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WO2007001080A1 - Boisson/aliment et produit pharmaceutique comprenant un extrait de feuille de néflier du japon - Google Patents

Boisson/aliment et produit pharmaceutique comprenant un extrait de feuille de néflier du japon Download PDF

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Publication number
WO2007001080A1
WO2007001080A1 PCT/JP2006/313197 JP2006313197W WO2007001080A1 WO 2007001080 A1 WO2007001080 A1 WO 2007001080A1 JP 2006313197 W JP2006313197 W JP 2006313197W WO 2007001080 A1 WO2007001080 A1 WO 2007001080A1
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WO
WIPO (PCT)
Prior art keywords
action
tea
food
group
fraction
Prior art date
Application number
PCT/JP2006/313197
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English (en)
Japanese (ja)
Inventor
Yusuke Sakata
Makoto Fujii
Takayuki Nakano
Norioki Ko
Fumio Hashimoto
Original Assignee
Kagoshima University
Totsukawanoujou Corporation
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Filing date
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Application filed by Kagoshima University, Totsukawanoujou Corporation filed Critical Kagoshima University
Priority to JP2007524050A priority Critical patent/JP4974116B2/ja
Publication of WO2007001080A1 publication Critical patent/WO2007001080A1/fr

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Classifications

    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention includes, for example, an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibiting action, a cancer cell apoptosis inducing action, a reactive oxygen species producing action, a hyperglycemic lowering action and / or an antioxidative action or
  • the present invention relates to pharmaceuticals and their manufacturing methods.
  • Bean leaves are used as a favorite drink of bean tea and are not only popular as bean tea but also used as a medicine blended in traditional Chinese medicine.
  • Non-patent Document 1 cytotoxic activity of polyphenol components contained in birch leaves against human tumor cells
  • Non-Patent Documents the anti-carcinogenic promotion effect of Megastigman glycosides contained in birch leaves
  • Non-patent document 3 anti-oxidant action of birch leaves
  • Patent Document 1 filed by the present applicant discloses a method and production equipment for producing a large amount of flavored bean tea using bean leaves.
  • the present applicant uses the bean-cha tea manufacturing facility described in Patent Document 1 to produce screw mebiwa tea by roasting tourmaline stone.
  • Concerning the manufactured Nemebiwa tea consumers say that “the blood sugar level of diabetes has decreased” and “the blood pressure of hypertension has returned to normal”, but empirical evidence has been confirmed. (Non-Patent Document 5).
  • Patent Document 1 states that “The leaves of bean koji, which is a material for producing conventional koji tea, use the cultivated varieties with the aim of harvesting the fruits. In the past, there was a disadvantage that the original effect was not obtained even if it was used for medicinal purposes because the nutrients collected in the fruit and not enough nutrients were distributed to the birch leaves. Was not sufficiently effective for medicinal purposes.
  • bean has the following activities or usage methods. ing.
  • Patent Document 2 discloses that koji or an extract thereof has a matrix meta-mouth proteinase (MMP-1) inhibitory activity.
  • MMP-1 matrix meta-mouth proteinase
  • Patent Document 3 discloses that the leaves of birch trees are baked into charcoal to make a powdered food material.
  • Patent Document 4 the raw leaves of bean paste are cut, steamed with steam, squeezed while sending hot air, dried, shaped, and then dried again to increase the aroma and taste.
  • a characteristic method for producing Kampo tea is disclosed.
  • Patent Document 5 discloses a method for producing health tea, which is obtained by mixing leaf green health tea of Gramineae plant and * leaf of birch and containing more ⁇ -aminobutyric acid.
  • Patent Document 6 discloses that a koji extract excluding a fruit part has a sarcoidase inhibitory activity.
  • Patent Document 7 discloses a composition for promoting nourishing tonic health in which shakiyota and bibi leaf are mixed.
  • Patent Document 8 discloses a health tea obtained by mixing roasted yellow persimmon leaves with bean tea.
  • Patent Document 9 discloses a health tea composed of a mixture of sweet tea and bean paste.
  • Patent Document 1 Q discloses that a composition containing an organic extract of the genus Rosaceae inhibits C0X-2 activity in living organisms.
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-154108
  • Patent Document 4 JP 2004-105036 A
  • Patent Document 5 Japanese Patent Application Laid-Open No. 2002-0652 7
  • Patent Document 6 Japanese Patent Application Laid-Open No. 2001-163795
  • Patent Document 7 JP 2001-163792 A
  • Patent Document 8 Japanese Patent Application Laid-Open No. 7-274832
  • Patent Document 9 Japanese Patent Laid-Open No. 5-056772 Patent Document 1 o Special Table 2004-529079
  • Non-Patent Document 1 Ito, H. et al., “Chem. Pharm. Bull.” 2000, pp. 48, p. 687-693
  • Non-Patent Document 2 Takashi Yoshida et al., “Bio Industry”, 2003, 20th 27, 33
  • Non-Patent Document 3 Ito, H. et al., “J. Agric. Food Chem.”, 2002, 50th, p. 2400-2403
  • Non-Patent Document 4 Jung, H. et al., “Arch. Pharm. Res. J, 1999, Vol. 22, p. 213-218
  • Non-Patent Document 5 Totsukawa Farm, Agricultural Production Corporation,“ Nemebiwa Tea Pamphlet ” , 2004 Invention Disclosure
  • birch leaves have a hypoglycemic-lowering action, an antihypertensive action, and an inhibition of cancer cell growth. It has not been known to have effects such as action, cancer cell apoptosis-inducing action, reactive oxygen species producing action, and antihyperlipidemic action. If these effects can be confirmed, biliary leaves can be used for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension, and cancer.
  • the present invention uses an anti-hyperlipidemic action, a hypertension-inhibiting action, a cancer cell proliferation-inhibiting action, a cancer cell apoptosis-inducing action, a reactive oxygen species producing action, a hyperglycemia using biliary leaves.
  • the object is to provide a food or drink or medicine having a lowering action and / or antioxidant action and a method for producing the same.
  • the present inventors have reduced the blood glucose level by subjecting bean tea to hot water extraction and feeding the obtained extract to model mice or rats. It was also found that the weight of serum lipids such as cholesterol and triglycerides and adipose tissue is reduced. It was also found that the crude fraction obtained from the extract of bime tea has a strong antioxidant effect.
  • the present invention includes the following.
  • (1) Contains an extract or purified product of bime leaves or bime tea as an active ingredient, and also has an antihyperlipidemic effect, an antihypertensive effect, an anticancer effect on cancer cell proliferation, an inducement effect on cancer cell apoptosis, an active oxygen species
  • a food or drink or a pharmaceutical product having one or more actions selected from the group consisting of a production action, a hypoglycemic action and an antioxidant action.
  • the purified product is one or more selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferol 3-rhamnoside, quenorece'tin 3-rhamnoside, 2oc_hydroxyursolic acid and ursolic acid
  • the food / beverage product or medicine according to (1) characterized in that it does not contain a compound of
  • the above food or drink is selected from the group consisting of antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species production action, hyperglycemia lowering action and antioxidant action.
  • a method for producing a food or drink or pharmaceutical having one or more actions selected from the group consisting of cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemic lowering action and antioxidant action.
  • the purified product is selected from the group consisting of chlorogenic acid, taercetin 3-sambubioside, methyl chlorogenic acid, kaempferonole 3-rhamnoside, quercetin 3-rhamnoside, 2 ⁇ -hydroxyursolic acid and ursolic acid 1 Above (6) The production method according to (6), wherein the compound is not contained.
  • the food / beverage product or pharmaceutical product according to the present invention contains an extract or purified product of bean leaf or bean tea as an active ingredient, and has an antihyperlipidemic effect, an antihypertensive effect, an anticancer effect on cancer cell growth, It has one or more actions selected from the group consisting of cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemic lowering action and antioxidant action. Hyperlipidemia and hypertension can be prevented or treated by ingesting or administering the food or drink or pharmaceutical product according to the present invention to animals such as humans.
  • the growth of cancer cells is suppressed, apoptosis is induced in cancer cells, and reactive oxygen species are produced in cancer cells. It can be shown.
  • the blood glucose level can be lowered and active oxygen or the like can be removed.
  • the antihyperlipidemic effect means that the cholesterol in the blood is decreased.
  • the antihypertensive activity means to suppress the increase of the 'blood pressure ⁇
  • the cancer cell growth inhibitory action means killing cancer cells and suppressing their growth.
  • the cancer cell apoptosis-inducing action means that cancer cells are induced to induce apoptosis and kill the cancer cells.
  • the reactive oxygen species producing action means producing reactive oxygen species in cancer cells and killing the cancer cells.
  • hypoglycemic action means that the blood sugar level is lowered.
  • antioxidation means removing active oxygen in the body.
  • koji leaf or koji tea is used.
  • bibi leaves fresh leaves or dried leaves can be used as they are.
  • bean tea any bean tea can be used, but for example, Nejimebiwa tea (trade name), ⁇ Nemebiwacha is a product that is more scorched by high-temperature heating with tourmaline stone. It can be prepared by the method described in Patent Document 1 described above.
  • Nemebiwa tea Neiso-cha
  • the extract used for the food or drink or the pharmaceutical product according to the present invention can be obtained by subjecting the birch leaves or bibi tea to hot water extraction or solvent extraction.
  • bean leaves or bean tea Is subjected to extraction using hot water, and this is repeated once or several times (for example, 3 times) to obtain a bime leaf or bime tea extract.
  • bi-biba or bi-bicha extract can be obtained by subjecting bi-bipo or bi-bicha to extraction using water or an organic solvent such as lower alcohol Nyaseton.
  • bi koji leaf or koji tea extract means an extract or various solvent extracts obtained by the above extraction method, a diluted solution thereof, a concentrated solution thereof or a dried powder thereof.
  • a purified product obtained by removing impurities from the extract by subjecting the above-described bibi leaf or bime tea extract to purification means such as filtration, centrifugation, or purification treatment is used.
  • purification means include column chromatography, normal phase or reverse phase chromatography, ion exchange chromatography, and gel filtration, with column chromatography being particularly preferred.
  • an aqueous solution of bime leaf or bime tea is directly subjected to open column chromatography on a gel for reverse phase (eg, MCI gel CHP-20P (Mitsubishi Chemical), etc.). Chromatography is performed by using water as the liquid A and ethanol as the liquid B in the mobile phase, changing the ratio of the liquid A and the liquid B to 100: 0, 30:70, 70:30. Finally, use acetonitrile as solution C, and perform chromatography with the ratio of solution A and solution C set to 50:50.
  • the crude fraction that elutes at a ratio of A to B of 70:30 and the crude fraction that elutes at a ratio of 50 to 50 of A and C shows a strong hypoglycemic action. . Therefore, these crude fractions can be suitably used for foods and beverages or pharmaceuticals according to the present invention as purified products of piu leaves or boiled tea.
  • the residue is converted into a gel for reverse phase (for example, MCI gel CHP-2
  • chlorogenic acid quercetin 3-sanbubio It does not contain one or more of the following compounds: sid, methylcuccinic acid, kenwei chinolose 3-rhamnoside, quercetin 3-rhamnoside, 2-hydroxyursolic acid and ursolic acid. Therefore, it is preferable that these compounds are not contained in the koji leaf or koji tea purified product used for the food or drink or the pharmaceutical product according to the present invention.
  • the food or drink or the pharmaceutical product according to the present invention can be produced.
  • the food / beverage product according to the present invention has an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibitory action, a cancer cell apoptosis inducing action, a reactive oxygen species producing action, a hyperglycemic lowering action and / or an antioxidant action. It can be used as a food and drink, especially as a health supplement or food for specified health use. Preferably, it is a health supplement or special health food in the form of tablets, 'capsules, granules, drinks, PET bottles, etc.
  • Examples of food and drink include, but are not limited to, confectionery, retort food, juices, teas, and dairy products.
  • sweeteners, seasonings, emulsifiers, suspending agents, preservatives, etc. may be added to foods and drinks as necessary, or vitamins, nutrients, immune enhancers (for example, propolis) You can add mushroom extract etc.).
  • the amount of the extract or purified product of bime leaves or bime tea to the food and drink according to the present invention is an amount within a range corresponding to 0.1 to 200 mg per kg of adult body weight to be ingested or 50 mg to 1 item. lg, but not limited to this range.
  • the pharmaceutical product according to the present invention contains an effective amount of an extract or a purified product of bime leaves or bime tea.
  • the pharmaceutical agent according to the present invention is used as an antihyperlipidemic agent, a hypertension inhibitor, a cancer cell proliferation inhibitor, a cancer cell apoptosis inducer, a reactive oxygen species producing agent, a hyperglycemic agent or an antioxidant. Can do.
  • the medicinal product according to the present invention includes, in addition to an extract or purified product of bean leaf or bean tea, a pharmaceutically acceptable carrier (excipient or diluent), a binder, a bulking agent, and a lubricant.
  • Disintegrating agents, wetting agents, emulsifying agents, buffering agents, suspending agents, preservatives, coloring agents, flavoring agents and An additive appropriately selected from sweeteners and the like can be contained.
  • Carriers and additives that are generally used for formulation can be used for the production of the pharmaceutical product according to the present invention.
  • the binder include starch, polyvinyl chloride, hydroxypropylmethylcellulose, and the like.
  • bulking agents include ratatoose and microcrystalline cellulose.
  • lubricants examples include talc, silica, and magnesium stearate.
  • disintegrants examples include starch and sodium starch glycolate.
  • wetting agents include sodium lauryl sulfate.
  • emulsifiers include cell mouth derivatives, sorbitol and the like.
  • preservatives include methyl-P-hydroxybenzoate and sorbic acid.
  • the additives that can be used in the present invention are not limited to these additive examples.
  • the pharmaceutical product according to the present invention is formulated for oral administration or parenteral administration (intravenous, intraarterial, intraperitoneal, intrarectal, subcutaneous, intramuscular, sublingual, intranasal, intravaginal, etc.) Can be.
  • parenteral administration intravenous, intraarterial, intraperitoneal, intrarectal, subcutaneous, intramuscular, sublingual, intranasal, intravaginal, etc.
  • the form of the preparation is not particularly limited, and examples thereof include solutions, tablets, powders, granules, capsules, suppositories, sprays, controlled release agents, suspensions and drinks. '
  • the dose of the extract or purified product of bean leaf or bean tea contained in the pharmaceutical product according to the present invention may vary depending on factors such as the patient's age, weight, sex, condition and severity.
  • the daily dose of the extract or purified product of bicucumber leaf or bibi tea administered to a patient is, for example, in the range of 0.1 to 200 mg, preferably 1 to 100 mg per kg of patient weight. It is not limited to. If necessary, the dose may be divided into several doses, for example, divided into 2-3 doses.
  • the pharmaceutical agent according to the present invention may have other antihyperlipidemic agents having the same or different therapeutic uses, hypertension inhibitors, cancer cell proliferation inhibitors, cancer cell apoptosis inducers, reactive oxygen species producing agents, hyperglycemic agents. And / or can be administered to a patient in combination with an antioxidant.
  • the food / beverage products or pharmaceutical products according to the present invention can be subjected to pharmacological evaluation as follows, for example.
  • Examples of the pharmacological evaluation of the antihyperlipidemic action of the food and drink or pharmaceutical product according to the present invention include, for example, ingesting the food and drink or pharmaceutical product according to the present invention in a model animal and A method for measuring tissue weight is mentioned.
  • the present invention When the amount of total cholesterol, HDL cholesterol and / or triglyceride in the blood is significantly reduced in the animals receiving the food / drinks / medicines according to the present invention compared to the animals not taking the food / drinks / medicines Therefore, it can be determined that the food or drink or pharmaceutical product according to the present invention has an antihyperlipidemic effect. Similarly, when the adipose tissue weight of the organ is significantly reduced, it can be determined that the food or drink or the pharmaceutical product according to the present invention has an antihyperlipidemic effect well.
  • examples of the pharmacological evaluation of the antihypertensive action of foods and beverages or pharmaceuticals according to the present invention include a method using males of SHR rats (natural hypertension rats).
  • the food or beverage or pharmaceutical product according to the present invention is fed to the SHR rat, and after the breeding is finished, the blood pressure of each rat is measured using an unheated cuff blood pressure meter (Muromachi Kikai Co., Ltd.).
  • the blood pressure is reduced as compared with a rat not fed with the food or drink or pharmaceutical product according to the present invention as measured by the apparatus, the food or food product or pharmaceutical product according to the present invention has a good antihypertensive product. I can judge.
  • the pharmacological evaluation of the cancer cell 'growth-inhibiting action, cancer cell apoptosis-inducing action and reactive oxygen species producing action of the food or drink or pharmaceutical product according to the present invention includes, for example, human acute
  • HL-60 cells promyelocytic leukemia disease cells
  • the food or drink or pharmaceutical product according to the present invention has a cancer cell apoptosis-inducing action. Furthermore, when HL-60 cells are cultured in the presence of a food or drink or pharmaceutical product according to the present invention, and the intracellular active oxygen is increased compared to the control group (in the absence of the food or food product or pharmaceutical product of the present invention) Therefore, it can be judged that the food or drink or the pharmaceutical product according to the present invention has a reactive oxygen species producing action on cancer cells.
  • a model animal for example, a KKAy mouse that is a type II diabetes model mouse
  • Blood glucose level during or after breeding The method to determine is mentioned.
  • the blood glucose level is significantly reduced in the animal that has taken the food or drink or medicine according to the present invention, compared to the animal that has not taken the food or drink or medicine according to the present invention, the food or drink according to the present invention Alternatively, it can be determined that the drug has good hypoglycemic action.
  • examples of the pharmacological evaluation of the antioxidant action of foods and beverages or pharmaceuticals according to the present invention include the method described in Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002. . That is, DPPH (1, ldiphenyl-2-picrylhydrazyl) is added to the food / beverage product or pharmaceutical product according to the present invention, the absorbance is measured, and the concentration of TrQlox is determined from the absorbance using the Trolox absorbance standard curve. Is converted. From the converted value, the radical scavenging rate is calculated from the 'Trolox radical scavenging standard curve. When the radical scavenging rate of the food or drink according to the present invention is equal to or higher than, for example, the positive control vitamin C or E, the food or drink according to the present invention has a good antioxidant effect. It can be judged that it has.
  • Non-roasted bean leaves are anti-hyperlipidemic, antihypertensive, cancer cell growth inhibitory, cancer cell apoptosis-inducing, reactive oxygen species producing, and hypoglycemic action Or, no antioxidant action is observed.
  • an extract or purified product of bean koji leaf or koji tea an anti-lipidemia action, an antihypertensive action, a cancer cell proliferation inhibiting action, a cancer cell apoptosis inducing action, an active oxygen It is possible to produce a food or drink or a pharmaceutical product having a seed production action, a hyperglycemic action and / or an antioxidant action.
  • Fig. 1 shows rats in the control group and the bi ⁇ tea group during the breeding period in Example 1. The result of having investigated the weight transition of is shown.
  • FIG. 2 shows the results of examining the total cholesterol level in the blood of the rats of the control group and the bime tea group in Example 1.
  • FIG. 3 shows the results of examining the amount of HDL cholesterol in the blood of the rats in the control group and the bime tea group in Example 1.
  • FIG. 4 shows the results of examining the amount of triglyceride in the blood of the rats of the control group and the green tea group in Example 1.
  • FIG. 5 shows the results of measuring the weights of the liver, the peripheral testicles, and the adipose tissue around the kidneys of the rats in the control group and the bicha tea group after the breeding in Example 1.
  • FIG. 6 shows the results of examining the body weight changes of the mice in the control group and the bi ⁇ tea group during the breeding period in Example 1.
  • FIG. 7 shows the results of measurement of the weight of the fat tissue around the kidney and the accessory testicles of the mice in the control group and the birch tea group after the breeding in Example 1.
  • FIG. 8 shows changes in blood glucose levels of mice in the control group and the bi ⁇ tea group during the breeding period in Example 2.
  • FIG. 9 shows the results of the glucose tolerance test of mice in the control mouth group and the bibi tea group after breeding in Example 2.
  • FIG. 10 shows the measurement results of blood HbAlC values in mice of the control group and the bi ⁇ tea group after the glucose tolerance test in Example 2.
  • FIG. 11 shows a thin-layer chromatograph of the dried crude fraction in Example 3.
  • FIG. 12 shows the transition of the blood glucose level of the mice in the control group and each rice bran and tea group during the breeding period in Example 3.
  • FIG. 13 shows the results of the glucose tolerance test of the mice in the control group after breeding in Example 3 and in the bi ⁇ tea group fed with fractions 3 and 4.
  • ⁇ -Fig. 14 shows the measurement results of blood HbAlC values in mice of the control group and each of the bicha tea groups after the glucose tolerance test in Example 3.
  • FIG. 15 shows a thin-layer chromatograph of the crude fraction in Example 4.
  • FIG. 16 shows the results of examining the antioxidant action of the crude fraction in Example 4.
  • Figure 17 shows the antioxidant activity of the crude fraction prepared in Example 3 in Example 5. The results of the investigation are shown.
  • FIG. 18 shows the results of examining the antioxidant action of the crude fraction further obtained from fraction 3 of Example 3 in Example 5.
  • FIG. 19 shows the results of examining the antihypertensive effect of Nemebiwa tea extract in Example 6.
  • FIG. 20 is a photograph of an agarose gel electrophoresis in Example 8, showing the results of examining the apoptosis-inducing action of the crude fraction of Example 3.
  • FIG. 21 is a photograph of an agarose gel electrophoresis in Example 8, showing the results of examining the apoptosis-inducing action of the crude fraction further obtained from fraction 3 in Example 3.
  • FIG. 22 shows the results of examining the effect of active oxygen species production on the cancer cells of the crude fraction of Example 3 in Example 9.
  • FIG. 23 shows ligation obtained by examining the effect of reactive oxygen species production on cancer cells of the crude fraction further obtained from fraction 3 of Example 3 in Example 9. Best Mode for Carrying Out the Invention ''
  • Nemebiwa tea used in the following examples is commercially available from Totsugawa Farm, Kagoshima Agricultural Production Corporation. -In addition, the results in the figure accompanying each example are shown as the average value or average soil standard deviation of each group.
  • Nejimebiwa tea 50 g sold with hot water (400 ml). This was repeated three times. The obtained hot water extract was subjected to freeze-drying (Tokyo Rikakikai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder.
  • the total dry weight of Nemebiwa tea used was 346.2 g, and the total dry powder amount of the hot water extract was 57.37 g. Therefore, the dry powder content of the hot water extract relative to Nemebiwa tea was 16.57%.
  • Wistar rats Japan SLC
  • KKAy mice Claire Japan
  • the materials weighed so that the proportions shown in Table 1 below were mixed, kneaded with distilled water, and then dried in a freeze dryer. It was used as a food (a rat or a mouse fed with the food was referred to as a “control mouth group” in this example). Also, add the dry powder of the hot water extract obtained in (1 ') above to the control food so that it becomes 1%, and add this to the Nemebiwa tea group food (the rat or mouse fed with the food). In this example, it was used as “biso tea group”.
  • Rats were weighed every 3 days during the breeding period. In addition, both groups used tap water for the water supply of the rats, and the water supply was free during the breeding period.
  • each animal was anesthetized with Nembutal, blood was collected from the heart, and serum lipids were analyzed. In addition, the organs of each group of rats were excised and weighed.
  • Figure 1 shows the results of rat body weight measurement.
  • Figure 1 shows the results of examining the weight transition of rats in both groups during the breeding period.
  • the black circles are the results for the rats in the control group
  • the white circles are the results for the rats in the bi ⁇ cha group.
  • FIGS. 2 to 4 The analysis results of serum lipids after breeding are shown in Figs.
  • Figure 2 shows the results of examining the total cholesterol content in the blood (m g / dl).
  • Figure 3 shows the results of examining the amount of HDL cholesterol in the blood (rag / dl).
  • Figure 4 shows the results of examining the amount of triglyceride (mg / dl) in the blood.
  • control is the result for the rats in the control group
  • “bi ⁇ cha” is the result for the rats in the bi ⁇ tea group.
  • FIG. 1 shows the results of measuring the weight of rat liver, rat testicles, and adipose tissue around the kidney after the breeding.
  • control is the result of the rats in the control group
  • b ⁇ cha is the result of the rats in the boiled tea group.
  • the weight of the rats in the bicha tea group compared with the weight of the rats in the control group. It can be seen that gag is lower. In particular, with regard to the weight of adipose tissue around the kidney, it can be seen that the weight of rats in the bi ⁇ cha group is significantly lower (* indicates P, 0.05 in Fig. 5).
  • mice were divided into a control group (10 animals) and a bime tea group (10 animals), and the control group mice were allowed to freely take control food.
  • mice in the bi ⁇ cha group were allowed to freely consume Nemebiwa tea group food. Both groups were raised for 7 weeks each.
  • mice were weighed every 3 days. 'In addition, both groups used tap water for the water supply, and the water supply was free during the breeding period.
  • each animal was anesthetized with Nembutal, and the organs of each group of mice were removed and their weights were measured.
  • FIG. Figure 6 shows the results of examining the weight transition of mice in both groups during the breeding period.
  • the black circles are the results for the control group of mice, and the white circles are the results for the Biwacha group of mice.
  • * indicates P and 0.05, and ** indicates P and 0.01. '
  • Fig. 7 shows the results of examining organ weights after breeding.
  • Fig. 7 shows the results of measuring the weight of adipose tissue around the kidney and the accessory testicle after the breeding.
  • control is the result of the mice in the control group
  • bi ⁇ cha is the result of the mice in the bi ⁇ tea group.
  • Nemebiwa tea 50 g was extracted with hot water (400 ml). This was repeated three times. The obtained hot water extract was freeze-dried (Tokyo Rikakikai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder.
  • the total dry weight of Nemebiwa tea used was 30.0 g, and the total dry powder amount of the hot water extract was 51.73 g. Therefore, the dry powder content of the hot water extract relative to Nemebiwa tea was 17.24%.
  • mice KKAy mice (CLEA Japan) were used as hyperglycemic (type I type I diabetes model) mice.
  • the ingredients weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried in a freeze dryer.
  • the mice fed with the food were referred to as “control group” in this example).
  • the dry powder of hot water extract obtained in (1) above was added to the control food so that it would be 1%, and this was added to the Nemebiwa tea group (the mice fed this food were In the example, it was used as “biso tea group”.
  • mice were divided into a control group (10 animals) and a bime tea group (10 animals), and the control group mice were allowed to freely ingest control food.
  • mice in the bi ⁇ cha group were given free access to Nemebiwa tea group food. Both groups were raised for 7 weeks each.
  • Tap water was used in both groups for water supply to mice, and free water was provided during the breeding period. During the breeding period, blood glucose levels of mice were measured every other week. The enzyme electrode method was used for blood glucose level measurement.
  • Figure 8 shows the changes in blood glucose levels during the breeding period.
  • the black circles are the results for the control group mice, and the white circles are the results for the Biwacha group mice.
  • each group of mice was fasted for 14 hours, and glucose was administered thereto.
  • the dose of glucose was 4 g / 10 ml of glucose (distilled water) with a mouse body weight of xl / 200 (ml). This dose was injected directly into the stomach of each mouse with a stainless steel sonde and forcibly administered. After administration, blood glucose levels were measured over time.
  • the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbAlC was measured.
  • Figure 9 shows the results of the glucose tolerance test after breeding.
  • the black circles are the results for the control group of mice, and the white circles are the results for the Biwacha group of mice.
  • * indicates P is 0.05.
  • control is the result of the mice in the control group
  • bi ⁇ cha is the result of the mice in the bi ⁇ tea group.
  • the blood HbAlC value of mice in the BY tea group was significantly higher than the blood HbAlC value of mice in the control group (in Fig. 10, ** is P + 0.01). It shows that it is low. '
  • Nemebiwa tea 50 g was extracted with hot water (400 ml). This was repeated three times. The resulting hot water extract was freeze-dried (Tokyo Rikakikai Co. 'LTD, Freeze Dryer FR-1) The dried powder was obtained. The total dry weight of Nemebiwa tea used was 950.0 g.
  • the obtained hot water extract was cooled and then sequentially applied to an open column chromatogram of MCI gel CHP-20P.
  • the amount of gel is preferably about 1 kg as a dry weight.
  • the glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass, acrylic material, or the like.
  • the solvent is eluted with water (2L), 30% aqueous ethanol (2L), 70% aqueous ethanol (2L) and water-acetonone (1: 1 ratio) (2L) to obtain 4 fractions. It was.
  • the yield of the four dry crude fractions was 90.0 g of the dry crude fraction eluted with water (2 L) (hereinafter referred to as “Fraction 1”) and the dry crude fraction eluted with 30% aqueous ethanol (2 L). 45.3 g of the dry crude fraction (hereinafter referred to as “Fraction 2”) and 18.3 g of the dry crude fraction (hereinafter referred to as “Fraction 3”) eluted with 70% aqueous ethanol (2 L).
  • the dry crude fraction (hereinafter referred to as “Fraction 4”) eluted with water-acetone (1: 1 ratio) (2 L) was 2. lg.
  • TLC Thin Layer Chromatography
  • fractions 1 and 2 contain a chlorogenic acid-like substance in the vicinity of an Rf value of about 0.1. It can also be seen that fractions 3 and 4 do not contain much organic and chlorogenic acid-like substances.
  • mice (CLEA Japan) were used as hyperglycemic (type II diabetes model) mice.
  • the ingredients weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried in a freeze dryer. Mice fed with the food were used as “control group” in this example.
  • Fraction 1, Fraction 2 or Fraction 3 and 4 obtained in (1) above. was added to the control bait to 1% each, and used as Nemebiwa tea group bait (the mice fed with the bait respectively were collectively referred to as the “boiled tea group” in this example). .
  • mice were used in 32 experiments. Give mice a control group (8 animals), feed for Nemebiwa tea group containing fraction 1 Bean tea group (eight animals), bean meal give food for Nemebiwa tea group containing fraction 2 Divided into 4 groups: 8 tea groups, Biso tea group (8 animals) fed with Nemebiwa tea group containing a mixture of fractions 3 and 4, and 1 day for mice in the control group At one time, they were fed 1,5g of control food, and the shortage was fed with mouse sales. In addition, mice in each bi ⁇ cha group were fed once a day with 1.5g of control food and 15mg of each fraction of Nemebiwa tea group feed. Was given a state of satiation. Each group was raised for 7 weeks. For each group, tap water was used for water supply and free water was used during the breeding period.
  • mice blood glucose levels of mice were measured every other week.
  • the enzyme electrode method was used for blood glucose level measurement.
  • Figure 12 shows the changes in blood glucose levels during the breeding period.
  • the black circles are the results for the mice in the control mouth group
  • the white squares are the mice from the Biwacha group that have been fed the Nemebiwa tea group food containing fraction 1.
  • Is the result of The white triangles are the results for the bime tea group mice fed with the food for screw mebiwa tea group containing fraction 2
  • the white circles are the screws containing fractions 3 and 4. This is the result of the mouse of the bime tea group fed with food for mebiwa tea group.
  • each of the 4 groups of mice was fasted for 14 hours and glucose was administered thereto.
  • the dose of buducose is 4g / 10ml of buducose (steamed) with mouse body weight X 1/200 (ml). Retained water). This dose was injected directly into the stomach of each mouse with a stainless steel sonde and forcibly administered. After administration, blood glucose levels were measured over time.
  • the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbAlC was measured.
  • Fig. 13 shows the results of the glucose tolerance test conducted on the Biwacha group fed with fractions 3 and 4 after the rearing.
  • the black circle is the result of the control group of mice
  • the white circle is the mouse of the Biwacha group fed with the Nemebiwa tea group containing fractions 3 and 4. Is the result of
  • the blood glucose level of the bi ⁇ cha group fed with fractions 3 and 4 was increased rapidly immediately after administration by glucose administration, and about 500 mg after 15 minutes. It rose to / dl. It can be seen that after 30 minutes, the control group continues to rise and then declines. On the other hand, it can be seen that in the green tea group fed with minutes 3 and 4, the blood glucose level began to decrease after 30 minutes and then decreased rapidly. In particular, after 30 and 60 minutes, it can be seen that the value is significantly lower (in Fig. 13, '* indicates P is 0.05).
  • FIG. 14 the measurement results of blood HbAlC levels after the glucose tolerance test are shown in FIG. In Fig. 14, “Control” is the result of the control group of mice, and “Biwacha 1” is the result of the Biwacha group fed with the Nemebiwa tea group containing fraction 1. Mouse results. In addition, it is the result of the mice of the Biwacha group that was fed with the feed for Nemebiwa tea group containing Fraction 2, and the Bibicha (3 + 4) force fraction 3 4 is a result of mice of the bi ⁇ cha group fed with Nemebiwa tea group food containing 4 and 4.
  • Nemebiwa tea (1247 g) was extracted with 80% aqueous acetone (5000 ml). This was repeated three times. From the obtained 80% aqueous acetone extract, the solvent was distilled off under reduced pressure to obtain a syrupy extract (448.2 g). Part of the resulting extract (278g) was collected And subjected to column chromatography of MCI gel CHP-20P. The amount of gel is preferably about 1 kg as dry weight.
  • the glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass or acrylic material. Chromatography was performed by using a linear gradient from solution A to solution B using water as the solution A and methanol as the solution B as the mobile phase.
  • crude fraction 1 was obtained by combining fraction solutions eluted from liquid A and liquid B at a ratio of 1: 0 (300 ml) and 95: 5 (300 ml).
  • the crude fraction 2 was obtained by combining the fraction solutions eluting A liquid and B liquid at 9: 1 ratio (300 ml), 8: 2 ratio (300 ml) and 7: 3 ratio (300 ml). did.
  • the fraction solution obtained by eluting the A solution and the B solution at a ratio of 6: 4 (300 ml) was designated as crude fraction 3.
  • the fraction solution obtained by eluting the A solution and the B solution at a ratio of 5: 5 (300 ml) was designated as crude fraction 4.
  • the fraction solution obtained by eluting the A solution and B solution at a ratio of 4: 6 (300 ml) was designated as crude fraction 5.
  • the fraction solution obtained by eluting the A solution and the B solution at a ratio of 3: 7 (300 ml) was designated as crude fraction 6.
  • the fraction solution in which A solution and B solution were eluted at a ratio of 2: 8 (300 ml) was designated as crude fraction 7.
  • the fraction solution in which A liquid and B liquid were eluted at a ratio of 1: 9 (300 m 1) was designated as crude fraction 8.
  • the fraction solution obtained by eluting the A solution and the B solution at a ratio of 0: 1 (300 ml) was designated as crude fraction 9.
  • crude fractions 1 to 9 were obtained.
  • the yield of the nine crude fractions is 12.3 g for crude fraction 1, 12.8 g for crude fraction 2, 0.4 g for crude fraction 3, 2.9 g for crude fraction 4, and crude fraction 5 was 8.3 g, crude fraction 6 was 6.7 g, crude fraction 7 was 19.9 g, crude fraction 8 was 5.0 g, and crude fraction 9 was 4.9 g.
  • the TLCs of crude fractions 2-8 are shown: Shown in 5.
  • numbers 2 to 8 indicate the results of crude fractions 2 to 8, respectively.
  • the developing solvent used for TLC was three types of solvents: benzene-ethyl formate-formic acid, and the ratio was mixed at a solution volume of 3: 6: 1.
  • the coloring reagent used was a solution in which about 5% of ferric chloride (FeCl 3 ) was dissolved in methanol, and the compound was detected by color spraying directly onto TLC. As shown in Fig. 15, it can be seen that the substance that develops color from green to blue is a phenolic substance.
  • the antioxidant activity experiment was performed using the method described in the following literature (Harwat, K. S. M. et al., Free Radical Res., 36: 177-187, 2002).
  • the sample was prepared by adjusting each of the nine crude fractions obtained in (1) above to a concentration of 10 g / ml.
  • dry powder of hot water extract of Biso tea was used at a concentration of 10 g / ml.
  • a prepared product a standard product prepared by adjusting vitamin C (ascorbic acid) at a concentration of 5 g / m 1 and vitamin E adjusted at a concentration of S / zg / ml was used. Place these samples in a flat-bottom plate (96-well), add DPPH (1, 1-dipheny l-2-picrylhydrazyl, adjusted to the concentration of micromolar) 190 ⁇ and mix for 10 seconds. , Protected from light and left for 30 minutes.
  • FIG. 16 shows the experimental results of the antioxidant activity.
  • the sample numbers 1-9 correspond to the crude fractions 1-9, respectively.
  • Sample No. 10 is a dry powder of hot water extract of bean tea.
  • VC is vitamin C
  • VE is vitamin E.
  • crude fractions 6, 8 and 9 have a strong antioxidant effect. The degree is about the same as or higher than vitamin E, and about half that of vitamin C.
  • the antioxidant effect of crude fractions 6, 8 and 9 is about 3 times stronger than the dry powder of hot water extract of bi ⁇ cha.
  • Samples were the four crude fractions obtained in Example 3; fractions 1 to 4 were each adjusted to a concentration of 10 ⁇ g / ml, and Nejimebiwa tea obtained in Example 1 as a control.
  • the dry powder of hot water extract used was adjusted at a concentration of 10 / g / m l.
  • These samples were applied to a flat-bottomed plate (96wel l) [1 and DPPH (1, l_diphenyl-2-picrylhydrazyl, adjusted at the concentration of the mouth mouth mora) to 190 ⁇ 1. In addition, after mixing for 10 seconds, it was left for 30 minutes in the dark.
  • the reaction solution of each well was measured with a microplate reader using the absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using the Trolox absorbance standard curve. From the converted values, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve.
  • Figure 17 shows the experimental results of the antioxidant activity.
  • the vertical axis represents DPPH radical scavenging rate as%
  • the horizontal axis fraction 1 to fraction 4 samples are the fractions 1 to 4 prepared in Example 3, respectively.
  • Bi ⁇ cha is a dried powder of Nemebiwacha hot water extract obtained in Example 1.
  • Fraction 2 Fraction 3 and Fraction 4 have a strong antioxidant effect. It can be seen that the degree is greater than the effect of Nemebiwa tea hot water extract obtained in Example 1. In addition, it can be seen that the antioxidant effect of Fraction 3 is about 1.6 times stronger than the dry powder of Nemebiwa Chatoyu extract obtained in Example 1.
  • a dry powder of fraction 3 obtained in Example 3 (5.0 g) was weighed, dissolved in an appropriate amount of water, and subjected to open column chromatography.
  • a solution mobile phase (H 2 0) Methanol (MeOH) was used as the B liquid, and various chromatographies were performed by increasing the content of the B liquid from the A liquid.
  • the solvent is 80%
  • Samples were obtained in Example 3 as a control and the 9 crude fractions obtained in (2) above: fractions 3-1 to 3-9 adjusted to a concentration of 10 ⁇ g / ml, respectively.
  • a dry powder prepared from fraction 3 at a concentration of 10 ⁇ g / ml was used.
  • fractions 3-1, 3-2 and 3-4 have a strong antioxidant effect. It can be seen that the degree is larger than the effect of fraction 3 prepared in Example 3. In particular, it can be seen that the antioxidant effect of fraction 3-1 is about 1.37 times stronger than the dry powder of fraction 3.
  • SHR rats rats with spontaneous hypertension males (5 weeks old) (manufactured by SLC, Japan) were used to measure blood pressure using two I. These rats were divided into a control group (6 animals) and a bi ⁇ cha group (6 animals), and the control group rats were allowed to freely ingest control food. Rats in the bi ⁇ cha group were given free access to Nemebiwa tea group food. Both groups were reared for 50 days each.
  • the breeding room was 23 ° C and .12 hours lighting (7: 00-19: 00) '.
  • an unheated force-type blood pressure measuring device (Muromachi Kikai Co., Ltd.) was used as a blood pressure measuring device.
  • Figure 19 shows the changes in blood pressure in rats in both groups during the breeding period.
  • the open circle is the label for the control group.
  • the black circles are the results for the rats of the bi ⁇ cha group.
  • the blood pressure (maximum blood pressure) was measured from a certain time (around 10:00 in the morning). The measurement was repeated 10 times on average for each individual and expressed as the average value. Vertical bars indicate standard error.
  • blood pressure values fluctuate very easily, and can fluctuate very easily depending on the mental state and environmental conditions of the rat. Went under.
  • the average value of the maximum blood pressure in the control group and the bi-tea group was about 134 mmHg at the start.
  • the blood pressure in the control group increased over 10 days with the passage of the breeding period, and remained fairly high after 20 days.
  • the Biwacha group did not increase until 21 days, and was significantly different from the control group on the 21st (P ⁇ 0. 05).
  • the force S increased with the progress of the experiment days, and the blood pressure was lower by 10 mmHg or less than that of the control group, and the effect of Nejimewa tea extract on the suppression of hypertension was recognized.
  • Nemebiwa tea extract suppresses an increase in blood pressure.
  • HL-60 cells human acute promyelocytic leukemia disease cells
  • HL-60 cells were cultured using RPMI1640 medium containing 10% fetal bovine serum. Concentrate 0, 25, 50, 75, 100, 125, 150, ug / ml of dry powder of Nemebiwacha hot water extract obtained in Example 1 and fractions 1-4 prepared in Example 3
  • HL-60 cells were treated with 0.1% DMS0 (dimethyl sulfoxide) solution and treated for 6 hours.
  • DMS0 dimethyl sulfoxide
  • the extract in Table 3 is the result of dry powder of Nemebiwa tea hot water extract obtained in Example 1.
  • fractions 2-4 had a relatively strong HL-60 cell growth inhibitory effect on the dried powder of Nemebiwa-cha hot water extract. Among them, it can be seen that Fraction 4 has the strongest HL-60 cell growth inhibitory effect.
  • HL-60 cells The 50% viability of HL-60 cells was measured by the method described in the following literature (Mo-smann, T., J. Immunol. Methods, 65: 55-63, 1983).
  • HL-60 cells were cultured using RPMI1640 medium containing 10% fetal bovine serum. Dry powder of Nemebiwa tea hot water extract obtained in Example 1 and fractions 3-1 to 3-9 prepared in Example 5 were added at 0, 25, 50, 75, 100, 125, 150 ⁇ g.
  • HL-60 cells were treated for 6 hours in 0.1% DMS0 (dimethyl sulfoxide) solution to contain at a concentration of / ml.
  • DMS0 dimethyl sulfoxide
  • fractions 3-1, 3-2 and 3-4 had a relatively strong HL-60 cell growth inhibitory effect. Among them, it can be seen that Fraction 3-4 has the strongest HL-60 cell growth inhibitory effect.
  • Example 8 Effect of the crude fraction of Nemebiwa tea on apoptosis induction of cancer cells
  • DNA fragmentation of human acute promyelocytic leukemia disease cells was measured by the method described in the following literature (Hou, DX et al., Int. J. Oncol., 23: 705-712). , 2003).
  • Fraction 2, fraction 3 and fraction 4 prepared in Example 3 were adjusted to three concentrations of 250, 500 and 750 g / ml, and HL-60 cells were treated for 6 hours to extract DNA from the cell nucleus. did.
  • the obtained DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG. 'In Fig. 20, M is a DNA marker, and C is a control at a concentration of O / ig / ml.
  • M is a DNA marker
  • C is a control at a concentration of O / ig / ml.
  • DNA ladders were observed at concentrations of 500 ig / ml and 75'C ig / inl in fraction 3, indicating that apoptos
  • ⁇ is a DNA marker and C is a control at a concentration of 0 / g / ml.
  • DNA ladders were observed in fractions 3-1, 3-2, 3-4, and 3-6, indicating that apoptosis was induced.
  • strong DNA ladders are observed in fractions 3-2 and 3-4, indicating that apoptosis is strongly induced.
  • HL-60 cells Human acute promyelocytic leukemia disease cells (HL-60 cells) were passaged at 2.0 x 10 6 cel ls I ml. This was diluted 4-fold with 'RPMI1640 medium to give 2.0 ⁇ 10 5 cells / 400 1. The required amount is 400 ⁇ cell fluid per well, so 20 ml of cell fluid was prepared per plate. This was cultured for 24 hours at 37 ° C. in a 5% CO 2 stream while gently shaking the plate to prevent cell bias. -
  • control represents a control.
  • the extract is Nemebiwa tea hot water extract prepared in Example 1.
  • Fraction 3 is HL-60 thin It can be seen that it has a strong ability to produce active oxygen in the vesicle.
  • HL-60 cells were passaged at 2.0 ⁇ 10 6 cel ls / ml. This was diluted 4-fold with RPMI1640 medium to 2.0 ⁇ 10 5 cells / 400 ⁇ 1. The required amount was 400 1 cell solution per wel l, so 20 ml cell solution was prepared per plate. This was cultured for 24 hours at 37 ° C. in a 5% CO 2 stream while gently shaking the plate to prevent cell bias.
  • fractions 3-1 to 3_9 prepared in Example 5 were added to the culture solution so as to have a concentration of 500 ⁇ g / ml. This was incubated for 15 minutes at 37 ° C, of 5% C0 2 incubator. After culture, MTT solution (5 mM DCFH-DA: 1 mg of DCFH-DA dissolved in 413 ⁇ 1 of ethanol; DCFA-DA, dichlorofluorescin 2 acetic acid) 1.6 / 1 each dispensed, subsequently, it was incubated 30 min further at 37 ° C, 5% C0 2 Inkyubeta one under a stream of air.
  • MTT solution 5 mM DCFH-DA: 1 mg of DCFH-DA dissolved in 413 ⁇ 1 of ethanol; DCFA-DA, dichlorofluorescin 2 acetic acid
  • chondrol indicates a control.
  • fractions 3-3-2, 3-3 and 3-5 have a stronger ability to produce active oxygen than the control.
  • Fraction 3-1 has about 2.6 times stronger active and reproductive oxygen producing capacity than HL-60 cells compared to the control.
  • Industrial Applicability-According to the present invention antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species producing action, hyperglycemia lowering action and / or anti A food / beverage product or a pharmaceutical product having an oxidizing action and containing an extract or purified product of bime leaf or bime tea is provided.
  • the food / beverage products (especially health supplements or foods for specified health use) or pharmaceutical products according to the present invention have an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibiting action, Anti-hyperlipidemic agent, antihypertensive agent, cancer cell proliferation inhibitor, cancer cell apoptosis inducer, because it has a cell apoptosis inducing action, reactive oxygen species producing action, hyperglycemic lowering action and / or antioxidant action It can be used as a reactive oxygen species generator, a hyperglycemic agent, and an antioxidant.

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Abstract

La présente invention concerne une boisson, un aliment ou un produit pharmaceutique qui comprend une feuille de néflier du Japon ou un extrait ou produit purifié de feuille de néflier du Japon en tant que matière active et qui présente un ou plusieurs effets choisis dans le groupe composé d'un effet anti-hyperlipémique, d'un effet anti-hypertension, d'un effet d'inhibition de la prolifération d'une cellule cancéreuse, d'un effet d'induction de l'apoptose d'une cellule cancéreuse, d'un effet de production d'une espèce active d'oxygène, d'un effet anti-hyperglycémique et d'un effet anti-oxydant.
PCT/JP2006/313197 2005-06-27 2006-06-27 Boisson/aliment et produit pharmaceutique comprenant un extrait de feuille de néflier du japon WO2007001080A1 (fr)

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JP2009232823A (ja) * 2008-03-28 2009-10-15 Totsukawa Nojo:Kk 養殖魚介類用飼料
CN103082365A (zh) * 2013-01-31 2013-05-08 福建省闽中有机食品有限公司 一种稳定无沉淀整果取汁加工枇杷果汁饮料的方法
JP2014101286A (ja) * 2012-11-16 2014-06-05 Totsukawa Nojo:Kk ビワ茶の抗腫瘍作用と肝機能改善作用
JP2016132633A (ja) * 2015-01-19 2016-07-25 農業生産法人 有限会社十津川農場 ねじめびわ茶のアルツハイマー症予防又は治療作用
CN108717095A (zh) * 2018-05-30 2018-10-30 四川新绿色药业科技发展有限公司 一种枇杷叶或含枇杷叶原料的药物的检测方法及鉴别、含量测定方法
CN113080456A (zh) * 2021-05-18 2021-07-09 中国疾病预防控制中心营养与健康所 一种含枇杷叶提取物的组合物及其制备方法和应用
CN114306439A (zh) * 2022-02-25 2022-04-12 浙江大学 一种枇杷叶绒毛提取物及应用

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JP2000342229A (ja) * 1999-06-04 2000-12-12 Totsukawa Kk ビワ茶の製造方法及びビワ茶の製造設備
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WO2006013866A1 (fr) * 2004-08-03 2006-02-09 Nagasaki Prefectural Government Thé fermenté obtenu en frottant de jeunes feuilles de thé et des feuilles de nèfle japonaise et composition ayant un extrait contenu dans le the thé fermenté comme ingrédient actif
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