JPWO2007001080A1 - Foods and beverages and pharmaceuticals containing loquat leaf extract - Google Patents
Foods and beverages and pharmaceuticals containing loquat leaf extract Download PDFInfo
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- JPWO2007001080A1 JPWO2007001080A1 JP2007524050A JP2007524050A JPWO2007001080A1 JP WO2007001080 A1 JPWO2007001080 A1 JP WO2007001080A1 JP 2007524050 A JP2007524050 A JP 2007524050A JP 2007524050 A JP2007524050 A JP 2007524050A JP WO2007001080 A1 JPWO2007001080 A1 JP WO2007001080A1
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- tea
- food
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- loquat
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Classifications
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- 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
Abstract
本発明は、ビワ葉又はビワ茶の抽出物又は精製物を有効成分として含有し、且つ抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び抗酸化作用から成る群から選択される１以上の作用を有する飲食品又は医薬品に関する。The present invention contains an extract or purified product of loquat leaf or loquat tea as an active ingredient, and 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 production The present invention relates to a food or drink or a pharmaceutical product having at least one action selected from the group consisting of action, hypoglycemic action and antioxidant action.
Description
本発明は、例えば抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有する飲食品又は医薬品及びその製造方法に関する。 The present invention relates to a food or drink or pharmaceutical having, for example, 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 hyperglycemic-lowering action and / or an antioxidant action And a manufacturing method thereof.
ビワ葉は、ビワ茶の嗜好飲料に利用され、ビワ茶として親しまれているのみならず、古くから漢方に配合される薬として用いられている。
近年、ビワ葉の有する機能性が明らかにされつつある。例えば、ビワ葉に含まれているポリフェノール成分のヒト口腔癌細胞に対する細胞傷害活性（非特許文献１）、ビワ葉に含まれるメガスチグマン配糖体の抗発癌プロモーション作用（非特許文献２）、ビワ葉の抗腫瘍活性作用（非特許文献３）及び抗酸化作用（非特許文献４）などが報告されている。
本出願人が出願した特許文献１には、ビワ葉を利用して風味のあるビワ茶を多量に製造する方法及び製造設備が開示されている。本出願人は特許文献１に記載のビワ茶の製造設備を用いて、トルマリン石焙煎によりねじめびわ茶を製造している。製造したねじめびわ茶については、「糖尿の血糖値が下がった」、「高血圧の血圧が正常になった」などの声が消費者より寄せられているが、その実証的な証拠は確認されていなかった（非特許文献５）。
一方、特許文献１の段落番号「０００４」に「従来のビワ茶製造の材料となるビワの葉は、果実の収穫を目的とした果実栽培種が利用されているため、栄養分は果実に集まり、ビワの葉に栄養分が十分に行き渡らないため、薬用として利用しても本来の効果は得られないという欠点がある」という記載があるように、従来では、ビワ葉は薬用として十分に効果を発揮していなかった。
従来において、ビワには以下のような活性又は利用方法があることが開示されている。
特許文献２には、ビワ又はその抽出物がマトリックスメタロプロテイナーゼ（ＭＭＰ−１）阻害活性を有することが開示されている。
特許文献３には、ビワの木の葉を焼いて炭にして粉末状食品素材とすることが開示されている。
特許文献４には、ビワの生葉を切断し、蒸気で蒸し、熱風を送りつつ揉み、乾燥させ、整形した後、再度乾燥させることにより香気と旨味とを増すように仕上げることを特徴とした漢方茶の製造方法が開示されている。
特許文献５には、イネ科植物の葉緑健康茶とビワの茶葉を混合し、より多くのγ−アミノ酪酸を含有する健康茶の製造方法が開示されている。
特許文献６には、果実部を除くビワの抽出物がα−グルコシダーゼ阻害活性を有することが開示されている。
特許文献７には、シャキョクとビワ葉を混合した滋養強壮健康増進用組成物が開示されている。
特許文献８には、黄杞の葉を焙煎処理したものをビワ茶と混合してなる健康茶が開示されている。
特許文献９には、甘茶とビワの混合物で構成する健康茶が開示されている。
特許文献１０には、バラ科ビワ属の有機抽出物を含む組成物が生物におけるＣＯＸ−２活性を阻害することが開示されている。
In recent years, the functionality of loquat leaves has been clarified. For example, the cytotoxic activity of polyphenol components contained in loquat leaves against human oral cancer cells (Non-patent Document 1), the anti-carcinogenic promotion action of Megastigman glycosides contained in loquat leaves (Non-patent Document 2), loquat leaves Have reported antitumor activity (Non-patent Document 3) and antioxidant activity (Non-patent Document 4).
Patent Document 1 filed by the present applicant discloses a method and production equipment for producing a large amount of flavored loquat tea using loquat leaves. The present applicant uses the production equipment for loquat tea described in Patent Document 1 to produce screw mebiwa tea by roasting tourmaline stone. Concerning manufactured Nemebiwa tea, the voices such as “the blood sugar level of diabetes has decreased” and “the blood pressure of hypertension has become normal” have been received from consumers, but empirical evidence has been confirmed. (Non-Patent Document 5).
On the other hand, in paragraph No. "0004" of Patent Document 1, "The loquat leaf used as a material for conventional loquat tea production uses fruit cultivars for the purpose of fruit harvesting, so nutrients gather in the fruit, Traditionally, loquat leaves are sufficiently effective for medicinal use, as described in the statement that the original effect cannot be obtained even if used as a medicinal product because the nutrients are not sufficiently distributed to the loquat leaves. I did not.
Conventionally, it has been disclosed that loquat has the following activity or utilization method.
Patent Document 2 discloses that loquat or an extract thereof has a matrix metalloproteinase (MMP-1) inhibitory activity.
Patent Document 3 discloses that loquat leaves are baked into charcoal to make a powdered food material.
Patent Document 4 is a Chinese medicine characterized by cutting raw loquat leaves, steaming with steam, scouring while sending hot air, drying, shaping, and then drying again to increase aroma and umami. A method for producing tea is disclosed.
Patent Document 5 discloses a method for producing health tea, which is a mixture of leafy green health tea and loquat tea leaves, and contains more γ-aminobutyric acid.
Patent Document 6 discloses that loquat extracts excluding fruit parts have α-glucosidase inhibitory activity.
Patent Document 7 discloses a composition for enhancing nutritional tonic health in which peony and loquat leaves are mixed.
Patent Document 8 discloses a healthy tea obtained by mixing roasted yellow persimmon leaves with loquat tea.
Patent Document 9 discloses a health tea composed of a mixture of sweet tea and loquat.
Patent Document 10 discloses that a composition containing an organic extract of the genus Rosaceae inhibits COX-2 activity in an organism.
上述したように、従来ではビワ葉に含まれる種々の生物学的活性や活性化合物の存在が明らかにされたものの、ビワ葉が高血糖値の降下作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、抗高脂血症作用等の効果を有することは知られていなかった。これらの効果が確認できれば、ビワ葉を糖尿病、高脂血症、高血圧症、癌等の疾患の予防又は治療に使用できるものと考えられる。
そこで、本発明は、上述した実情に鑑み、ビワ葉を用いた、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有する飲食品又は医薬品及びその製造方法を提供することを目的とする。
本発明者らは、上記課題を解決するため鋭意研究を行った結果、ビワ茶を熱水抽出に供し、得られた抽出物をモデルマウス又はラットに給餌することで、血糖値を低下させ、またコレステロール及びトリグリセリド等の血清脂質並びに脂肪組織の重量を低下させることを見出した。また、ビワ茶の抽出物から得られた粗製分画物は強い抗酸化作用を有することを見出した。
更に、ビワ茶を熱水抽出に供し、得られた抽出物をモデルマウス又はラットに給餌することで、血圧の上昇を抑制し、また、癌細胞に直接作用させることによってその増殖を抑制し、更にまた、癌細胞に対してアポトーシス誘導すること及び／又は活性酸素種を産生させることを見出し、本発明を完成するに至った。
本発明は以下を包含する。
（１）ビワ葉又はビワ茶の抽出物又は精製物を有効成分として含有し、且つ抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び抗酸化作用から成る群から選択される１以上の作用を有する飲食品又は医薬品。
（２）上記ビワ茶がねじめびわ茶であることを特徴とする、（１）記載の飲食品又は医薬品。
（３）上記精製物がクロロゲン酸、クエルセチン３−サンブビオシド、メチルクロロゲン酸、ケンフェロール３−ラムノシド、クエルセチン３−ラムノシド、２α−ヒドロキシウルソール酸及びウルソール酸から成る群から選択される１以上の化合物を含まないことを特徴とする、（１）記載の飲食品又は医薬品。
（４）上記飲食品が抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び抗酸化作用から成る群から選択される１以上の作用を有する旨の表示を有するものであることを特徴とする、（１）記載の飲食品又は医薬品。
（５）ビワ葉又はビワ茶を熱水抽出又は溶媒抽出に供し、抽出物を得る工程を含むことを特徴とする、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び抗酸化作用から成る群から選択される１以上の作用を有する飲食品又は医薬品の製造方法。
（６）上記抽出物を精製手段に供し、精製物を得る工程をさらに含むことを特徴とする、（５）記載の製造方法。
（７）上記精製手段がカラムクロマトグラフィーであることを特徴とする、（６）記載の製造方法。
（８）上記ビワ茶がねじめびわ茶であることを特徴とする（５）記載の製造方法。
（９）上記精製物がクロロゲン酸、クエルセチン３−サンブビオシド、メチルクロロゲン酸、ケンフェロール３−ラムノシド、クエルセチン３−ラムノシド、２α−ヒドロキシウルソール酸及びウルソール酸から成る群から選択される１以上の化合物を含まないことを特徴とする、（６）記載の製造方法。
以下、本発明を詳細に説明する。
本発明に係る飲食品又は医薬品は、ビワ葉又はビワ茶の抽出物又は精製物を有効成分として含有し、且つ抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び抗酸化作用から成る群から選択される１以上の作用を有するものである。本発明に係る飲食品又は医薬品をヒト等の動物に摂取又は投与することにより、高脂血症や高血圧症を予防又は治療することができる。また、本発明に係る飲食品又は医薬品をヒト等の動物に摂取又は投与することにより、癌細胞の増殖を抑制し、癌細胞にアポトーシスを誘導せしめ、癌細胞に活性酸素種を産生せしめることができる。さらに、本発明に係る飲食品又は医薬品をヒト等の動物に摂取又は投与することにより、血糖値を低下させ、また活性酸素等を取り除くことができる。
ここで、抗高脂血症作用とは、血中のコレステロールやトリグリセリドを低下させることを意味する。また、高血圧抑制作用とは、血圧の上昇を抑えることを意味する。
癌細胞増殖抑制作用とは、癌細胞を死滅させ、その増殖を抑えることを意味する。また、癌細胞アポトーシス誘導作用とは、癌細胞にアポトーシスを誘導せしめ、癌細胞を死滅させることを意味する。さらに、活性酸素種産生作用とは、癌細胞中に活性酸素種を産生し、癌細胞を死滅させることを意味する。
また、高血糖降下作用とは、血糖値を低下させることを意味する。さらに、抗酸化作用とは、体内の活性酸素を取り除くことを意味する。
本発明においては、ビワ葉又はビワ茶を用いる。ビワ葉としては、新鮮葉又は乾燥葉をそのまま使用することができる。ビワ茶としては、いずれのビワ茶であってもよいが、例えばねじめびわ茶（商品名）が挙げられる。ねじめびわ茶は、トルマリン石による高温加熱により焦がしたもので、上述した特許文献１に記載の方法によって調製することができる。なお、ねじめびわ茶（根占枇杷茶）は、鹿児島県農業生産法人有限会社十津川農場から市販されている。
本発明に係る飲食品又は医薬品に使用する抽出物は、ビワ葉又はビワ茶を熱水抽出又は溶媒抽出に供することで得ることができる。例えば、ビワ葉又はビワ茶を、熱湯を用いた抽出に供し、これを１回又は数回（例えば３回）繰り返すことでビワ葉又はビワ茶抽出物を得ることができる。あるいは、例えば、ビワ葉又はビワ茶を水、低級アルコールやアセトンなどの有機溶媒を用いた抽出に供することで、ビワ葉又はビワ茶抽出物を得ることができる。なお、本発明においては、ビワ葉又はビワ茶抽出物とは、上記抽出方法で得られた抽出液もしくは各種溶媒抽出液、その希釈液、その濃縮液又はその乾燥粉末を意味する。
また、本発明においては、上述したビワ葉又はビワ茶抽出物を濾過、遠心分離又は精製処理等の精製手段に供することで、当該抽出物から夾雑物を除去した精製物を用いることができる。精製手段としては、例えば、カラムクロマトグラフィー、順相又は逆相クロマトグラフィー、イオン交換クロマトグラフィー及びゲル濾過が挙げられるが、カラムクロマトグラフィーが特に好ましい。
例えば、ビワ葉又はビワ茶の水抽出溶液を直接、逆相用のゲル（例えば、ＭＣＩ ｇｅｌ ＣＨＰ−２０Ｐ（三菱化学）など）のオープンカラムクロマトグラフィーに付す。移動相にＡ液として水、Ｂ液としてエタノールを用い、Ａ液とＢ液との比率を１００：０、３０：７０、７０：３０と変えて、クロマトグラフィーを行う。最後にＣ液としてアセトンを用い、Ａ液とＣ液との比率を５０：５０にしてクロマトグラフィーを行う。Ａ液とＢ液との比率が７０：３０で溶出する粗製分画物並びにＡ液とＣ液との比率が５０：５０で溶出する粗製分画物には、強い高血糖降下作用が見られる。従って、これら粗製分画物は、ビワ葉又はビワ茶の精製物として好適に本発明に係る飲食品又は医薬品に使用することができる。
あるいは、８０％水性アセトンで抽出したビワ葉又はビワ茶の抽出溶液を減圧エバポレータにて溶媒を留去した後、残差を逆相用のゲル（例えば、ＭＣＩ ｇｅｌ ＣＨＰ−２ＯＰ（三菱化学）など）のオープンカラムクロマトグラフィーに付す。移動相にＡ液として水、Ｂ液としてメタノールを用い、Ａ液からＢ液へのリニアーグラジエントによってクロマトグラフィーを行う。最後にＣ液としてアセトンを用い、Ａ液とＣ液との比率を５０：５０にしてクロマトグラフィーを行う。この方法で溶出する粗製分画物には、強い抗酸化作用が見られる。従って、この粗製分画物は、ビワ葉又はビワ茶の精製物として好適に本発明に係る飲食品又は医薬品に使用することができる。なお、この粗製分画物には、クロロゲン酸、クエルセチン３−サンブビオシド、メチルクロロゲン酸、ケンフェロール３−ラムノシド、クエルセチン３−ラムノシド、２α−ヒドロキシウルソール酸及びウルソール酸のうち１以上の化合物が含まれない。従って、本発明に係る飲食品又は医薬品に使用するビワ葉又はビワ茶精製物には、これら化合物が含まれないことが好ましい。
以上のように説明したビワ葉又はビワ茶抽出物又は精製物を有効成分として用いることで、本発明に係る飲食品又は医薬品を製造することができる。
本発明の抽出物又は精製物の有効量を、錠剤、カプセル、顆粒、ドリンク、ペットボトルなどの任意の形態に添加又は封入するか、あるいは任意の食品に添加することで、本発明に係る飲食品を得ることができる。本発明に係る飲食品は、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有する飲食品、特に、健康補助食品又は特定保健用食品として使用することができる。好ましくは、錠剤、カプセル、顆粒、ドリンク、ペットボトルなどの形態の健康補助食品又は特定保健用食品とするのがよい。
飲食品には、例えば、菓子類、レトルト食品、ジュース類、お茶類、乳製品などが含まれるが、これらに限定されない。また、飲食品には、必要に応じて甘味剤、調味料、乳化剤、懸濁化剤、防腐剤などを添加してもよいし、あるいはビタミン類、栄養剤、免疫増強剤（例えば、プロポリスやきのこ抽出物など）などを添加してもよい。
本発明に係る飲食品に対するビワ葉又はビワ茶の抽出物又は精製物の添加量は、摂取する成人体重１ｋｇあたり０．１〜２００ｍｇに相当する範囲内の量又は１製品あたり例えば５０ｍｇ〜１ｇであるが、この範囲に限定されない。
一方、本発明に係る医薬品は、ビワ葉又はビワ茶の抽出物又は精製物の有効量を含む。本発明に係る医薬品は、抗高脂血症用剤、高血圧抑制剤、癌細胞増殖抑制剤、癌細胞アポトーシス誘導剤、活性酸素種産生剤、高血糖降下剤又は抗酸化剤として使用することができる。
本発明に係る医薬品には、ビワ葉又はビワ茶の抽出物又は精製物以外に、さらに製薬上許容可能な担体（賦形剤もしくは希釈剤）並びに結合剤、増量剤、滑沢剤、崩壊剤、湿潤剤、乳化剤、緩衝剤、懸濁化剤、保存剤、着色剤、風味剤及び甘味剤などから適宜選択される添加剤を含有させることができる。担体及び添加剤は、製剤化のために一般的に使用されるものを、本発明に係る医薬品の製造に使用することができる。例えば、結合剤の例としては、デンプン、ポリビニルピロリドン、ヒドロキシプロピルメチルセルロースなどが挙げられる。増量剤の例としては、ラクトース、微結晶セルロースなどが挙げられる。滑沢剤の例としては、タルク、シリカ、ステアリン酸マグネシウムなどが挙げられる。崩壊剤の例としては、デンプン、デンプングリコール酸ナトリウムなどが挙げられる。湿潤剤の例としては、ラウリル硫酸ナトリウムなどが挙げられる。乳化剤の例としては、セルロース誘導体、ソルビトールなどが挙げられる。また、保存剤の例としては、メチル−ｐ−ヒドロキシベンゾエート、ソルビン酸などが挙げられる。ただし、本発明に使用できる添加剤は、これら添加剤の例に限定されない。
本発明に係る医薬品は、例えば経口投与又は非経口投与（静脈内、動脈内、腹腔内、経直腸内、皮下、筋肉内、舌下、経鼻腔内、経膣内など）用に製剤化され得る。製剤の形態としては、特に限定されないが、例えば溶液剤、錠剤、粉末剤、顆粒剤、カプセル剤、座剤、噴霧剤、制御放出剤、懸濁剤及びドリンク剤などが挙げられる。
本発明に係る医薬品に含まれるビワ葉又はビワ茶の抽出物又は精製物の用量は、患者の年齢、体重、性別、状態、重篤度などの要因によって変化しうる。患者に投与されるビワ葉又はビワ茶の抽出物又は精製物の１日用量は、例えば患者の体重１ｋｇあたり０．１〜２００ｍｇ、好ましくは１〜１００ｍｇの範囲であるが、この範囲に限定されない。必要に応じて、用量を数回、例えば２〜３回に分けて分割投与してもよい。また、本発明に係る医薬品は治療用途の同じ又は異なる他の抗高脂血症用剤、高血圧抑制剤、癌細胞増殖抑制剤、癌細胞アポトーシス誘導剤、活性酸素種産生剤、高血糖降下剤及び／又は抗酸化剤と併用して患者に投与することもできる。
本発明に係る飲食品又は医薬品は、例えば、以下のように薬理評価を行うことができる。
本発明に係る飲食品又は医薬品の抗高脂血症作用の薬理評価としては、例えばモデル動物に本発明に係る飲食品又は医薬品を摂取させ、飼育中又は飼育後に、血清脂質分析又は臓器の脂肪組織重量測定を行う方法が挙げられる。本発明に係る飲食品又は医薬品を摂取していない動物と比較して、本発明に係る飲食品又は医薬品を摂取した動物において、血中の総コレステロール、ＨＤＬコレステロール及び／又はトリグリセリド量が有意に低下した場合には、本発明に係る飲食品又は医薬品が良好に抗高脂血症作用を有すると判断することができる。また同様に、臓器の脂肪組織重量が有意に低下した場合には、本発明に係る飲食品又は医薬品が良好に抗高脂血症作用を有すると判断することができる。
また、本発明に係る飲食品又は医薬品の高血圧抑制作用の薬理評価としては、例えば、ＳＨＲラット（高血圧自然発症ラット）の雄を用いる方法が挙げられる。すなわち、本発明に係る飲食品又は医薬品を当該ＳＨＲラットに給餌し、飼育終了後、各ラットの血圧を、無加温型カフ式血圧測定器（室町機械（株））などの血圧測定装置で測定し、本発明に係る飲食品又は医薬品を給餌していないラットと比較して、血圧が下がった場合には、本発明に係る飲食品又は医薬品が良好に高血圧抑制作用を有すると判断できる。
さらに、本発明に係る飲食品又は医薬品の癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用及び活性酸素種産生作用の薬理評価としては、例えば、ヒト急性前骨髄性白血病疾患細胞（ＨＬ−６０細胞）を用いる方法が挙げられる。すなわち、本発明に係る飲食品又は医薬品存在下でＨＬ−６０細胞を培養し、対照群（本発明に係る飲食品又は医薬品不在下）と比較して、その増殖が抑制された場合には、本発明に係る飲食品又は医薬品が良好に癌細胞増殖抑制作用を有すると判断できる。また、本発明に係る飲食品又は医薬品存在下でＨＬ−６０細胞を培養し、対照群（本発明に係る飲食品又は医薬品不在下）と比較して、その５０％生存率が抑制された場合には、本発明に係る飲食品又は医薬品が良好に癌細胞アポトーシス誘導作用を有すると判断できる。さらに、本発明に係る飲食品又は医薬品存在下でＨＬ−６０細胞を培養し、対照群（本発明に係る飲食品又は医薬品不在下）と比較して、細胞内の活性酸素が上昇した場合には、本発明に係る飲食品又は医薬品が良好に癌細胞に対する活性酸素種産生作用を有すると判断できる。
また、本発明に係る飲食品又は医薬品の高血糖降下作用の薬理評価としては、例えば、モデル動物（例えば、ＩＩ型糖尿病モデルマウスであるＫＫＡｙマウスなど）に本発明に係る飲食品又は医薬品を摂取させ、飼育中又は飼育後に、血糖値を測定する方法が挙げられる。本発明に係る飲食品又は医薬品を摂取していない動物と比較して、本発明に係る飲食品又は医薬品を摂取した動物において、血糖値が有意に低下した場合には、本発明に係る飲食品又は医薬品が良好に高血糖降下作用を有すると判断することができる。同様に、モデル動物を用いたブドウ糖負荷試験において、本発明に係る飲食品又は医薬品を摂取した動物において、血糖値又はＨｂＡ１Ｃ値が有意に低下した場合には、本発明に係る飲食品又は医薬品が良好に高血糖降下作用を有すると判断することができる。
さらに、本発明に係る飲食品又は医薬品の抗酸化作用の薬理評価としては、例えば、Ｈａｒｗａｔ，Ｋ．Ｓ．Ｍ．ｅｔ ａｌ．，Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓ．，３６：１７７−１８７，２００２に記載の方法が挙げられる。すなわち、本発明に係る飲食品又は医薬品にＤＰＰＨ（１，１−ｄｉｐｈｅｎｙｌ−２−ｐｉｃｒｙｌｈｙｄｒａｚｙｌ）を加えて反応させ、吸光度を測定し、その吸光度の値からＴｒｏｌｏｘの吸光度標準曲線を用いてＴｒｏｌｏｘ濃度を換算する。その換算値より、Ｔｒｏｌｏｘのラジカル消去能標準曲線からラジカル消去率を算出する。本発明に係る飲食品又は医薬品のラジカル消去率が、例えば陽性対照であるビタミンＣ又はＥと同程度かそれ以上である場合に、本発明に係る飲食品又は医薬品が良好に抗酸化作用を有すると判断することができる。
焙煎ビワ葉や非焙煎ビワ葉には、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用又は抗酸化作用は全く認められない。一方、本発明においては、ビワ葉又はビワ茶の抽出物又は精製物を用いることで、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有する飲食品又は医薬品を製造できる。これら飲食品又は医薬品は糖尿病、高脂血症、高血圧症、癌等の疾患の予防又は治療に有用である。
本明細書は本願の優先権の基礎である日本国特許出願２００５−１８７１３３号の明細書及び／又は図面に記載される内容を包含する。As described above, conventionally, various biological activities and active compounds contained in loquat leaves have been clarified, but loquat leaves have a high blood glucose lowering action, an antihypertensive action, a cancer cell proliferation inhibiting action, It has not been known to have effects such as cancer cell apoptosis-inducing action, reactive oxygen species producing action, and antihyperlipidemic action. If these effects can be confirmed, loquat leaves can be used for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension and cancer.
Therefore, in view of the above-described circumstances, the present invention uses anti-hyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species producing action, hypoglycemia lowering using loquat leaves. It aims at providing the food-drinks or pharmaceutical which have an effect | action and / or an antioxidant effect | action, and its manufacturing method.
As a result of conducting intensive research to solve the above problems, the present inventors have provided loquat tea for hot water extraction, feeding the resulting extract to model mice or rats, thereby reducing the blood glucose level, It has also been found to reduce the weight of serum lipids such as cholesterol and triglycerides and adipose tissue. Moreover, it discovered that the crude fraction obtained from the extract of Biwa tea had a strong antioxidant action.
Furthermore, by subjecting loquat tea to hot water extraction and feeding the resulting extract to model mice or rats, it suppresses the increase in blood pressure, and suppresses its growth by acting directly on cancer cells, Furthermore, the inventors have found that apoptosis is induced in cancer cells and / or that reactive oxygen species are produced, and the present invention has been completed.
The present invention includes the following.
(1) Contains loquat leaf or loquat tea extract or purified product 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 producing effect A food or drink or a pharmaceutical product having one or more actions selected from the group consisting of a hypoglycemic action and an antioxidant action.
(2) The food or drink or medicine according to (1), wherein the loquat tea is Nememebiwa tea.
(3) One or more compounds selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferol 3-rhamnoside, quercetin 3-rhamnoside, 2α-hydroxyursolic acid and ursolic acid The food or drink or medicine according to (1), which is not contained.
(4) The food or drink is selected from the group consisting of 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 an antioxidant action. The food / beverage product or the pharmaceutical product according to (1), wherein the food / beverage product or pharmaceutical product has a display indicating that it has one or more actions.
(5) An antihyperlipidemic effect, an antihypertensive effect, an antihypertensive effect, a cancer cell proliferation inhibitory effect, a cancer cell, comprising a step of subjecting loquat leaves or loquat tea to hot water extraction or solvent extraction to obtain an extract A method for producing a food or drink or a pharmaceutical product having at least one action selected from the group consisting of an apoptosis-inducing action, a reactive oxygen species producing action, a hyperglycemic lowering action and an antioxidant action.
(6) The method according to (5), further comprising a step of subjecting the extract to a purification means to obtain a purified product.
(7) The method according to (6), wherein the purification means is column chromatography.
(8) The method according to (5), wherein the loquat tea is Nememebiwa tea.
(9) One or more compounds selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferol 3-rhamnoside, quercetin 3-rhamnoside, 2α-hydroxyursolic acid and ursolic acid. The production method according to (6), which is not included.
Hereinafter, the present invention will be described in detail.
The food / beverage product or pharmaceutical product according to the present invention contains an extract or purified product of loquat leaf or loquat tea as an active ingredient, and has an antihyperlipidemic effect, an antihypertensive effect, an anticancer effect on cancer cell proliferation, and an induction of cancer cell apoptosis. It has one or more actions selected from the group consisting of action, reactive oxygen species production action, hyperglycemia 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. In addition, by ingesting or administering the food or drink or medicine according to the present invention to an animal such as a human, it is possible to suppress the growth of cancer cells, induce apoptosis in cancer cells, and produce reactive oxygen species in cancer cells. it can. Furthermore, by ingesting or administering the food or drink or pharmaceutical product according to the present invention to an animal such as a human, the blood glucose level can be lowered and active oxygen or the like can be removed.
Here, the antihyperlipidemic action means lowering blood cholesterol and triglycerides. Moreover, the antihypertensive action means suppressing an increase in blood pressure.
The cancer cell growth inhibitory action means killing cancer cells and suppressing their growth. In addition, cancer cell apoptosis-inducing action means that cancer cells are induced to induce apoptosis and kill cancer cells. Furthermore, the reactive oxygen species producing action means producing reactive oxygen species in cancer cells and killing the cancer cells.
Moreover, the hyperglycemic lowering action means lowering the blood sugar level. Furthermore, the antioxidant action means removing active oxygen in the body.
In the present invention, loquat leaves or loquat tea is used. As loquat leaves, fresh leaves or dried leaves can be used as they are. As the loquat tea, any loquat tea may be used, for example, Nemebiwa tea (trade name). Nemebiwa tea is scorched by high-temperature heating with tourmaline stone, and can be prepared by the method described in Patent Document 1 described above. Nemebiwa tea (Neuro-cha) is commercially available from Totsukawa Farm, Kagoshima Prefectural Agricultural Production Corporation.
The extract used for the food or drink or pharmaceutical product according to the present invention can be obtained by subjecting loquat leaves or loquat tea to hot water extraction or solvent extraction. For example, loquat leaves or loquat tea is subjected to extraction using hot water, and this is repeated once or several times (for example, 3 times) to obtain loquat leaves or loquat tea extracts. Alternatively, for example, loquat leaves or loquat tea can be obtained by subjecting loquat leaves or loquat tea to extraction using water, an organic solvent such as lower alcohol or acetone. In the present invention, loquat leaf or loquat 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 dry powder thereof.
In the present invention, the above-described loquat leaf or loquat tea extract is subjected to purification means such as filtration, centrifugation, or purification treatment, whereby a purified product from which impurities are removed can be used. Examples of the purification means include column chromatography, normal phase or reverse phase chromatography, ion exchange chromatography, and gel filtration, and column chromatography is particularly preferable.
For example, an aqueous solution of loquat leaves or loquat tea is directly subjected to open column chromatography on a gel for reverse phase (for example, 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 and changing the ratio of the liquid A and the liquid B to 100: 0, 30:70, and 70:30. Finally, the chromatography is performed using acetone as the C liquid and the ratio of the A liquid and the C liquid at 50:50. A crude fraction eluted at a ratio of A to B of 70:30 and a crude fraction eluted at a ratio of A to C of 50:50 have 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 loquat leaves or loquat tea.
Alternatively, the solvent of the extract of loquat leaf or loquat tea extracted with 80% aqueous acetone is distilled off with a vacuum evaporator, and then the residual is subjected to a reverse phase gel (for example, MCI gel CHP-2OP (Mitsubishi Chemical)). ) To open column chromatography. Chromatography is performed by using a linear gradient from liquid A to liquid B using water as liquid A and methanol as liquid B in the mobile phase. Finally, the chromatography is performed using acetone as the C liquid and the ratio of the A liquid and the C liquid at 50:50. The crude fraction eluted by this method has a strong antioxidant effect. Therefore, this crude fraction can be suitably used for a food or drink or a pharmaceutical product according to the present invention as a purified product of loquat leaves or loquat tea. This crude fraction contains one or more compounds of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferol 3-rhamnoside, quercetin 3-rhamnoside, 2α-hydroxyursolic acid and ursolic acid. Absent. Therefore, it is preferable that these compounds are not contained in the loquat leaf or the loquat tea purified product used in the food or drink or the pharmaceutical product according to the present invention.
By using the loquat leaf or loquat tea extract or purified product described above as an active ingredient, the food or drink or pharmaceutical product according to the present invention can be produced.
The effective amount of the extract or purified product of the present invention can be added or enclosed in any form such as tablets, capsules, granules, drinks, PET bottles, etc. Goods can be obtained. The food and drink according to the present invention has 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 antioxidant action. It can be used as a product, in particular as a health supplement or a food for specified health use. Preferably, it is a health supplement or specific health food in the form of tablets, capsules, granules, drinks, PET bottles and the like.
Examples of the food and drink include, but are not limited to, confectionery, retort food, juices, teas, and dairy products. In addition, sweeteners, seasonings, emulsifiers, suspending agents, preservatives, etc. may be added to foods and drinks as necessary, or vitamins, nutrients, immune enhancers (for example, propolis or Mushroom extract etc.) may be added.
The addition amount of the extract or purified product of loquat leaf or loquat 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 g per product, for example. Although not limited to this range.
On the other hand, the pharmaceutical product according to the present invention includes an effective amount of an extract or purified product of loquat leaf or loquat tea. The pharmaceutical agent according to the present invention can be used as an antihyperlipidemic agent, a hypertension inhibitor, a cancer cell proliferation inhibitor, a cancer cell apoptosis inducer, a reactive oxygen species generator, a hyperglycemic agent or an antioxidant. it can.
In addition to the loquat leaf or loquat tea extract or purified product, the pharmaceutical product according to the present invention further includes a pharmaceutically acceptable carrier (excipient or diluent), a binder, a bulking agent, a lubricant, and a disintegrant. , Wetting agents, emulsifying agents, buffering agents, suspending agents, preservatives, coloring agents, flavoring agents, sweetening agents and the like may be added as appropriate. Carriers and additives that are commonly used for formulation can be used in the production of the pharmaceutical product according to the present invention. For example, examples of the binder include starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose and the like. Examples of the bulking agent include lactose and microcrystalline cellulose. Examples of lubricants include talc, silica, magnesium stearate and the like. Examples of disintegrants include starch and sodium starch glycolate. Examples of the wetting agent include sodium lauryl sulfate. Examples of emulsifiers include cellulose derivatives and sorbitol. Examples of preservatives include methyl-p-hydroxybenzoate and sorbic acid. However, the additives that can be used in the present invention are not limited to the examples of these additives.
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.). obtain. Although it does not specifically limit as a form of a formulation, For example, a solution, a tablet, a powder, a granule, a capsule, a suppository, a spray, a controlled release agent, a suspension agent, a drink, etc. are mentioned.
The dose of the loquat leaf or loquat tea extract or purified product contained in the pharmaceutical product according to the present invention may vary depending on factors such as the age, weight, sex, condition, and severity of the patient. The daily dose of loquat leaf or loquat tea extract or purified product administered to a patient is, for example, in the range of 0.1 to 200 mg, preferably 1 to 100 mg per kg of the patient's body weight, but is not limited to this range . If necessary, the dose may be divided and administered in several divided doses, for example, 2-3 times. In addition, the pharmaceuticals according to the present invention are 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.
As the pharmacological evaluation of the antihyperlipidemic action of the food or drink or pharmaceutical product according to the present invention, for example, a model animal is fed the food or food product or pharmaceutical product according to the present invention, and during or after breeding, serum lipid analysis or organ fat A method for measuring tissue weight is mentioned. Compared with animals that have not taken the food or drink or medicine according to the present invention, the amount of total cholesterol, HDL cholesterol and / or triglyceride in the blood is significantly reduced in the animals that have taken the food or drink or medicine according to the present invention In that case, 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 pharmaceutical product according to the present invention has an antihyperlipidemic effect well.
Moreover, as a pharmacological evaluation of the hypertension inhibitory action of the food / beverage products or the pharmaceutical which concerns on this invention, the method using the male of a SHR rat (hypertensive spontaneously hypertensive rat) is mentioned, for example. That is, the food or beverage or pharmaceutical product according to the present invention is fed to the SHR rat, and after the breeding, the blood pressure of each rat is measured with a blood pressure measuring device such as an unheated cuff blood pressure measuring instrument (Muromachi Kikai Co., Ltd.). It can be judged that the food and drink or the pharmaceutical product according to the present invention has a good antihypertensive effect when the blood pressure is reduced as compared with the rat that is measured and not fed the food or beverage or pharmaceutical product according to the present invention.
Furthermore, as pharmacological evaluation of the cancer cell proliferation inhibitory 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, for example, human acute promyelocytic leukemia disease cells (HL-60 cells) The method using is mentioned. That is, when HL-60 cells are cultured in the presence of a food or drink or pharmaceutical product according to the present invention and compared with the control group (in the absence of the food or food product or pharmaceutical product according to the present invention), It can be judged that the food or drink or the pharmaceutical product according to the present invention has a cancer cell growth inhibitory effect. In addition, when HL-60 cells are cultured in the presence of a food or drink or pharmaceutical product according to the present invention, and compared with the control group (in the absence of the food or beverage product or pharmaceutical product according to the present invention), the 50% survival rate is suppressed. Therefore, it can be determined that 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 the 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 according to the present invention). It can be determined that the food or drink or pharmaceutical product according to the present invention has a reactive oxygen species producing action on cancer cells.
The pharmacological evaluation of the hyperglycemic action of the food or drink or pharmaceutical product according to the present invention includes, for example, ingesting the food or beverage or pharmaceutical product according to the present invention into a model animal (for example, a KKAy mouse that is a type II diabetes model mouse). And a method of measuring the blood glucose level during or after breeding. When the blood glucose level is significantly reduced in an animal that has ingested the food or drink or medicine according to the present invention as compared to an animal that has not ingested the food or food 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 a good hypoglycemic effect. Similarly, in a glucose tolerance test using a model animal, if the blood glucose level or HbA1C value is significantly reduced in an animal ingesting the food or drink according to the present invention, the food or drink according to the present invention It can be determined that it has a good hypoglycemic effect.
Furthermore, as pharmacological evaluation of the antioxidant effect of the food or drink or pharmaceutical product according to the present invention, for example, Harwat, K. et al. S. M.M. et al. , Free Radical Res. 36: 177-187,2002. That is, DPPH (1,1-diphenyl-2-picrylhydrazyl) is added to the food and drink or pharmaceutical product according to the present invention to react, and the absorbance is measured. From the absorbance value, the Trolox concentration is calculated using the Trolox absorbance standard curve. Convert. 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 determined.
Roasted loquat leaves and non-roasted loquat leaves have antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemic lowering action or antioxidant action Is not allowed at all. On the other hand, in the present invention, by using an extract or purified product of loquat leaf or loquat tea, antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species production It is possible to produce a food or drink or a pharmaceutical product having an action, a hypoglycemic action and / or an antioxidant action. These foods and drinks or pharmaceuticals are useful for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension and cancer.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2005-187133, which is the basis of the priority of the present application.
図１は、実施例１において飼育期間中のコントロール群及びビワ茶群のラットの体重推移を調べた結果を示す。
図２は、実施例１においてコントロール群及びビワ茶群のラットの血中の総コレステロール量を調べた結果を示す。
図３は、実施例１においてコントロール群及びビワ茶群のラットの血中のＨＤＬコレステロール量を調べた結果を示す。
図４は、実施例１においてコントロール群及びビワ茶群のラットの血中のトリグリセリド量を調べた結果を示す。
図５は、実施例１において飼育終了後、コントロール群及びビワ茶群のラットの肝臓、副睾丸周辺及び腎臓周辺の脂肪組織の重量を測定した結果を示す。
図６は、実施例１において飼育期間中のコントロール群及びビワ茶群のマウスの体重推移を調べた結果を示す。
図７は、実施例１において飼育終了後、コントロール群及びビワ茶群のマウスの腎臓周辺及び副睾丸周辺の脂肪組織の重量を測定した結果を示す。
図８は、実施例２において飼育期間中のコントロール群及びビワ茶群のマウスの血糖値の推移を示す。
図９は、実施例２における飼育後のコントロール群及びビワ茶群のマウスの耐糖能試験の結果を示す。
図１０は、実施例２における耐糖能試験後のコントロール群及びビワ茶群のマウスの血中ＨｂＡ１Ｃ値の測定結果を示す。
図１１は、実施例３における乾燥粗製分画物の薄層クロマトグラフを示す。
図１２は、実施例３における飼育期間中のコントロール群及び各ビワ茶群のマウスの血糖値の推移を示す。
図１３は、実施例３において飼育後のコントロール群及び分画３と４を給餌したビワ茶群のマウスの耐糖能試験の結果を示す。
図１４は、実施例３において耐糖能試験後のコントロール群及び各ビワ茶群のマウスの血中ＨｂＡ１Ｃ値の測定結果を示す。
図１５は、実施例４における粗製分画物の薄層クロマトグラフを示す。
図１６は、実施例４において粗製分画物の抗酸化作用を調べた結果を示す。
図１７は、実施例５において、実施例３で調製した粗製分画物の抗酸化作用を調べた結果を示す。
図１８は、実施例５において、実施例３の分画３から更に得られた粗製分画物の抗酸化作用を調べた結果を示す。
図１９は、実施例６においてねじめびわ茶抽出物の高血圧抑制作用を調べた結果を示す。
図２０は、実施例８におけるアガロースゲル電気泳動写真であり、実施例３の粗製分画物のアポトーシス誘導の作用を調べた結果を示す。
図２１は、実施例８におけるアガロースゲル電気泳動写真であり、実施例３の分画３から更に得られた粗製分画物のアポトーシス誘導の作用を調べた結果を示す。
図２２は、実施例９において、実施例３の粗製分画物の癌細胞に対する活性酸素種産生の作用を調べた結果を示す。
図２３は、実施例９において、実施例３の分画３から更に得られた粗製分画物の癌細胞に対する活性酸素種産生の作用を調べた結果を示す。FIG. 1 shows the results of examining the weight transition of rats in the control group and the loquat tea group during the breeding period in Example 1.
FIG. 2 shows the results of examining the total cholesterol level in the blood of rats of the control group and the loquat tea group in Example 1.
FIG. 3 shows the results of examining the amount of HDL cholesterol in the blood of rats of the control group and the loquat tea group in Example 1.
FIG. 4 shows the results of examining the blood triglyceride levels in rats of the control group and the loquat tea group in Example 1.
FIG. 5 shows the results of measurement of the weights of the adipose tissue around the liver, the epididymis and the kidneys of the rats in the control group and the loquat tea group after the end of the breeding in Example 1.
FIG. 6 shows the results of examining the weight transition of mice in the control group and the loquat tea group during the breeding period in Example 1.
FIG. 7 shows the results of measuring the weight of adipose tissue around the kidney and around the epididymis of mice in the control group and the loquat tea group after the end of the breeding in Example 1.
FIG. 8 shows changes in blood glucose levels of mice in the control group and the loquat tea group during the breeding period in Example 2.
FIG. 9 shows the results of the glucose tolerance test of mice in the control group and the loquat tea group after breeding in Example 2.
FIG. 10 shows the measurement results of blood HbA1C values of mice in the control group and the loquat 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 changes in blood glucose levels of mice in the control group and each loquat tea group during the breeding period in Example 3.
FIG. 13 shows the results of a glucose tolerance test of mice in the control group after breeding in Example 3 and mice in the Biwa tea group fed with fractions 3 and 4.
FIG. 14 shows the measurement results of blood HbA1C values of mice in the control group and each loquat tea group 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.
FIG. 17 shows the results of examining the antioxidant effect of the crude fraction prepared in Example 3 in Example 5.
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 an agarose gel electrophoresis photograph in Example 8, showing the results of examining the apoptosis-inducing action of the crude fraction of Example 3.
FIG. 21 is an agarose gel electrophoresis photograph 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 reactive oxygen species production on the cancer cells of the crude fraction of Example 3 in Example 9.
FIG. 23 shows the results of examining the action of reactive oxygen species production on cancer cells of the crude fraction further obtained from fraction 3 of Example 3 in Example 9.
以下、本発明を実施例により具体的に説明するが、本発明の技術的範囲はこれらの実施例に限定されないものとする。
なお、下記の実施例における抗酸化作用の実験は、以下の文献に記載の方法を用いて行った（Ｈａｒｗａｔ，Ｋ．Ｓ．Ｍ．ｅｔ ａｌ．，Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓ．，３６：１７７−１８７，２００２）。
また、下記の実施例で使用するねじめびわ茶は、鹿児島県農業生産法人有限会社十津川農場から市販されているものである。
さらに、各実施例に伴う図中の結果は、各群の平均値又は平均値±標準偏差で示す。
［実施例１］ねじめびわ茶抽出物のダイエット効果
（１）ねじめびわ茶の抽出物の調製
市販のねじめびわ茶（５０ｇ）を熱湯（４００ｍｌ）で抽出した。これを３回繰り返した。得られた熱湯抽出液を凍結乾燥（Ｔｏｋｙｏ Ｒｉｋａｋｉｋａｉ Ｃｏ．，ＬＴＤ，Ｆｒｅｅｚｅ Ｄｒｙｅｒ ＦＲ−１）に供し、乾燥粉末を得た。
用いたねじめびわ茶の総乾燥重量は３４６．２ｇであり、熱湯抽出液の総乾燥粉末量は５７．３７ｇであった。従って、ねじめびわ茶に対する熱湯抽出液の乾燥粉末含量は、１６．５７％であった。
（２）動物の準備
Ｗｉｓｔａｒ系ラット（日本ＳＬＣ社製）及びＫＫＡｙマウス（日本クレア社製）を用いた。飼養試験には、下記の表１に示す割合となるように各材料を計量したものを混合し、これを蒸留水にて混練した後、凍結乾燥機にて乾燥させたものをコントロール用餌（当該餌を給餌したラット又はマウスを本実施例では「コントロール群」という）として用いた。また、上記（１）で得られた熱湯抽出液の乾燥粉末を１％となるようにコントロール用餌に加え、これをねじめびわ茶群用餌（当該餌を給餌したラット又はマウスを本実施例では「ビワ茶群」という）として用いた。
Ｗｉｓｔａｒ系ラット（日本ＳＬＣ社製）を１２匹用いて実験した。これらラットをコントロール群（６匹）とビワ茶群（６匹）とに分け、コントロール群のラットにはコントロール用餌を自由摂取させた。また、ビワ茶群のラットにはねじめびわ茶群用餌を自由摂取させた。双方の群を、それぞれ１ヶ月間飼育した。
飼育期間中、３日おきにラットの体重測定を行った。また、ラットの給水には両群とも水道水を用い、飼育期間中は自由給水とした。
飼育終了後、各動物をネンブタールで麻酔し、心臓より採血を行い血清脂質の分析を行った。また、各群のラットの臓器を摘出し、その重量を測定した。
ラットの体重測定の結果を図１に示す。図１は、飼育期間中の両群のラットの体重推移を調べた結果である。図１中、黒塗りの丸がコントロール群のラットの結果であり、白抜きの丸がビワ茶群のラットの結果である。
図１に示すように、飼育期間中、両群のラットの体重は増加したが、３週目より両群間で徐々に体重差が現れ、ビワ茶群のラットの体重はコントロール群のラットの体重に比べて、その増加が抑制されることが分かる。
飼育終了後の血清脂質の分析結果を図２〜図４に示す。図２は、血中の総コレステロール量（ｍｇ／ｄｌ）を調べた結果である。図３は、血中のＨＤＬコレステロール量（ｍｇ／ｄｌ）を調べた結果である。また、図４は、血中のトリグセリド量（ｍｇ／ｄｌ）を調べた結果である。なお、図２〜４において、「コントロール」はコントロール群のラットの結果であり、「ビワ茶」はビワ茶群のラットの結果である。
図２及び３に示すように、総コレステロールとＨＤＬコレステロールの値は、コントロール群のラットと比較してビワ茶群のラットはやや低い値になることが分かる。トリグリセリドについて、図４に示すように、コントロール群のラットに比べ、ビワ茶群のラットは低い水準（図４中、＊はＰ＜０．０１であることを示す）で推移することが分かる。
さらに飼育後の臓器重量を調べた結果を図５に示す。図５は、飼育終了後、ラットの肝臓、副睾丸周辺及び腎臓周辺の脂肪組織の重量を測定した結果である。図５において、「コントロール」はコントロール群のラットの結果であり、「ビワ茶」はビワ茶群のラットの結果である。
図５に示すように、肝臓、副睾丸周辺および腎臓周辺の脂肪組織の重量を比較した結果、コントロール群のラットの重量に比べ、ビワ茶群のラットの重量のほうがより低くなることが分かる。特に、腎臓周辺の脂肪組織の重量については、ビワ茶群のラットの重量は有意に低い（図５中、＊はＰ＜０．０５であることを示す）ことが分かる。
（４）マウスの体重及び臓器重量の測定：
ＫＫＡｙマウス（日本クレア社製）を２０匹用いて実験した。これらマウスをコントロール群（１０匹）とビワ茶群（１０匹）とに分け、コントロール群のマウスにはコントロール用餌を自由摂取させた。また、ビワ茶群のマウスにはねじめびわ茶群用餌を自由摂取させた。双方の群を、それぞれ７週間飼育した。
飼育期間中、３日おきにマウスの体重測定を行った。また、マウスの給水には両群とも水道水を用い、飼育期間中は自由給水とした。
飼育終了後、各動物をネンブタールで麻酔し、各群のマウスの臓器を摘出し、その重量を測定した。
マウスの体重測定の結果を図６に示す。図６は、飼育期間中の両群のマウスの体重推移を調べた結果である。図６中、黒塗りの丸がコントロール群のマウスの結果であり、白抜きの丸がビワ茶群のマウスの結果である。なお、図６において、＊はＰ＜０．０５を示し、＊＊はＰ＜０．０１を示す。
図６に示すように、飼育期間中、両群のマウスの体重は増加したが、ビワ茶群のマウスの体重はコントロール群のマウスの体重に比べて、その増加が有意に（Ｐ＜０．０１）抑制されることが分かる。
飼育後の臓器重量を調べた結果を図７に示す。図７は、飼育終了後、マウスの腎臓周辺及び副睾丸周辺の脂肪組織の重量を測定した結果である。図７において、「コントロール」はコントロール群のマウスの結果であり、「ビワ茶」はビワ茶群のマウスの結果である。
図７に示すように、腎臓周辺及び副睾丸周辺の脂肪組織の重量を比較した結果、コントロール群のマウスの重量に比べ、ビワ茶群のマウスの重量のほうがより低くなることが分かる。特に、腎臓周辺の脂肪組織の重量については、ビワ茶群のマウスの重量は有意に低い（図７中、＊はＰ＜０．０５であることを示す）ことが分かる。
［実施例２］ねじめびわ茶抽出物の高血糖値抑制効果
（１）ねじめびわ茶の抽出物の調製
市販のねじめびわ茶（５０ｇ）を熱湯（４００ｍｌ）で抽出した。これを３回繰り返した。得られた熱湯抽出液を凍結乾燥（Ｔｏｋｙｏ Ｒｉｋａｋｉｋａｉ Ｃｏ．，ＬＴＤ，Ｆｒｅｅｚｅ Ｄｒｙｅｒ ＦＲ−１）に供し、乾燥粉末を得た。
用いたねじめびわ茶の総乾燥重量は３００．０ｇであり、熱湯抽出液の総乾燥粉末量は５１．７３ｇであった。従って、ねじめびわ茶に対する熱湯抽出液の乾燥粉末含量は、１７．２４％であった。
（２）動物の準備
高血糖の（ＩＩ型糖尿病モデル）マウスとしてＫＫＡｙマウス（日本クレア社製）を用いた。飼養試験には上記の表１に示す割合となるように各材料を計量したものを混合し、これを蒸留水にて混練した後、凍結乾燥機にて乾燥させたものをコントロール用餌（当該餌を給餌したマウスを本実施例では「コントロール群」という）として用いた。また、上記（１）で得られた熱湯抽出液の乾燥粉末を１％となるようにコントロール用餌に加え、これをねじめびわ茶群用餌（当該餌を給餌したマウスを本実施例では「ビワ茶群」という）として用いた。
（３）ＩＩ型糖尿病モデルマウスの血糖値の測定
ＩＩ型糖尿病モデルマウスとしてＫＫＡｙマウス（日本クレア社製）を２０匹用いて実験した。マウスをコントロール群（１０匹）とビワ茶群（１０匹）とに分け、コントロール群のマウスにはコントロール用餌を自由摂取させた。また、ビワ茶群のマウスにはねじめびわ茶群用餌を自由摂取させた。双方の群をそれぞれ７週間飼育した。マウスの給水には両群とも水道水を用い、飼育期間中は自由給水とした。
飼育期間中、１週間おきにマウスの血糖値を測定した。血糖値の測定には酵素電極法を用いた。
飼育期間中の血糖値の推移を図８に示す。図８中、黒塗りの丸がコントロール群のマウスの結果であり、白抜きの丸がビワ茶群のマウスの結果である。
図８に示すように、血糖値は両群の動物とも徐々に上昇し、実験開始１週目より血糖値に差が現れることが分かる。すなわち、コントロール群のマウスに比べ、ビワ茶群のマウスは血糖値の上昇が抑えられたことが分かる。特に、３、５、６週目の血糖値については、コントロール群のマウスに比べ、ビワ茶群のマウスの血糖値は有意（図８中、＊はＰ＜０．０５であることを示す）に低くなることが分かる。
（４）ＩＩ型糖尿病モデルマウスのブドウ糖負荷試験
上記（３）での飼育終了後、各群のマウスを１４時間絶食させ、これにブドウ糖を投与した。ブドウ糖の投与量は、マウス体重×１／２００（ｍｌ）のブドウ糖４ｇ／１０ｍｌ（蒸留水）であった。この投与量をステンレス製ゾンデにて各マウスの胃に直接注入し、強制的に投与した。投与後、経時的に血糖値を測定した。
測定後、マウスをネンブタールで麻酔し、心臓より採血を行い、ＨｂＡ１Ｃの値を測定した。
飼育後の耐糖能試験の結果を図９に示す。図９中、黒塗りの丸がコントロール群のマウスの結果であり、白抜きの丸がビワ茶群のマウスの結果である。なお、図９中、＊はＰ＜０．０５であることを示す。
図９に示すように、血糖値はブドウ糖投与により、両群とも速やかな上昇を示し、１５分後にはおよそ４００ｍｇ／ｄｌの高い値となった。３０分後、コントロール群のマウスの血糖値はさらに上昇を続け、その後は減少することが分かる。これに対して、ビワ茶群のマウスの血糖値については、３０分後の値がコントロール群の値に比べて低く抑えられ（Ｐ＜０．０５水準で有意差あり）、その後はコントロール群の値と同様に血糖値は低下することが分かる。また、各測定時間におけるビワ茶群のマウスの血糖値は、コントロール群の血糖値に比べて、常により低い値をとって推移していくことが分かる。
さらに、耐糖能試験後の血中ＨｂＡ１Ｃ値の測定結果を図１０に示す。図１０において、「コントロール」はコントロール群のマウスの結果であり、「ビワ茶」はビワ茶群のマウスの結果である。
図１０に示すように、コントロール群のマウスの血中ＨｂＡ１Ｃ値に比べ、ビワ茶群のマウスの血中ＨｂＡ１Ｃ値が有意に（図１０中、＊＊はＰ＜０．０１であることを示す）低いことが分かる。
［実施例３］ねじめびわ茶の粗製分画物の血糖値上昇の抑制効果
（１）ねじめびわ茶の粗製分画物の調製
市販のねじめびわ茶（５０ｇ）を熱湯（４００ｍｌ）で抽出した。これを３回繰り返した。得られた熱湯抽出液を凍結乾燥（Ｔｏｋｙｏ Ｒｉｋａｋｉｋａｉ Ｃｏ．，ＬＴＤ，Ｆｒｅｅｚｅ Ｄｒｙｅｒ ＦＲ−１）に供し、乾燥粉末を得た。なお、用いたねじめびわ茶の総乾燥重量は９５０．０ｇであった。
得られた熱湯抽出液を、冷後、順次、ＭＣＩ ｇｅｌ ＣＨＰ−２０Ｐのオープンカラムクロマトに付した。なお、ゲルの量は乾燥重量として、１ｋｇ前後量を使用することが好ましい。また、使用したガラスカラムのサイズは内径８ｃｍであった。本実施例では、ガラスカラムを使用したが、カラムの材質はガラス、アクリル材質などに限定されなくても良い。溶媒を、水（２Ｌ）、３０％エタノール水溶液（２Ｌ）、７０％エタノール水溶液（２Ｌ）及び水−アセトン（１：１の比率）（２Ｌ）をそれぞれ用いて溶出させ、４つの分画を得た。４つの乾燥粗製分画物の収量は、水（２Ｌ）で溶出した乾燥粗製分画物（以下、「分画１」という）が９８．０ｇ、３０％エタノール水溶液（２Ｌ）で溶出した乾燥粗製分画物（以下、「分画２」という）が４５．３ｇ、７０％エタノール水溶液（２Ｌ）で溶出した乾燥粗製分画物（以下、「分画３」という）が１８．３ｇ、水−アセトン（１：１の比率）（２Ｌ）で溶出した乾燥粗製分画物（以下、「分画４」という）が２．１ｇであった。
これら分画１〜４の薄層クロマトグラフ（Ｔｈｉｎ Ｌａｙｅｒ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ、以下「ＴＬＣ」という）を図１１に示す。図１１中、１〜４の番号はそれぞれ分画１〜４の結果を示す。なお、ＴＬＣに用いた展開溶媒は、ベンゼン−ギ酸エチル−ギ酸の３種の溶媒を用い、その比率を３：６：１の溶液用量で混合したものを用いた。発色試薬は、塩化第二鉄（ＦｅＣｌ３）を５％程度メタノールに溶解したものを用い、ＴＬＣへ直接噴霧することにより、化合物を発色検出した。
図１１に示すように、分画１及び２には、クロロゲン酸様の物質が、Ｒｆ値約０．１付近にあることが分かる。また、分画３及び４には、有機酸及びクロロゲン酸様の物質があまり含まれていないことが分かる。
（２）動物の準備
高血糖の（ＩＩ型糖尿病モデル）マウスとしてＫＫＡｙマウス（日本クレア社製）を用いた。飼養試験には、上記の表１に示す割合となるように各材料を計量したものを混合し、これを蒸留水にて混練した後、凍結乾燥機にて乾燥させたものをコントロール用餌（当該餌を給餌したマウスを本実施例では「コントロール群」という）として用いた。また、上記（１）で得られた分画１、分画２又は分画３と４をコントロール用餌に対して１％となるようにそれぞれ加え、ねじめびわ茶群用餌（当該餌をそれぞれ給餌したマウスを本実施例では合わせて「ビワ茶群」という）として用いた。
（３）ＩＩ型糖尿病モデルマウスの血糖値の測定
ＩＩ型糖尿病モデルＫＫＡｙマウス（日本クレア社製）を３２匹用いて実験した。マウスをコントロール群（８匹）、分画１を含有するねじめびわ茶群用餌を与えるビワ茶群（８匹）、分画２を含有するねじめびわ茶群用餌を与えるビワ茶群（８匹）、分画３と４を混合して含有するねじめびわ茶群用餌を与えるビワ茶群（８匹）、の４群に分け、コントロール群のマウスには１日１回、１．５ｇのコントロール用餌を給餌し、不足分はマウス用市販餌を飽食状態となるよう与えた。また、各ビワ茶群のマウスには１日１回、１．５ｇのコントロール用餌に１５ｍｇの各分画を含むねじめびわ茶群用餌を給餌し、不足分はマウス用市販餌を飽食状態となるよう与えた。各群をそれぞれ７週間飼育した。マウスの給水には各群とも水道水を用い、飼育期間中は自由給水とした。
飼育期間中、１週間おきにマウスの血糖値を測定した。血糖値の測定には酵素電極法を用いた。
飼育期間中の血糖値の推移を図１２に示す。図１２中、黒塗りの丸がコントロール群のマウスの結果であり、白抜きの四角が、分画１を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果である。また、白抜きの三角が、分画２を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果であり、白抜きの丸が、分画３と４を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果である。
図１２に示すように、飼育期間中、血糖値はコントロール群及び各ビワ茶群のマウスの血糖値は上昇した。しかし、分画３と４を給餌したビワ茶群の血糖値は実験開始３週目より上昇が頭打ちとなり、コントロール群の血糖値に比べて低い値で推移することが分かる。
（４）ＩＩ型糖尿病モデルマウスのブドウ糖負荷試験
上記（３）での飼育終了後、各４群のマウスを１４時間絶食させ、これにブドウ糖を投与した。ブドウ糖の投与量は、マウス体重×１／２００（ｍｌ）のブドウ糖４ｇ／１０ｍｌ（蒸留水）であった。この投与量をステンレス製ゾンデにて各マウスの胃に直接注入し、強制的に投与した。投与後、経時的に血糖値を測定した。
測定後、マウスをネンブタールで麻酔し、心臓より採血を行い、ＨｂＡ１Ｃの値を測定した。
分画３と４を給餌したビワ茶群について、飼育終了後に耐糖能試験を行った結果を図１３に示す。図１３中、黒塗りの丸がコントロール群のマウスの結果であり、白抜きの丸が、分画３と４を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果である。
図１３に示すように、分画３と４を給餌したビワ茶群の血糖値はブドウ糖投与により、コントロール群と同様に投与直後から速やかな上昇が認められ、１５分後にはおよそ５００ｍｇ／ｄｌと上昇した。３０分後には、コントロール群はさらに上昇を続け、その後、減少することが分かる。これに対し、分画３と４を給餌したビワ茶群では、３０分後既に血糖値の低下が始まり、その後、速やかに下降することが分かる。特に、３０、６０分後では有意に（図１３中、＊はＰ＜０．０５であることを示す）低いことが分かる。
さらに、耐糖能試験後の血中ＨｂＡ１Ｃ値の測定結果を図１４に示す。図１４において、「コントロール」はコントロール群のマウスの結果であり、「ビワ茶１」は、分画１を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果である。また、「ビワ茶２」が、分画２を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果であり、「ビワ茶（３＋４）」が、分画３と４を含有するねじめびわ茶群用餌を給餌したビワ茶群のマウスの結果である。
図１４に示すように、コントロール群のマウスの血中ＨｂＡ１Ｃ値に比べ、分画３と４を給餌したビワ茶群のマウスの血中ＨｂＡ１Ｃ値が有意に（図１４中、＊はＰ＜０．０５であることを示す）低いことが分かる。
［実施例４］ねじめびわ茶の粗製分画物の抗酸化作用の効果
（１）ねじめびわ茶の粗製分画物の調製
市販のねじめびわ茶（１２４７ｇ）を８０％水性アセトン（５０００ｍｌ）で抽出した。これを３回繰り返した。得られた８０％水性アセトン抽出液は、減圧下で溶媒を留去することで、シロップ状のエキス（４４８．２ｇ）を得た。得られたエキスの一部（２７８ｇ）を分取し、ＭＣＩ ｇｅｌ ＣＨＰ−２０Ｐのカラムクロマトに付した。なお、ゲルの量は乾燥重量として、１ｋｇ前後量を使用することが好ましい。また、使用したガラスカラムのサイズは内径８ｃｍであった。本実施例では、ガラスカラムを使用したが、カラムの材質はガラス、アクリル材質などに限定されなくても良い。移動相にＡ液として水、Ｂ液としてメタノールを用い、Ａ液からＢ液へのリニアーグラジエントによってクロマトグラフィーを行った。より詳しくは、Ａ液とＢ液とを１：０の比率（３００ｍｌ）及び９５：５の比率（３００ｍｌ）で溶出した分画溶液をまとめたものを粗製分画１とした。Ａ液とＢ液とを９：１の比率（３００ｍｌ）、８：２の比率（３００ｍｌ）及び７：３の比率（３００ｍｌ）で溶出した分画溶液をまとめたものを粗製分画２とした。Ａ液とＢ液とを６：４の比率（３００ｍｌ）で溶出した分画溶液を粗製分画３とした。Ａ液とＢ液とを５：５の比率（３００ｍｌ）で溶出した分画溶液を粗製分画４とした。Ａ液とＢ液とを４：６の比率（３００ｍｌ）で溶出した分画溶液を粗製分画５とした。Ａ液とＢ液とを３：７の比率（３００ｍｌ）で溶出した分画溶液を粗製分画６とした。Ａ液とＢ液とを２：８の比率（３００ｍｌ）で溶出した分画溶液を粗製分画７とした。Ａ液とＢ液とを１：９の比率（３００ｍｌ）で溶出した分画溶液を粗製分画８とした。Ａ液とＢ液とを０：１の比率（３００ｍｌ）で溶出した分画溶液を粗製分画９とした。最後にＣ液としてアセトンを用い、Ａ液とＣ液との比率を５０：５０（１Ｌ）にしてクロマトグラフィーを行った。なお、このＡ液とＣ液との比率を５０：５０（１Ｌ）にしてクロマトグラフィーを行い、得ることができる分画溶液は、上記の粗製分画９と混合した。この結果、粗製分画１〜９と称する９つの粗製分画物を得た。９つの粗製分画物の収量は、粗製分画１が１２．３ｇ、粗製分画２が１２．８ｇ、粗製分画３が０．４ｇ、粗製分画４が２．９ｇ、粗製分画５が８．３ｇ、粗製分画６が６．７ｇ、粗製分画７が１９．９ｇ、粗製分画８が５．０ｇ、粗製分画９が４．９ｇ、であった。
これら９つの粗製分画物のうち粗製分画２〜８のＴＬＣを図１５に示す。図１５中、２〜８の番号はそれぞれ粗製分画２〜８の結果を示す。なお、ＴＬＣに用いた展開溶媒は、ベンゼン−ギ酸エチル−ギ酸の３種の溶媒を用い、その比率を３：６：１の溶液用量で混合したものを用いた。発色試薬は、塩化第二鉄（ＦｅＣｌ３）を５％程度メタノールに溶解したものを用い、ＴＬＣへ直接噴霧することにより、化合物を発色検出した。
図１５に示すように、緑色から青色に発色するものはフェノール性の物質であることが分かる。
（２）抗酸化作用の実験
抗酸化作用の実験は、以下の文献に記載の方法を用いて行った（Ｈａｒｗａｔ，Ｋ．Ｓ．Ｍ．ｅｔ ａｌ．，Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓ．，３６：１７７−１８７，２００２）。
サンプルは、上記（１）で得られた９つの粗製分画物をそれぞれ１０μｇ／ｍｌの濃度で調整したもの、コントロールとしてビワ茶の熱湯抽出物の乾燥粉末を１０μｇ／ｍｌの濃度で調整したもの、標準品として、ビタミンＣ（アスコルビン酸）を５μｇ／ｍｌの濃度で調整したもの、ビタミンＥを５μｇ／ｍｌの濃度で調整したものを用いた。
これらのサンプルを平底のプレート（９６ｗｅｌｌ）に加え、さらにＤＰＰＨ（１，１−ｄｉｐｈｅｎｙｌ−２−ｐｉｃｒｙｌｈｙｄｒａｚｙｌ、マイクロモーラーの濃度で調整したもの）を１９０μｌ加え、１０秒間混合した後、遮光して３０分間放置した。この反応後、各ウエルの反応溶液を、４９０ｎｍの波長の吸光度を用いてマイクロプレートリーダーで測定し、その吸光度の値からＴｒｏｌｏｘの吸光度標準曲線を用いてＴｒｏｌｏｘ濃度を換算した。その換算値より、Ｔｒｏｌｏｘのラジカル消去能標準曲線からラジカル消去率を算出した。
抗酸化作用の実験結果を図１６に示す。図１６中、１〜９のサンプルナンバーは、それぞれの粗製分画１〜９に対応する。また、サンプルナンバー１０はビワ茶の熱湯抽出物の乾燥粉末である。ＶＣはビタミンＣであり、ＶＥはビタミンＥを示す。
図１６に示すように、粗製分画６、８及び９に強い抗酸化作用があることが分かる。その程度は、ビタミンＥと同程度かそれ以上であり、ビタミンＣの２分の１程度である。また、粗製分画６、８及び９の抗酸化作用は、ビワ茶の熱湯抽出物の乾燥粉末に比べ３倍程度強くなることが分かる。
［実施例５］ねじめびわ茶の粗製分画物の抗酸化作用の効果２
（１）実施例３で調製した分画１〜４の抗酸化作用の実験
抗酸化作用の実験は、以下の文献に記載の方法を用いて行った（Ｈａｒｗａｔ，Ｋ．Ｓ．Ｍ．ｅｔ ａｌ．，Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓ．，３６：１７７−１８７，２００２）。
サンプルは、実施例３で得られた４つの粗製分画物：分画１〜４をそれぞれ１０μｇ／ｍｌの濃度で調整したもの、コントロールとして実施例１で得られたねじめびわ茶熱湯抽出液の乾燥粉末を１０μｇ／ｍｌの濃度で調整したものを用いた。
これらのサンプルを平底のプレート（９６ｗｅｌｌ）に加え、さらにＤＰＰＨ（１，１−ｄｉｐｈｅｎｙｌ−２−ｐｉｃｒｙｌｈｙｄｒａｚｙｌ、マイクロモーラーの濃度で調整したもの）を１９０μｌ加え、１０秒間混合した後、遮光して３０分間放置した。この反応後、各ウエルの反応溶液を、４９０ｎｍの波長の吸光度を用いてマイクロプレートリーダーで測定し、その吸光度の値からＴｒｏｌｏｘの吸光度標準曲線を用いてＴｒｏｌｏｘ濃度を換算した。その換算値より、Ｔｒｏｌｏｘのラジカル消去能標準曲線からラジカル消去率を算出した。
抗酸化作用の実験結果を図１７に示す。図１７中、縦軸はＤＰＰＨラジカル消去率を％として表示し、横軸の分画１〜分画４のサンプルは、実施例３で調製したそれぞれの分画１〜４に対応する。また、ビワ茶は実施例１で得られたねじめびわ茶熱湯抽出液の乾燥粉末である。
図１７に示すように、分画２、分画３及び分画４に強い抗酸化作用があることが分かる。その程度は、実施例１で得られたねじめびわ茶熱湯抽出液の効果より大きいことがわかる。また、分画３の抗酸化作用は、実施例１で得られたねじめびわ茶熱湯抽出液の乾燥粉末に比べ１．６倍程度強いことが分かる。
（２）ねじめびわ茶の粗製分画物の調製
実施例３によって得られた分画３の乾燥粉末５．０ｇを秤量し、これを適量の水に溶解した後、オープンカラムクロマトグラフィーに付した。オープンカラムクロマトグラフィーの条件は、固定相にクロマトレックスＯＤＳ（Ｃｈｒｏｍａｔｏｒｅｘ ＯＤＳ、富士シリシア化学株式会社、Ｆｕｊｉ Ｓｉｌｙｓｉａ Ｃｈｅｍｉｃａｌ ＬＴＤ．）を用い、移動層にＡ液として蒸留水（Ｈ２Ｏ）、Ｂ液としてメタノール（ＭｅＯＨ）を用い、Ａ液からＢ液の含量を増やすことによって各種クロマトグラフィーを行った。具体的には、溶媒を、８０％Ｈ２Ｏ−２０％ＭｅＯＨ（４００ｍｌ）、７０％Ｈ２Ｏ−３０％ＭｅＯＨ（４００ｍｌ）、６０％Ｈ２Ｏ−４０％ＭｅＯＨ（４００ｍｌ）、５０％Ｈ２Ｏ−５０％ＭｅＯＨ（４００ｍｌ）、４０％Ｈ２Ｏ−６０％ＭｅＯＨ（４００ｍｌ）、３０％Ｈ２Ｏ−７０％ＭｅＯＨ（４００ｍｌ）、２０％Ｈ２Ｏ−８０％ＭｅＯＨ（４００ｍｌ）、１０％Ｈ２Ｏ−９０％ＭｅＯＨ（４００ｍｌ）、１００％ＭｅＯＨ（４００ｍｌ）で溶出させ、各４００ｍｌ溶出画分を分取し、３−１から３−９までの計９個の分画を得た。各分画の収量と収率は次のとおりであった。分画３−１（０．１９ｇ、３．９％）、分画３−２（０．２８ｇ、５．７％）、分画３−３（０．５７ｇ、１１．６％）、分画３−４（０．８５ｇ、１７．３％）、分画３−５（０．６４ｇ、１３．１％）、分画３−６（０．７９ｇ、１６．１％）、分画３−７（０．５４ｇ、１１．０％）、分画３−８（０．７８ｇ、１５．９％）、分画３−９（０．１７ｇ、３．５％）。
（３）実施例３の分画３から更に得られた分画３−１〜３−９の抗酸化作用の実験
抗酸化作用の実験は、以下の文献に記載の方法を用いて行った（Ｈａｒｗａｔ，Ｋ．Ｓ．Ｍ．ｅｔ ａｌ．，Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓ．，３６：１７７−１８７，２００２）。
サンプルは、上記（２）で得た９つの粗製分画物：分画３−１〜３−９をそれぞれ１０μｇ／ｍｌの濃度で調整したものと、コントロールとして実施例３で得られた分画３の乾燥粉末を１０μｇ／ｍｌの濃度で調整したものを用いた。
これらのサンプルを平底のプレート（９６ｗｅｌｌ）に加え、さらにＤＰＰＨ（１，１−ｄｉｐｈｅｎｙｌ−２−ｐｉｃｒｙｌｈｙｄｒａｚｙｌ、マイクロモーラーの濃度で調整したもの）を１９０μｌ加え、１０秒間混合した後、遮光して３０分間放置した。この反応後、各ウエルの反応溶液を、４９０ｎｍの波長の吸光度を用いてマイクロプレートリーダーで測定し、その吸光度の値からＴｒｏｌｏｘの吸光度標準曲線を用いてＴｒｏｌｏｘ濃度を換算した。その換算値より、Ｔｒｏｌｏｘのラジカル消去能標準曲線からラジカル消去率を算出した。
抗酸化作用の実験結果を図１８に示す。図１８中、縦軸にはＤＰＰＨラジカル消去率を％の値として表示し、横軸の３−１〜３−９のサンプルは、調製したそれぞれの分画３−１〜３−９に対応する。また、分画３は実施例３で調製した分画３である。
図１８に示すように、分画３−１、３−２及び３−４に強い抗酸化作用があることが分かる。その程度は、実施例３で調製した分画３の効果より大きいことがわかる。特に、分画３−１の抗酸化作用は、分画３の乾燥粉末に比べ１．３７倍程度強いことが分かる。
［実施例６］ねじめびわ茶抽出物の高血圧抑制の効果
（１）動物の準備
ＳＨＲラット（高血圧自然発症ラット）の雄（５週齢）を１２匹（日本ＳＬＣ社製）用いた。飼養試験には、下記の表２に示す割合となるように各材料を計量したものを混合し、これを蒸留水にて混練した後、凍結乾燥機にて乾燥させたものをコントロール用餌（当該餌を給餌したラットを本実施例では「コントロール群」という）として用いた。また、実施例１で得られた熱湯抽出液の乾燥粉末を０．２％となるようにコントロール用餌に加え、これをねじめびわ茶群用餌（当該餌を給餌したラットを本実施例では「ビワ茶群」という）として用いた。
ＳＨＲラット（高血圧自然発症ラット）雄（５週齢）（日本ＳＬＣ社製）を１２匹用いて、血圧を測定した。これらラットをコントロール群（６匹）とビワ茶群（６匹）とに分け、コントロール群のラットにはコントロール用餌を自由摂取させた。また、ビワ茶群のラットにはねじめびわ茶群用餌を自由摂取させた。双方の群を、それぞれ５０日間飼育した。
飼育期間中、飼料、飲水は自由摂食、自由飲水とした。また飼育室は２３℃とし、１２時間照明（７：００〜１９：００）とした。
飼育終了後、各動物の血圧を測定するために、血圧測定装置として無加温型カフ式血圧測定器（室町機械（株））を用いた。
ラットの血圧測定の結果を図１９に示す。図１９は、飼育期間中の両群のラットの血圧推移を調べた結果である。図１９中、白抜きの丸がコントロール群のラットの結果であり、黒塗りの丸がビワ茶群のラットの結果である。また、血圧（最大血圧）は一定時刻（午前中１０時頃）より測定した。測定は各個体について平均１０回繰り返し、その平均値で表記した。縦棒は標準誤差を示す。血圧測定の実験的な困難さとして、血圧の値は大変容易に変動し、ラットの精神状態、環境条件で極めて容易に変動することがあるので、実験はラットを出来るだけ落ち着かせ、静かな環境下で行った。
図１９に示すように、スタート時にはコントロール群とビワ茶群の最大血圧の平均値はともに１３４ｍｍＨｇ程度であった。コントロール群の血圧は飼育期間の経過に伴って１０日過ぎより上昇し、２０日以降はかなり高めに推移した。それに対し、ビワ茶群は２１日までほとんど上昇せず、特に２１日ではコントロール群と有意差が認められた（Ｐ＜０．０５）。その後は実験日数の進展に伴って上昇していくが、コントロール群に較べて１０ｍｍＨｇ以下低い血圧を示し、ねじめびわ茶抽出物の高血圧を抑制する効果が認められた。この結果、ねじめびわ茶抽出物は血圧の上昇を抑制することがわかる。
［実施例７］ねじめびわ茶の粗製分画物の癌細胞増殖抑制作用の効果
（１）実施例３で調製した分画１〜４の癌細胞増殖抑制効果
ヒト急性前骨髄性白血病疾患細胞（ＨＬ−６０細胞）の５０％生存率は以下の文献記載の方法で測定した（Ｍｏｓｍａｎｎ，Ｔ．，Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ，６５：５５−６３，１９８３）。ＨＬ−６０細胞は、１０％の牛胎児血清を含むＲＰＭＩ１６４０培地を用いて培養した。実施例１で得たねじめびわ茶熱湯抽出液の乾燥粉末と、実施例３で調製した分画１〜４を、０、２５、５０、７５、１００、１２５、１５０μｇ／ｍｌの濃度で含むように、０．１％ＤＭＳＯ（ジメチルスルフォキシド）溶液に溶解し、ＨＬ−６０細胞を６時間処理した。
処理後、細胞を遠心分離法により回収し、細胞溶解緩衝液により細胞膜を取り除き、ＲＮＡ分解酵素によりＲＮＡを分解し、さらにタンパク質分解酵素によりタンパク質を除去した。得られたＤＮＡ断片を２％アガロースゲル電気泳動法により分離し、染色後、ＵＶトランスイルミネーターにより検出し、ＤＮＡの断片率を算出した。４８時間後のＨＬ−６０細胞生存率に対する５０％抑制濃度（ＩＣ５０）を算出した。その結果を表３に示した。なお、表３における抽出物は、実施例１で得たねじめびわ茶熱湯抽出液の乾燥粉末の結果である。
（２）実施例５で調製した分画３−１〜３−９の癌細胞増殖抑制効果
上記（１）と同様の方法で、実施例５で調製した分画３−１〜３−９のＨＬ−６０細胞増殖抑制効果を検討した。
ＨＬ−６０細胞の５０％生存率は以下の文献記載の方法で測定した（Ｍｏｓｍａｎｎ，Ｔ．，Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ，６５：５５−６３，１９８３）。ＨＬ−６０細胞は、１０％の牛胎児血清を含むＲＰＭＩ１６４０培地を用いて培養した。実施例１で得たねじめびわ茶熱湯抽出液の乾燥粉末と、実施例５で調製した分画３−１〜３−９を、０、２５、５０、７５、１００、１２５、１５０μｇ／ｍｌの濃度で含むように、０．１％ＤＭＳＯ（ジメチルスルフォキシド）溶液に溶解し、ＨＬ−６０細胞を６時間処理した。
処理後、細胞を遠心分離法により回収し、細胞溶解緩衝液により細胞膜を取り除き、ＲＮＡ分解酵素によりＲＮＡを分解し、さらにタンパク質分解酵素によりタンパク質を除去した。得られたＤＮＡ断片を２％アガロースゲル電気泳動法により分離し、染色後、ＵＶトランスイルミネーターにより検出し、ＤＮＡの断片率を算出した。４８時間後のＨＬ−６０細胞生存率に対する５０％抑制濃度（ＩＣ５０）を算出した。その結果を表４に示した。なお、ねじめびわ茶熱湯抽出液の乾燥粉末についての結果は、表３に示す結果である。
［実施例８］ねじめびわ茶の粗製分画物の癌細胞アポトーシス誘導作用の効果
（１）実施例３で調製した分画１〜４の癌細胞アポトーシス誘導効果
ヒト急性前骨髄性白血病疾患細胞（ＨＬ−６０細胞）のＤＮＡ断片化については以下の文献記載の方法で測定した（Ｈｏｕ，Ｄ．Ｘ．ｅｔ ａｌ．，Ｉｎｔ．Ｊ．Ｏｎｃｏｌ．，２３：７０５−７１２，２００３）。実施例３で調製した各分画２、分画３及び分画４を２５０、５００、７５０μｇ／ｍｌの３つの濃度に調整し、ＨＬ−６０細胞を６時間処理し、細胞核からＤＮＡを抽出した。得られたＤＮＡについてアガロースゲル電気泳動を行い、その結果を図２０に示した。
図２０中、ＭはＤＮＡマーカー、Ｃは濃度０μｇ／ｍｌのコントロールを示す。この結果、分画３の５００μｇ／ｍｌと７５０μｇ／ｍｌの濃度にＤＮＡラダーが観察され、アポトーシスを誘導することが分かる。
（２）実施例５で調製した分画３−１〜３−９の癌細胞アポトーシス誘導効果
上記（１）と同様の方法で、実施例５で調製した分画３−１〜３−９の癌細胞アポトーシス誘導効果を検討した。
ＨＬ−６０細胞のＤＮＡ断片化については以下の文献記載の方法で測定した（Ｈｏｕ，Ｄ．Ｘ．ｅｔ ａｌ．，Ｉｎｔ．Ｊ．Ｏｎｃｏｌ．，２３：７０５−７１２，２００３）。実施例５にて調製した各分画３−１〜３−９を５００μｇ／ｍｌの濃度に調整し、ＨＬ−６０細胞を６時間処理し、細胞核からＤＮＡを抽出した。得られたＤＮＡについてアガロースゲル電気泳動を行い、その結果を図２１に示した。
図２１中、ＭはＤＮＡマーカー、Ｃは濃度０μｇ／ｍｌのコントロールを示す。この結果、分画３−１、３−２、３−４及び３−６にＤＮＡラダーが観察され、アポトーシスを誘導することが分かる。特に、分画３−２と３−４には強いＤＮＡラダーが観察され、アポトーシスを強く誘導することが分かる。
［実施例９］ねじめびわ茶の粗製分画物の活性酸素種産生作用の効果
（１）実施例３で調製した分画１〜４の活性酸素種産生効果
ヒト急性前骨髄性白血病疾患細胞（ＨＬ−６０細胞）を２．０×１０６ｃｅｌｌｓ／ｍｌで継代した。これをＲＰＭＩ１６４０培地で４倍に希釈し、２．０×１０５ｃｅｌｌｓ／４００μｌとした。必要量は１ｗｅｌｌあたり４００μｌ細胞液であり、従って、１ｐｌａｔｅあたり２０ｍｌの細胞液を準備した。プレートを軽く振って細胞の偏りを防ぎながら、これを、３７℃、５％ＣＯ２気流下で２４時間培養した。
２４時間の培養後、この培養液に、実施例３で調製した分画１〜４を５００μｇ／ｍｌの濃度になるように添加した。また、比較のために、実施例１で調製したねじめびわ茶熱湯抽出液を同様に添加した。これを３７℃、５％ＣＯ２のインキュベーターで１５分間培養した。培養後、それぞれのｗｅｌｌにＭＴＴ液（５ｍＭ ＤＣＦＨ−ＤＡ：ＤＣＦＨ−ＤＡの１ｍｇをエタノール４１３μｌで溶かしたもの；ＤＣＦＡ−ＤＡ、ジクロロフルオレシン２酢酸）を１．６μｌずつ分注し、引き続き、３７℃、５％ＣＯ２気流下のインキュベーターでさらに３０分間培養した。
培養後、各ｗｅｌｌをマイクロプレートリーダー（ｅｘｃｉｔａｔｉｏｎ波長：４８５ｎｍ，ｅｍｉｓｓｉｏｎ波長：５３０ｎｍ）により測定し、コントロールと比較して、吸光度からＨＬ−６０細胞内で産生された活性酸素の量を算出した。その結果を図２２に示す。なお、コントロールはＨＬ−６０細胞のみで試験を行った場合の結果である。
図２２中、コントロールは対照を示す。また、図２２において、抽出物は、実施例１で調製したねじめびわ茶熱湯抽出液である。この結果、分画３はＨＬ−６０細胞内に対して強い活性酸素産生能を有することが分かる。
（２）実施例５で調製した分画３−１〜３−９の活性酸素種産生効果
上記（１）と同様の方法で、実施例５で調製した分画３−１〜３−９の活性酸素種産生効果を検討した。
ＨＬ−６０細胞を２．０×１０６ｃｅｌｌｓ／ｍｌで継代した。これをＲＰＭＩ１６４０培地で４倍に希釈し、２．０×１０５ｃｅｌｌｓ／４００μｌとした。必要量は１ｗｅｌｌあたり４００μｌ細胞液であり、従って、１ｐｌａｔｅあたり２０ｍｌの細胞液を準備した。プレートを軽く振って細胞の偏りを防ぎながら、これを、３７℃、５％ＣＯ２気流下で２４時間培養した。
２４時間の培養後、この培養液に、実施例５で調製した分画３−１〜３−９を５００μｇ／ｍｌの濃度になるように添加した。これを３７℃、５％ＣＯ２のインキュベーターで１５分間培養した。培養後、それぞれのｗｅｌｌにＭＴＴ液（５ｍＭ ＤＣＦＨ−ＤＡ：ＤＣＦＨ−ＤＡの１ｍｇをエタノール４１３μｌで溶かしたもの；ＤＣＦＡ−ＤＡ、ジクロロフルオレシン２酢酸）を１．６μｌずつ分注し、引き続き、３７℃、５％ＣＯ２気流下のインキュベーターでさらに３０分間培養した。
培養後、各ｗｅｌｌをマイクロプレートリーダー（ｅｘｃｉｔａｔｉｏｎ波長：４８５ｎｍ，ｅｍｉｓｓｉｏｎ波長：５３０ｎｍ）により測定し、コントロールと比較して、吸光度からＨＬ−６０細胞内で産生された活性酸素の量を算出した。その結果を図２３に示す。なお、コントロールはＨＬ−６０細胞のみで試験を行った場合の結果である。
図２３中、コントロールは対照を示す。この結果、分画３−１、３−２、３−３及び３−５がコントロールより強い活性酸素産生能を有することが分かる。特に、分画３−１はＨＬ−６０細胞内に対してコントロールと比較して約２．６倍程度強い活性酸素産生能を有することが分かる。EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited to these examples.
In addition, the experiment of the antioxidant effect | action in the following Example was performed using the method as described in the following literature (Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).
Further, Nemebiwa tea used in the following examples is commercially available from Totsukawa Farm, Kagoshima Prefectural Agricultural Production Corporation.
Furthermore, the result in the figure accompanying each Example is shown by the average value or average value +/- standard deviation of each group.
[Example 1] Diet effect of Nemebiwa tea extract
(1) Preparation of extract of Nemebiwa tea
Commercial Nejimebiwa tea (50 g) was extracted with hot water (400 ml). This was repeated three times. The obtained hot water extract was subjected to freeze-drying (Tokyo Ryokai 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 with respect to Nemebiwa tea was 16.57%.
(2) Animal preparation
Wistar rats (Nihon SLC) and KKAy mice (Claire Japan) were used. In the feeding test, the materials weighed so as to have the ratios shown in Table 1 below were mixed, kneaded with distilled water, and then dried with a freeze dryer. Rats or mice fed with the food were used as “control group” in this example. In addition, the dry powder of the hot water extract obtained in (1) above was added to the control food so as to be 1%, and this was added to the Nemebiwa tea group food (the rat or mouse fed with the food). In the example, it was used as “biwa tea group”).
Experiments were carried out using 12 Wistar rats (Japan SLC). These rats were divided into a control group (6 animals) and a loquat tea group (6 animals), and the control group rats were allowed to freely ingest control food. Rats in the Biwa tea group were given free access to Nemebiwa tea group food. Both groups were raised for 1 month each.
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.
After the breeding, 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 removed and their weights were measured.
The results of rat body weight measurement are shown in FIG. FIG. 1 shows the results of examining the weight transition of rats in both groups during the breeding period. In FIG. 1, the black circles are the results for the rats in the control group, and the white circles are the results for the rats in the Biwa tea group.
As shown in FIG. 1, during the breeding period, the weights of the rats in both groups increased, but gradually a difference in weights appeared between the two groups from the third week. It can be seen that the increase is suppressed compared to body weight.
The analysis result of the serum lipid after completion | finish of breeding is shown in FIGS. FIG. 2 shows the results of examining the total cholesterol level (mg / dl) in blood. FIG. 3 shows the results of examining the amount of HDL cholesterol in blood (mg / dl). FIG. 4 shows the results of examining the amount of triglyceride (mg / dl) in blood. 2 to 4, “control” is the result for the rats in the control group, and “biwa tea” is the result for the rats in the Biwa tea group.
As shown in FIGS. 2 and 3, it can be seen that the values of total cholesterol and HDL cholesterol are slightly lower in the rats of the loquat tea group than in the rats of the control group. About triglyceride, as shown in FIG. 4, it turns out that the rat of the Biwa tea group changes in a low level (in FIG. 4, * shows that P <0.01) compared with the rat of a control group.
Furthermore, the result of having investigated the organ weight after breeding is shown in FIG. FIG. 5 shows the results of measuring the weight of the adipose tissue around the liver, the accessory testicles, and the kidneys after the breeding. In FIG. 5, “control” is the result of the rats in the control group, and “biwa tea” is the result of the rats in the Biwa tea group.
As shown in FIG. 5, as a result of comparing the weights of the adipose tissues around the liver, the accessory testicles, and the kidneys, it can be seen that the weight of the rats in the Biwa tea group is lower than the weight of the rats in the control group. In particular, regarding the weight of adipose tissue around the kidney, it can be seen that the weight of rats in the Biwa tea group is significantly low (in FIG. 5, * indicates P <0.05).
(4) Measurement of mouse body weight and organ weight:
Experiments were performed using 20 KKAy mice (CLEA Japan). These mice were divided into a control group (10 animals) and a loquat tea group (10 animals), and the control group mice were allowed to freely ingest control food. In addition, mice in the Biwa tea group were given free access to Nemebiwa tea group food. Both groups were raised for 7 weeks each.
During the breeding period, mice were weighed every 3 days. In addition, both groups used tap water for the water supply of the mice, and the water supply was free during the breeding period.
After the breeding, each animal was anesthetized with Nembutal, and the organs of each group of mice were removed and their weights were measured.
The result of measuring the body weight of the mouse is shown in FIG. FIG. 6 shows the results of examining the weight transition of mice in both groups during the breeding period. In FIG. 6, black circles are the results of the control group of mice, and white circles are the results of the loquat brown group of mice. In FIG. 6, * indicates P <0.05, and ** indicates P <0.01.
As shown in FIG. 6, the weights of the mice in both groups increased during the breeding period, but the weights of the mice in the Biwa tea group increased significantly (P <0. 01) It turns out that it is suppressed.
The results of examining the organ weight after breeding are shown in FIG. FIG. 7 shows the results of measurement of the weight of the adipose tissue around the kidney and the accessory testicle after the breeding. In FIG. 7, “control” is the result of the mice in the control group, and “biwa tea” is the result of the mice in the Biwa tea group.
As shown in FIG. 7, as a result of comparing the weights of the adipose tissue around the kidney and around the accessory testicle, it can be seen that the weight of the mouse in the Biwa tea group is lower than the weight of the mouse in the control group. In particular, regarding the weight of the adipose tissue around the kidney, it can be seen that the weight of the mice in the Biwa tea group is significantly low (in FIG. 7, * indicates P <0.05).
[Example 2] High blood sugar level inhibitory effect of Nemebiwa tea extract
(1) Preparation of extract of Nemebiwa tea
Commercial Nejimebiwa tea (50 g) was extracted with hot water (400 ml). This was repeated three times. The obtained hot water extract was subjected to freeze-drying (Tokyo Ryokai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder.
The total dry weight of Nemebiwa tea used was 300.0 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 with respect to Nemebiwa tea was 17.24%.
(2) Animal preparation
A KKAy mouse (manufactured by Claire Japan) was used as a hyperglycemic (type II diabetes model) mouse. In the feeding test, the ingredients weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried with a freeze dryer. Mice fed with food were used as “control group” in this example. In addition, the dry powder of the hot water extract obtained in (1) above is added to the control bait so as to be 1%, and this is added to the Nemebiwa tea group bait (the mice fed with the bait in this example). Used as “biwa tea group”).
(3) Measurement of blood glucose level in type II diabetes model mice
Experiments were carried out using 20 KKAy mice (manufactured by CLEA Japan) as type II diabetes model mice. The mice were divided into a control group (10 animals) and a loquat tea group (10 animals), and the control group mice were allowed to freely ingest control food. In addition, mice in the Biwa tea group were given free access to Nemebiwa tea group food. Both groups were raised for 7 weeks each. In both groups, tap water was used for water supply to the mice, and free water was provided during the breeding period.
During the breeding period, the blood glucose level of the mice was measured every other week. The enzyme electrode method was used for blood glucose level measurement.
The transition of blood glucose level during the breeding period is shown in FIG. In FIG. 8, the black circles are the results for the mice in the control group, and the white circles are the results for the mice in the Biwa tea group.
As shown in FIG. 8, the blood glucose level gradually increases in both groups of animals, and it can be seen that a difference appears in the blood glucose level from the first week of the experiment. That is, it can be seen that the increase in blood glucose level was suppressed in the Biwa tea group mice compared to the control group mice. In particular, regarding blood glucose levels at 3, 5, and 6 weeks, the blood glucose level of the mice in the Biwa tea group was significant compared to the mice in the control group (in FIG. 8, * indicates that P <0.05). It turns out that it becomes low.
(4) Glucose tolerance test in type II diabetes model mice
After completion of the breeding in (3) above, each group of mice was fasted for 14 hours, and glucose was administered thereto. The dosage of glucose was 4 g / 10 ml of glucose (distilled water) of mouse body weight × 1/200 (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.
After the measurement, the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbA1C was measured.
The results of the glucose tolerance test after breeding are shown in FIG. In FIG. 9, the black circles are the results for the mice in the control group, and the white circles are the results for the mice in the Biwa tea group. In FIG. 9, * indicates that P <0.05.
As shown in FIG. 9, the blood glucose level rapidly increased in both groups by glucose administration, and reached a high value of about 400 mg / dl after 15 minutes. After 30 minutes, it can be seen that the blood glucose level of the mice in the control group continues to rise and then decreases. On the other hand, the blood glucose level of mice in the Biwa tea group was suppressed to a value lower than that of the control group after 30 minutes (there was a significant difference at the P <0.05 level). It turns out that a blood glucose level falls similarly to a value. Moreover, it turns out that the blood glucose level of the mouse | mouth of a loquat tea group in each measurement time always takes a lower value compared with the blood glucose level of a control group.
Furthermore, the measurement result of the blood HbA1C value after a glucose tolerance test is shown in FIG. In FIG. 10, “control” is the result of the mice in the control group, and “biwa tea” is the result of the mice in the Biwa tea group.
As shown in FIG. 10, the blood HbA1C value of the mice in the Biwa tea group was significantly higher than the blood HbA1C value of the mice in the control group (in FIG. 10, ** indicates P <0.01). ) I can see it is low
[Example 3] Inhibitory effect of blood sugar level rise of crude fraction of Nemebiwa tea
(1) Preparation of crude fraction of Nemebiwa tea
Commercial Nejimebiwa tea (50 g) was extracted with hot water (400 ml). This was repeated three times. The obtained hot water extract was subjected to freeze-drying (Tokyo Ryokai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder. The total dry weight of Nemebiwa tea used was 950.0 g.
The obtained hot water extract was cooled and subjected to MCI gel CHP-20P open column chromatography sequentially. 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, acrylic material, or the like. The solvent is eluted with water (2 L), 30% aqueous ethanol (2 L), 70% aqueous ethanol (2 L) and water-acetone (1: 1 ratio) (2 L), respectively, to obtain 4 fractions. It was. The yield of the four dry crude fractions was 98.0 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 a fraction (hereinafter referred to as “Fraction 2”), 18.3 g of a dry crude fraction (hereinafter referred to as “Fraction 3”) eluted with 70% aqueous ethanol (2 L), The dry crude fraction eluted with acetone (1: 1 ratio) (2 L) (hereinafter referred to as “Fraction 4”) was 2.1 g.
The thin layer chromatographs (Thin Layer Chromatography, hereinafter referred to as “TLC”) of these fractions 1 to 4 are shown in FIG. In FIG. 11, the numbers 1 to 4 indicate the results of fractions 1 to 4, respectively. In addition, the developing solvent used for TLC used the 3 types of solvent of benzene-ethyl formate-formic acid, and mixed the ratio by the solution dose of 3: 6: 1. The coloring reagent is ferric chloride (FeCl 3 ) Was dissolved in about 5% methanol and sprayed directly onto TLC to detect the color of the compound.
As shown in FIG. 11, it can be seen that 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 acid and chlorogenic acid-like substances.
(2) Animal preparation
A KKAy mouse (manufactured by Claire Japan) was used as a hyperglycemic (type II diabetes model) mouse. In the feeding test, the materials weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried with a freeze dryer. Mice fed with the food were used as “control group” in this example. In addition, the fraction 1, fraction 2 or fractions 3 and 4 obtained in (1) above were added to 1% of the control bait, respectively, and the Nemebiwa tea group bait (the bait) The mice fed with each were used together in this example as “biwa tea group”).
(3) Measurement of blood glucose level in type II diabetes model mice
Experiments were performed using 32 type II diabetes model KKAy mice (CLEA Japan). Control group (8 animals), Biwa tea group (8 animals) that gives food for Nemebiwa tea group containing fraction 1, Biwa tea group that gives food for Nemebiwa tea group containing fraction 2 (8 animals), Biwa tea group (8 animals) that gives food for Nemebiwa tea group containing a mixture of fractions 3 and 4, divided into 4 groups, once a day for mice in the control group, 1.5g of control food was fed, and the shortage was given a commercial food for mice to be in a satiety state. In addition, mice in each Biwa tea group were fed once a day with 1.5g of control food and 15 mg of each fraction containing Nejimebiwa tea group food, and the deficiency was fed with commercial food for mice. Given to be in a state. Each group was raised for 7 weeks. Tap water was used for each group of mice, and water was supplied freely during the breeding period.
During the breeding period, the blood glucose level of the mice was measured every other week. The enzyme electrode method was used for blood glucose level measurement.
The transition of blood glucose level during the breeding period is shown in FIG. In FIG. 12, the black circles are the results of the control group of mice, and the open squares are the results of the loquat tea group of mice fed with the Nemebiwa tea group containing fraction 1. The white triangle is the result of the Biwa tea group mice fed with the Nejibiwa tea group food containing fraction 2, and the white circle is the screw type containing fractions 3 and 4. It is the result of the mouse | mouth of the Biwa tea group which fed the food for Biwa tea groups.
As shown in FIG. 12, during the breeding period, the blood glucose level of the mice in the control group and each loquat tea group increased. However, it can be seen that the blood sugar level of the Biwa tea group fed with fractions 3 and 4 peaked at the third week after the start of the experiment, and remained at a lower value than that of the control group.
(4) Glucose tolerance test in type II diabetes model mice
After completion of the breeding in (3) above, each of the 4 groups of mice was fasted for 14 hours, and glucose was administered thereto. The dosage of glucose was 4 g / 10 ml of glucose (distilled water) of mouse body weight × 1/200 (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.
After the measurement, the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbA1C was measured.
FIG. 13 shows the results of the glucose tolerance test conducted on the loquat tea group fed with fractions 3 and 4 after the rearing. In FIG. 13, the black circles are the results of the control group mice, and the white circles are the results of the Biwa tea group mice fed the Nemebiwa tea group containing fractions 3 and 4. is there.
As shown in FIG. 13, the blood sugar level of the loquat tea group fed with fractions 3 and 4 was rapidly increased immediately after administration by glucose administration, and about 500 mg / dl after 15 minutes. Rose. It can be seen that after 30 minutes, the control group continues to rise and then decreases. On the other hand, in the loquat tea group fed with fractions 3 and 4, it can be seen that after 30 minutes, the blood sugar level started to decrease and then decreased rapidly. In particular, it can be seen that it is significantly lower after 30 and 60 minutes (in FIG. 13, * indicates that P <0.05).
Furthermore, the measurement result of the blood HbA1C value after a glucose tolerance test is shown in FIG. In FIG. 14, “control” is the result of the control group of mice, and “biwa tea 1” is the result of the loquat tea group of mice fed with the Nemebiwa tea group containing fraction 1. In addition, “Biwa tea 2” is the result of the mice of the Biwa tea group fed with the food for Nemebiwa tea group containing fraction 2, and “Biwa tea (3 + 4)” is the fractions 3 and 4. It is the result of the mouse | mouth of the loquat tea group which supplied the food for Nemebiwa tea group containing.
As shown in FIG. 14, the blood HbA1C value of mice in the Biwa tea group fed with fractions 3 and 4 was significantly higher than the blood HbA1C value of mice in the control group (in FIG. 14, * indicates P <0 .05 indicating low).
[Example 4] Effect of antioxidant effect of crude fraction of Nemebiwa tea
(1) Preparation of crude fraction of Nemebiwa tea
Commercial Nemebiwa tea (1247 g) was extracted with 80% aqueous acetone (5000 ml). This was repeated three times. The obtained 80% aqueous acetone extract was distilled off the solvent under reduced pressure to obtain a syrupy extract (448.2 g). A part (278 g) of the obtained extract 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, acrylic material, or the like. Chromatography was performed by using a linear gradient from liquid A to liquid B using water as liquid A and methanol as liquid B in the mobile phase. More specifically, crude fraction 1 was obtained by collecting fraction solutions in which A solution and B solution were eluted at a ratio of 1: 0 (300 ml) and a ratio of 95: 5 (300 ml). Crude fraction 2 was obtained by combining fraction solutions eluted with A liquid and B liquid at a ratio of 9: 1 (300 ml), a ratio of 8: 2 (300 ml), and a ratio of 7: 3 (300 ml). . 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 in which A liquid and B liquid were eluted at a ratio of 5: 5 (300 ml) was designated as crude fraction 4. The fraction solution in which A liquid and B liquid were eluted at a ratio of 4: 6 (300 ml) was designated as crude fraction 5. The fraction solution in which A liquid and B liquid were eluted at a ratio of 3: 7 (300 ml) was designated as crude fraction 6. The fraction solution in which A liquid and B liquid 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 ml) was designated as crude fraction 8. The fraction solution in which A liquid and B liquid were eluted at a ratio of 0: 1 (300 ml) was designated as crude fraction 9. Finally, acetone was used as the C liquid, and the ratio of the A liquid and the C liquid was 50:50 (1 L) for chromatography. It should be noted that chromatography was carried out at a ratio of this A liquid to C liquid of 50:50 (1 L), and the fraction solution obtained was mixed with the crude fraction 9 described above. This resulted in nine crude fractions referred to as crude fractions 1-9. The yields of the nine crude fractions were 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 5 for 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.
Of these nine crude fractions, TLCs of crude fractions 2-8 are shown in FIG. In FIG. 15, the numbers 2 to 8 indicate the results of the crude fractions 2 to 8, respectively. In addition, the developing solvent used for TLC used the 3 types of solvent of benzene-ethyl formate-formic acid, and mixed the ratio by the solution dose of 3: 6: 1. The coloring reagent is ferric chloride (FeCl 3 ) Was dissolved in about 5% methanol and sprayed directly onto TLC to detect the color of the compound.
As shown in FIG. 15, it can be seen that what develops color from green to blue is a phenolic substance.
(2) Antioxidant action experiment
The antioxidant activity experiment was performed using the method described in the following literature (Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).
The sample was prepared by adjusting the nine crude fractions obtained in (1) above at a concentration of 10 μg / ml, and as a control, a dry powder of Biwa tea hot water extract adjusted at a concentration of 10 μg / ml. As a standard product, one prepared by adjusting vitamin C (ascorbic acid) at a concentration of 5 μg / ml and one prepared by adjusting vitamin E at a concentration of 5 μg / ml were used.
These samples were added to a flat-bottom plate (96 well), and further DPPH (1,1-diphenyl-2-picrylhydrazyl, adjusted with the concentration of micromolar) 190 μl was added, mixed for 10 seconds, then shielded from light for 30 minutes. I left it alone. After this reaction, the reaction solution in each well was measured with a microplate reader using absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using a Trolox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve.
The experimental results of the antioxidant action are shown in FIG. In FIG. 16, sample numbers 1 to 9 correspond to the crude fractions 1 to 9, respectively. Sample number 10 is a dry powder of hot water extract of Biwa tea. VC is vitamin C, and VE indicates vitamin E.
As shown in FIG. 16, it can be seen that the 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. Moreover, it turns out that the antioxidant effect of the crude fractions 6, 8, and 9 becomes about 3 times stronger than the dry powder of the hot water extract of Biwa tea.
[Example 5] Effect 2 of antioxidant effect of crude fraction of Nemebiwa tea
(1) Experiment of antioxidant action of fractions 1 to 4 prepared in Example 3
The antioxidant activity experiment was performed using the method described in the following literature (Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).
Samples were the four crude fractions obtained in Example 3: fractions 1 to 4 each adjusted to a concentration of 10 μg / ml, and Nemebiwa tea hot water extract obtained in Example 1 as a control. A dry powder prepared at a concentration of 10 μg / ml was used.
These samples were added to a flat-bottom plate (96 well), and further DPPH (1,1-diphenyl-2-picrylhydrazyl, adjusted with the concentration of micromolar) 190 μl was added, mixed for 10 seconds, then shielded from light for 30 minutes. I left it alone. After this reaction, the reaction solution in each well was measured with a microplate reader using absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using a Trolox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve.
The experimental results of the antioxidant action are shown in FIG. In FIG. 17, the vertical axis represents the DPPH radical elimination rate as%, and the samples of fractions 1 to 4 on the horizontal axis correspond to the respective fractions 1 to 4 prepared in Example 3. Biwa tea is a dry powder of Nemebiwa tea hot water extract obtained in Example 1.
As shown in FIG. 17, it can be seen that 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. Moreover, it turns out that the antioxidant effect of the fraction 3 is about 1.6 times stronger than the dry powder of Nemebiwa tea hot water extract obtained in Example 1.
(2) Preparation of crude fraction of Nemebiwa tea
The dry powder 5.0 g of fraction 3 obtained in Example 3 was weighed, dissolved in an appropriate amount of water, and then subjected to open column chromatography. The conditions for the open column chromatography were as follows: Chromatorex ODS (Chromatorex ODS, Fuji Silysia Chemical Ltd., Fuji Silysia Chemical LTD.) Was used as the stationary phase, and distilled water (H 2 O), 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. Specifically, the solvent is 80% H 2 O-20% MeOH (400 ml), 70% H 2 O-30% MeOH (400 ml), 60% H 2 O-40% MeOH (400 ml), 50% H 2 O-50% MeOH (400 ml), 40% H 2 O-60% MeOH (400 ml), 30% H 2 O-70% MeOH (400 ml), 20% H 2 O-80% MeOH (400 ml), 10% H 2 Elution was performed with O-90% MeOH (400 ml) and 100% MeOH (400 ml), and each 400 ml elution fraction was fractionated to obtain a total of 9 fractions from 3-1 to 3-9. The yield and yield of each fraction were as follows. Fraction 3-1 (0.19 g, 3.9%), Fraction 3-2 (0.28 g, 5.7%), Fraction 3-3 (0.57 g, 11.6%), Fraction 3-4 (0.85 g, 17.3%), fraction 3-5 (0.64 g, 13.1%), fraction 3-6 (0.79 g, 16.1%), fraction 3- 7 (0.54 g, 11.0%), fraction 3-8 (0.78 g, 15.9%), fraction 3-9 (0.17 g, 3.5%).
(3) Experiment of antioxidant action of fractions 3-1 to 3-9 further obtained from fraction 3 of Example 3
The antioxidant activity experiment was performed using the method described in the following literature (Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).
Samples were prepared by adjusting the 9 crude fractions obtained in (2) above: fractions 3-1 to 3-9 at a concentration of 10 μg / ml, and the fraction obtained in Example 3 as a control. 3 dry powder prepared at a concentration of 10 μg / ml was used.
These samples were added to a flat-bottom plate (96 well), and further DPPH (1,1-diphenyl-2-picrylhydrazyl, adjusted with the concentration of micromolar) 190 μl was added, mixed for 10 seconds, then shielded from light for 30 minutes. I left it alone. After this reaction, the reaction solution in each well was measured with a microplate reader using absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using a Trolox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve.
The experimental results of the antioxidant action are shown in FIG. In FIG. 18, the vertical axis represents the DPPH radical elimination rate as a value in%, and the samples of 3-1 to 3-9 on the horizontal axis correspond to the prepared fractions 3-1 to 3-9. . Fraction 3 is fraction 3 prepared in Example 3.
As shown in FIG. 18, it can be seen that fractions 3-1, 3-2 and 3-4 have a strong antioxidant effect. It can be seen that the degree is greater 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.
[Example 6] Effect of Nejimebiwa tea extract on hypertension suppression
(1) Animal preparation
Twelve males (5 weeks old) (manufactured by SLC Japan) were used as SHR rats (rats with spontaneous hypertension). In the feeding test, the materials weighed so as to have the ratios shown in Table 2 below were mixed, kneaded with distilled water, and then dried with a freeze dryer. Rats fed with the food were used as “control group” in this example. Further, the dry powder of the hot water extract obtained in Example 1 was added to the control food so as to be 0.2%, and this was added to Nejibiwa tea group food (the rat fed with the food was used in this Example). It was called “Biwa tea group”.
Blood pressure was measured using 12 male SHR rats (rats with spontaneous hypertension) (5 weeks old) (manufactured by SLC, Japan). These rats were divided into a control group (6 animals) and a loquat tea group (6 animals), and the rats in the control group were allowed to freely ingest control food. Rats in the Biwa tea group were given free access to Nemebiwa tea group food. Both groups were raised for 50 days each.
During the breeding period, feed and drinking water were free food and water. The breeding room was 23 ° C. and illuminated for 12 hours (7: 0 to 19:00).
After the breeding, in order to measure the blood pressure of each animal, an unheated cuff blood pressure measuring device (Muromachi Kikai Co., Ltd.) was used as a blood pressure measuring device.
The result of the blood pressure measurement of the rat is shown in FIG. FIG. 19 shows the results of examining blood pressure changes in rats in both groups during the breeding period. In FIG. 19, white circles are the results for the rats in the control group, and black circles are the results for the rats in the Biwa tea group. The blood pressure (maximum blood pressure) was measured from a certain time (around 10 am 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. As an experimental difficulty in measuring blood pressure, blood pressure values fluctuate very easily and can fluctuate very easily depending on the mental state and environmental conditions of the rat. Went under.
As shown in FIG. 19, at the start, the average value of the maximum blood pressure in the control group and the loquat tea group was both about 134 mmHg. The blood pressure of the control group increased from the past 10 days with the passage of the breeding period, and changed considerably after 20 days. In contrast, the Biwa tea group hardly increased until the 21st day, and a significant difference from the control group was observed especially on the 21st day (P <0.05). Thereafter, the blood pressure increased with the progress of the experiment days. However, the blood pressure was lower by 10 mmHg or less than that of the control group, and the effect of suppressing the hypertension of the Nejimewa tea extract was recognized. As a result, it can be seen that Nemebiwa tea extract suppresses an increase in blood pressure.
[Example 7] Effect of crude cell fraction of Nemebiwa tea on cancer cell proliferation inhibitory effect
(1) Cancer cell proliferation inhibitory effect of fractions 1 to 4 prepared in Example 3
The 50% survival rate of human acute promyelocytic leukemia disease cells (HL-60 cells) was measured by the method described in the following literature (Mosmann, T., J. Immunol. Methods, 65: 55-63, 1983). HL-60 cells were cultured using RPMI 1640 medium containing 10% fetal bovine serum. The dried powder of Nemebiwa tea hot water extract obtained in Example 1 and fractions 1 to 4 prepared in Example 3 are included at concentrations of 0, 25, 50, 75, 100, 125, and 150 μg / ml. As described above, HL-60 cells were treated with a 0.1% DMSO (dimethyl sulfoxide) solution and treated for 6 hours.
After the treatment, the cells were collected by centrifugation, the cell membrane was removed with a cell lysis buffer, RNA was degraded with RNase, and protein was further removed with proteolytic enzyme. The obtained DNA fragments were separated by 2% agarose gel electrophoresis, stained and then detected by a UV transilluminator, and the DNA fragment rate was calculated. 50% inhibitory concentration against HL-60 cell viability after 48 hours (IC 50 ) Was calculated. The results are shown in Table 3. In addition, the extract in Table 3 is the result of the dry powder of Nemebiwa tea hot water extract obtained in Example 1.
(2) Cancer cell proliferation inhibitory effect of fractions 3-1 to 3-9 prepared in Example 5
In the same manner as in the above (1), the HL-60 cell proliferation inhibitory effect of fractions 3-1 to 3-9 prepared in Example 5 was examined.
The 50% viability of HL-60 cells was measured by the method described in the following literature (Mosmann, T., J. Immunol. Methods, 65: 55-63, 1983). HL-60 cells were cultured using RPMI 1640 medium containing 10% fetal bovine serum. 0, 25, 50, 75, 100, 125, 150 μg / ml of dried powder of Nemebiwa tea hot water extract obtained in Example 1 and fractions 3-1 to 3-9 prepared in Example 5 The HL-60 cells were treated for 6 hours in a 0.1% DMSO (dimethyl sulfoxide) solution.
After the treatment, the cells were collected by centrifugation, the cell membrane was removed with a cell lysis buffer, RNA was degraded with RNase, and protein was further removed with proteolytic enzyme. The obtained DNA fragments were separated by 2% agarose gel electrophoresis, stained and then detected by a UV transilluminator, and the DNA fragment rate was calculated. 50% inhibitory concentration against HL-60 cell viability after 48 hours (IC 50 ) Was calculated. The results are shown in Table 4. In addition, the result about the dry powder of Nemebiwa tea hot water extract is the result shown in Table 3.
[Example 8] Effect of a crude fraction of Nemebiwa tea on inducing apoptosis of cancer cells
(1) Cancer cell apoptosis-inducing effect of fractions 1 to 4 prepared in Example 3
The DNA fragmentation of human acute promyelocytic leukemia disease cells (HL-60 cells) was measured by the method described in the following literature (Hou, DX et al., Int. J. Oncol., 23: 705). -712, 2003). Each fraction 2, fraction 3 and fraction 4 prepared in Example 3 were adjusted to three concentrations of 250, 500, and 750 μg / ml, HL-60 cells were treated for 6 hours, and DNA was extracted from the cell nucleus. . The obtained DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG.
In FIG. 20, M represents a DNA marker, and C represents a control with a concentration of 0 μg / ml. As a result, DNA ladder was observed at a concentration of 500 μg / ml and 750 μg / ml in fraction 3, indicating that apoptosis was induced.
(2) Cancer cell apoptosis-inducing effect of fractions 3-1 to 3-9 prepared in Example 5
The cancer cell apoptosis-inducing effect of fractions 3-1 to 3-9 prepared in Example 5 was examined by the same method as in (1) above.
The DNA fragmentation of HL-60 cells was measured by the method described in the following literature (Hou, DX et al., Int. J. Oncol., 23: 705-712, 2003). Fractions 3-1 to 3-9 prepared in Example 5 were adjusted to a concentration of 500 μg / ml, HL-60 cells were treated for 6 hours, and DNA was extracted from the cell nucleus. The obtained DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG.
In FIG. 21, M represents a DNA marker, and C represents a control at a concentration of 0 μg / ml. As a result, DNA ladders are observed in fractions 3-1, 3-2, 3-4, and 3-6, indicating that apoptosis is induced. In particular, strong DNA ladders are observed in fractions 3-2 and 3-4, indicating that apoptosis is strongly induced.
[Example 9] Effect of reactive oxygen species producing action of crude fraction of Nemebiwa tea
(1) Reactive oxygen species production effect of fractions 1 to 4 prepared in Example 3
2.0 × 10 human acute promyelocytic leukemia disease cells (HL-60 cells) 6 Passaged at cells / ml. This was diluted 4-fold with RPMI 1640 medium to 2.0 × 10 5 cells / 400 μl. The required amount is 400 μl cell fluid per well, so 20 ml cell fluid was prepared per plate. Shake the plate lightly to prevent cell bias, 2 The cells were cultured for 24 hours under an air stream.
After culturing for 24 hours, fractions 1 to 4 prepared in Example 3 were added to this culture solution so as to have a concentration of 500 μg / ml. For comparison, the extract of Nemebiwa tea hot water prepared in Example 1 was similarly added. This is 37 ° C, 5% CO 2 Incubator for 15 minutes. After culturing, 1.6 μl of MTT solution (1 mg of 5 mM DCFH-DA: DCFH-DA dissolved in 413 μl of ethanol; DCFA-DA, dichlorofluorescin diacetic acid) was dispensed into each well. 37 ° C, 5% CO 2 The culture was further continued for 30 minutes in an incubator under an air stream.
After culturing, each well was measured with a microplate reader (excitation wavelength: 485 nm, emission wavelength: 530 nm), and the amount of active oxygen produced in HL-60 cells was calculated from the absorbance compared with the control. The result is shown in FIG. In addition, control is a result at the time of testing only with HL-60 cells.
In FIG. 22, the control indicates a control. In FIG. 22, the extract is Nemebiwa tea hot water extract prepared in Example 1. As a result, it can be seen that Fraction 3 has a strong ability to produce active oxygen in HL-60 cells.
(2) Reactive oxygen species production effect of fractions 3-1 to 3-9 prepared in Example 5
The reactive oxygen species production effect of fractions 3-1 to 3-9 prepared in Example 5 was examined by the same method as in (1) above.
2.0 × 10 HL-60 cells 6 Passaged at cells / ml. This was diluted 4-fold with RPMI 1640 medium to 2.0 × 10 5 cells / 400 μl. The required amount is 400 μl cell fluid per well, so 20 ml cell fluid was prepared per plate. Shake the plate lightly to prevent cell bias, 2 The cells were cultured for 24 hours under an air stream.
After culturing for 24 hours, fractions 3-1 to 3-9 prepared in Example 5 were added to this culture solution so as to have a concentration of 500 μg / ml. This is 37 ° C, 5% CO 2 Incubator for 15 minutes. After culturing, 1.6 μl of MTT solution (1 mg of 5 mM DCFH-DA: DCFH-DA dissolved in 413 μl of ethanol; DCFA-DA, dichlorofluorescin diacetic acid) was dispensed into each well. 37 ° C, 5% CO 2 The culture was further continued for 30 minutes in an incubator under an air stream.
After culturing, each well was measured with a microplate reader (excitation wavelength: 485 nm, emission wavelength: 530 nm), and the amount of active oxygen produced in HL-60 cells was calculated from the absorbance compared with the control. The result is shown in FIG. In addition, control is a result at the time of testing only with HL-60 cells.
In FIG. 23, the control indicates a control. As a result, it can be seen that fractions 3-1, 3-2, 3-3 and 3-5 have a stronger ability to produce active oxygen than the control. In particular, it can be seen that Fraction 3-1 has an active oxygen production ability about 2.6 times stronger than that of the control in HL-60 cells.
本発明によれば、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有し、ビワ葉又はビワ茶の抽出物又は精製物を含有する飲食品又は医薬品が提供される。本発明に係る飲食品（特に、健康補助食品もしくは特定保健用食品）又は医薬品は、抗高脂血症作用、高血圧抑制作用、癌細胞増殖抑制作用、癌細胞アポトーシス誘導作用、活性酸素種産生作用、高血糖降下作用及び／又は抗酸化作用を有することから、抗高脂血症用剤、高血圧抑制剤、癌細胞増殖抑制剤、癌細胞アポトーシス誘導剤、活性酸素種産生剤、高血糖降下剤、抗酸化剤として使用できる。
本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。According to the present invention, it has 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 antioxidant action, A food or drink or a medicine containing an extract or purified product of leaf or loquat tea is provided. The food / beverage products (especially health supplement foods or foods for specified health use) or pharmaceutical products according to the present invention have antihyperlipidemic action, hypertension inhibiting action, cancer cell proliferation inhibiting action, cancer cell apoptosis inducing action, reactive oxygen species producing action Antihyperlipidemic agent, antihypertensive agent, cancer cell proliferation inhibitor, cancer cell apoptosis inducer, reactive oxygen species producing agent, hyperglycemic agent Can be used as an antioxidant.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
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| JP4704448B2 (en) * | 2008-03-28 | 2011-06-15 | 農業生産法人 有限会社十津川農場 | Feed for cultured seafood |
| JP2014101286A (en) * | 2012-11-16 | 2014-06-05 | Totsukawa Nojo:Kk | Antitumor action and liver function improvement action of loquat tea |
| CN103082365A (en) * | 2013-01-31 | 2013-05-08 | 福建省闽中有机食品有限公司 | Method for processing loquat juice beverage through stable and sediment-free juicing of whole loquats |
| JP2014177454A (en) * | 2013-02-18 | 2014-09-25 | Kagoshima Univ | Food and drink and pharmaceutical preparation containing nejime biwa tea extract |
| JP2016132633A (en) * | 2015-01-19 | 2016-07-25 | 農業生産法人 有限会社十津川農場 | Alzheimer's disease preventive or therapeutic action of nejime biwa tea |
| CN108717095B (en) * | 2018-05-30 | 2020-06-23 | 四川新绿色药业科技发展有限公司 | Detection method, identification method and content determination method of loquat leaves or medicine containing loquat leaf raw material |
| CN113080456A (en) * | 2021-05-18 | 2021-07-09 | 中国疾病预防控制中心营养与健康所 | Composition containing loquat leaf extract and preparation method and application thereof |
| CN114306439B (en) * | 2022-02-25 | 2023-05-02 | 浙江大学 | Loquat leaf villus extract and application thereof |
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- 2006-06-27 WO PCT/JP2006/313197 patent/WO2007001080A1/en active Application Filing
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| JP4974116B2 (en) | 2012-07-11 |
| JP2012051940A (en) | 2012-03-15 |
| WO2007001080A1 (en) | 2007-01-04 |
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