    （６）前記搾汁にさらにクエン酸を添加して酸度を調整する工程
    により得られたものであることを特徴とする、請求項１に記載の脳保護作用剤。     The purple koji fermentation broth is
    (1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing
    (2) thawing frozen purple koji,
    (3) steaming the thawed purple persimmon,
    (4) charging steamed purple rice bran and rice bran into a tank and fermenting purple rice cake at a temperature of 50 to 70 ° C. for 18 to 28 hours;
    (5) It is obtained by pressing the fermented purple koji and separating it into juice and solid residue, and (6) adding citric acid to the juice and adjusting the acidity. The brain protective agent according to claim 1, wherein
  3. 　前記紫芋発酵液が、
    （１）紫芋を剥皮し、剥いた紫芋の表面をクエン酸処理した後、冷凍する工程、
    （２）冷凍された紫芋を解凍する工程、
    （３）解凍した紫芋を圧搾し、搾汁と固形残渣とに分離する工程、および
    （４）搾汁液と米麹とをタンクに仕込み、５０～７０℃の温度で１８～２８時間、紫芋を発酵させる工程、
    （５）前記発酵液にさらにクエン酸を添加して酸度を調整する工程
    より得られたものであることを特徴とする、請求項１に記載の脳保護作用剤。     The purple koji fermentation broth is
    (1) Peeling purple koji, citric acid treatment of the surface of the peeled purple koji, and freezing
    (2) thawing frozen purple koji,
    (3) squeezing the thawed purple koji and separating it into squeezed juice and solid residue; and (4) charging the squeezed liquor and rice koji into a tank at a temperature of 50 to 70 ° C. for 18 to 28 hours. Fermenting the koji,
    (5) The brain protective agent according to claim 1, which is obtained from a step of adjusting acidity by further adding citric acid to the fermentation broth.
  4. 　さらに、前記紫芋発酵液を凍結乾燥し、粉末、錠剤またはカプセル状に加工したことを特徴とする、請求項２または３に記載の脳保護剤。 The brain protective agent according to claim 2 or 3, wherein the purple koji fermentation broth is freeze-dried and processed into a powder, a tablet or a capsule.
  5. 　前記脳保護作用が、脳梗塞の予防である、請求項１～４のいずれかに記載の脳保護作用剤。 The brain protective agent according to any one of claims 1 to 4, wherein the brain protective action is prevention of cerebral infarction.
PCT/JP2008/001521 2008-06-03 2008-06-13 Cerebroprotective agent WO2009147702A1 (en)

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JP2008145876A JP2009292747A (en) 2008-06-03 2008-06-03 Cerebroprotective agent

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JP6026148B2 (en) * 2012-06-19 2016-11-16 株式会社ファーマフーズ NO production promoting composition and chilling improving agent containing the same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003277271A (en) * 2002-03-26 2003-10-02 Norioki Ko Anticancer drug
JP2006246836A (en) * 2005-03-14 2006-09-21 Thosin Kk Processed food and drink of purple sweet potato, and method for producing the same
JP2008092912A (en) * 2006-10-16 2008-04-24 Thosin Kk Antiaging functional food derived from purple sweet potato

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003277271A (en) * 2002-03-26 2003-10-02 Norioki Ko Anticancer drug
JP2006246836A (en) * 2005-03-14 2006-09-21 Thosin Kk Processed food and drink of purple sweet potato, and method for producing the same
JP2008092912A (en) * 2006-10-16 2008-04-24 Thosin Kk Antiaging functional food derived from purple sweet potato

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