JP2019001725A - AGEs production inhibitor - Google Patents
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本発明は、AGEs産生抑制剤に関する。より具体的には、マンゴーの抽出物を含むAGEs産生抑制剤、およびAGEs産生抑制用食品などに関する。 The present invention relates to an AGE production inhibitor. More specifically, the present invention relates to an AGE production inhibitor containing a mango extract, a food for inhibiting AGE production, and the like.
AGEs（Advanced Glycation End Products の略語）は、タンパク質の糖化反応により生成される産物の総称であり、最終糖化産物又は後期糖化生成物などと言われる。AGEsは、体内に蓄積し，糖尿病、動脈硬化、神経性疾患など、加齢性慢性疾患等に関わる様々な症状を引き起こす又は症状を悪化させると報告されており、特に老化との関与が指摘されている。AGEsの例としては、例えばNε-カルボキシメチルリシン（CML）、Nε-カルボキシエチルリシン（CEL）、アルグピリミジン、ペントシジン（英語版）、ピラリン、クロスリン、GA-ピリジン、Nω-カルボキシメチルアルギニン（CMA）、フロイルフラニルイミダゾール、グルコスパン等多くの物質や、その中間体が知られている。 AGEs (abbreviation for Advanced Glycation End Products) is a general term for products produced by protein saccharification reaction, and is called final glycation product or late glycation product. AGEs have been reported to accumulate in the body and cause various symptoms related to age-related chronic diseases such as diabetes, arteriosclerosis, neurological diseases, etc. ing. Examples of AGEs include, for example, Nε-carboxymethyl lysine (CML), Nε-carboxyethyl lysine (CEL), argpyrimidine, pentosidine (English version), pyralin, croslin, GA-pyridine, Nω-carboxymethyl arginine (CMA) Many substances such as furoylfuranyl imidazole and glucospan, and intermediates thereof are known.
AGEsは体外、体内の双方で生じ得る。したがって、AGEsの体内での産生を抑制することは、多くの疾患の治療や予防に有用である。そして、従来より、抗糖化作用を有する化合物として、「アミノグアニジン」が知られている（例えば、特許文献１など）。「アミノグアニジン」は、糖化反応系の中間体と結合することにより、糖化反応の進行を抑制し、AGEs産生を抑制することが報告されている。しかし、臨床試験において貧血、肝障害およびビタミンB6欠乏症などの副作用を引き起こす可能性が指摘され、抗糖化作用を期待できる医薬品としての実用化には至っていない。 AGEs can occur both outside and inside the body. Therefore, suppressing the production of AGEs in the body is useful for the treatment and prevention of many diseases. Conventionally, “aminoguanidine” is known as a compound having an anti-glycation action (for example, Patent Document 1). It has been reported that “aminoguanidine” suppresses the progress of saccharification reaction and suppresses the production of AGEs by binding to an intermediate of a saccharification reaction system. However, it has been pointed out that clinical trials may cause side effects such as anemia, liver damage, and vitamin B6 deficiency, and it has not been put into practical use as a drug that can be expected to have an anti-glycation effect.
このような背景から、新規かつ安全性の高いAGE産生抑制剤（最終糖化産物産生抑制剤）の開発が切望されている。 From such a background, development of a novel and highly safe AGE production inhibitor (final glycation product production inhibitor) has been eagerly desired.
そこで、本願発明は新規かつ安全性の高いAGE産生抑制剤（最終糖化産物産生抑制剤）を提供することを目的とする。 Then, this invention aims at providing the novel and highly safe AGE production inhibitor (final glycation product production inhibitor).
上記課題を解決するため、本発明者は、鋭意研究の結果、マンゴーの抽出物にAGE産生抑制効果を見いだした。また、さらに研究を進め、マンゴーの葉抽出物に含有されるbenzophenone C-glycoside（ベンゾフェノンのＣ配糖体）、3-Glucosyl-2,4,4',6-tetrahydroxybenzophenone及びchlorophyll（クロロフィル）にAGE産生抑制効果を見いだして、本発明を完成させた。 In order to solve the above problems, the present inventor has found an AGE production inhibitory effect on mango extracts as a result of intensive studies. Furthermore, further research has been carried out, and benzophenone C-glycoside (benzoglycone C-glycoside), 3-Glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone and chlorophyll (chlorophyll) contained in mango leaf extract The production suppression effect was found and the present invention was completed.
そこで、本発明は、マンゴーの抽出物を含有するAGEs産生抑制用組成物、AGEs産生抑制用剤、AGEs産生抑制用食品からなる。また、抽出物は、有機溶媒による抽出物であることが好適である。また、これらは、特定保健用食品や機能性表示食品など、AGEs産生を抑制する旨が表示されている場合を含む。さらに、マンゴーの有機溶媒による抽出物、さらには葉の有機溶媒による抽出物であることが好適である。なお、本願において「食品」は飲食料品や経口用サプリメントを広く含み、固体だけではなく液体及び飲料形態等も含む。 Thus, the present invention comprises a composition for inhibiting AGEs production containing an extract of mango, an agent for inhibiting AGEs production, and a food for inhibiting AGEs production. In addition, the extract is preferably an extract using an organic solvent. Moreover, these include the case where it is indicated that the AGEs production is suppressed, such as food for specified health use and functional food. Further, an extract of mango with an organic solvent, and an extract of leaves with an organic solvent are preferred. In the present application, “food” widely includes foods and drinks and oral supplements, and includes not only solids but also liquids and beverages.
さらに、本発明は、benzophenone C-glycoside、3-glucosyl-2,4,4',6-tetrahydroxybenzophenone又はchlorophyllを有効成分とするAGEs産生抑制用組成物、AGEs産生抑制用剤、AGEs産生抑制用食品からなる。benzophenone C-glycoside はβ-グルコシド、α-グルコシドを含み、3-glucosyl-2,4,4',6-tetrahydroxybenzophenoneは、β-グルコシド、α-グルコシド、C-グルコシド、O-グルコシドを含む。好ましくは、3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneを有効成分とする。また、当該食品は、特定保健用食品や機能性表示食品など、AGEs産生を抑制する旨が表示されている場合を含む。 Furthermore, the present invention is a composition for inhibiting AGEs production comprising benzophenone C-glycoside, 3-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone or chlorophyll as an active ingredient, an agent for inhibiting AGEs production, a food for inhibiting AGEs production Consists of. Benzophenone C-glycoside includes β-glucoside and α-glucoside, and 3-glucosyl-2,4,4 ′, 6-tetrahydroxybenzophenone includes β-glucoside, α-glucoside, C-glucoside, and O-glucoside. Preferably, 3-C-β-D-glucosyl-2,4,4 ′, 6-tetrahydroxybenzophenone is an active ingredient. Moreover, the said foodstuff includes the case where the effect which suppresses AGEs production | generation is displayed, such as food for specified health use and a food with a functional indication.
マンゴー（Mangifera indica）は、ウルシ科マンゴー属の常緑高木の植物であり、古くから主として果実が食用とされている。本発明で使用するマンゴーは、アーウィン種、アルフォンソ種、カラバオ種など、ウルシ科マンゴー属のマンゴーが全て含まれる。本願で利用するマンゴーは、特定の部位に限られず「果肉」「果皮」「花」「種子」「葉」のいずれにおいてもAGEs産生抑制効果を有する。そして、植物部位における利用の優先性や採取量、採取効率、利用効率等を考慮すると、「葉」は好適である。本発明において「葉」は、所謂葉部分を意味し、葉身全体又はその一部を使用することができるが、葉茎など他部分を含んでいても良い。また、マンゴーは常緑樹ではあるが、成熟した緑色以外の若葉についてもAGEs産生抑制効果が認められたため、いずれの時期の葉についても使用することができる。 Mango (Mangifera indica) is an evergreen tree plant belonging to the genus Mango genus, and fruits have been mainly edible since ancient times. The mango used in the present invention includes all mangoes belonging to the genus Ursiaceae such as Irwin, Alfonso, and Carabao. The mango used in the present application is not limited to a specific site, and has an AGE production inhibitory effect in any of “fruit pulp”, “fruit skin”, “flower”, “seed”, and “leaf”. In view of the priority of use in plant parts, the amount collected, the collection efficiency, the use efficiency, etc., “leaves” are suitable. In the present invention, “leaf” means a so-called leaf portion, and the whole leaf blade or a part thereof can be used, but other portions such as a leaf stem may be included. In addition, although mango is an evergreen tree, AGEs production inhibitory effect was observed on young leaves other than mature green, so it can be used for leaves at any time.
本願発明の抽出溶媒としては、有機溶媒を用いることが好適である（有機溶媒を含んだ混合溶媒も含む。）。有機溶媒は、典型的にはメタノールやエタノールが挙げられるが、アセトン、ジエチルエーテル、酢酸エチル、ヘキサンなど、公知の有機溶媒を広く含む。benzophenone C-glycoside 又は3-Glucosyl-2,4,4',6-tetrahydroxybenzophenoneを抽出する場合にはエタノール又はメタノール、クロロフィルを抽出する場合には酢酸エチルやヘキサンを使用すると効率が良い。抽出温度や時間などは特に限定されないが、数時間以上抽出することが好ましい。また、抽出溶媒がメタノールである場合、抽出温度はたとえば１０〜７０℃、さらには室温（２０℃）〜５０℃前後程度であると好ましい。 As the extraction solvent of the present invention, it is preferable to use an organic solvent (including a mixed solvent containing an organic solvent). The organic solvent typically includes methanol and ethanol, but widely includes known organic solvents such as acetone, diethyl ether, ethyl acetate, and hexane. When extracting benzophenone C-glycoside or 3-Glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone, it is efficient to use ethanol or methanol, and when extracting chlorophyll, ethyl acetate or hexane. The extraction temperature and time are not particularly limited, but it is preferable to extract for several hours or more. When the extraction solvent is methanol, the extraction temperature is preferably about 10 to 70 ° C., and more preferably about room temperature (20 ° C.) to about 50 ° C.
本願発明のマンゴーの抽出物を含有する組成物は、一般的な処理手段に従って製造することができる。例えば、洗浄し、乾燥及び粉砕したマンゴーを使用することができる。この処理済みのマンゴー（いずれの部位も可能である）を有機溶媒で抽出し、液体として、またはその液体を乾燥させて、必要であれば添加物等を加えて、錠剤、散剤、顆粒剤、液剤、カプセル剤などとして、所望の態様にて組成物とすることが可能である。したがって、本願において「組成物」は液体及び個体のいずれの形態も含み、食用及び薬剤用のいずれをも少なくとも含む概念である。 The composition containing the mango extract of the present invention can be produced according to a general processing means. For example, washed, dried and crushed mango can be used. This treated mango (any part is possible) is extracted with an organic solvent, dried as a liquid, or dried, and if necessary, additives, etc. are added to tablets, powders, granules, As a liquid agent, a capsule, etc., it can be set as a composition in a desired aspect. Therefore, in the present application, the “composition” includes both liquid and solid forms, and is a concept including at least both edible and pharmaceutical.
具体的な態様としては、マンゴーの抽出物を、例えばマンゴー葉抽出物を茶飲料などとして、または抽出物を菓子や他の飲料中に含有させて、AGEs産生抑制用の食品（AGEs産生抑制効果のある表示をした特定保険用食品や機能性表示食品、サプリメント、健康食品）として提供することなどが可能である。さらに、抽出物を粉末状・錠型状・カプセル状などとし、AGEs産生抑制剤（医薬・医薬部外品等の薬剤など）として提供することも可能である。なお、抽出物を粉末・錠型・カプセル等や、食品・薬剤等とする工程は、他の素材や添加物の追加工程を含め、本願発明の効果を奏する限り、公知の様々な手段を採用することができるものであり、特に限定はされない。 As a specific aspect, for example, a mango extract, for example, a mango leaf extract as a tea beverage or the like, or an extract contained in a confectionery or other beverage, food for AGEs production inhibition (AGEs production inhibition effect) It is possible to provide foods for specific insurance, functionally labeled foods, supplements, health foods, etc., with a certain label. Further, the extract can be provided in the form of powder, tablets, capsules, etc., and can be provided as an AGE production inhibitor (pharmaceuticals such as drugs and quasi drugs). The process of making the extract into powder, tablets, capsules, foods, drugs, etc., including other steps of adding other ingredients and additives, adopts various known means as long as the effects of the present invention are achieved. There is no particular limitation.
さらに、benzophenone C-glycoside 及び3-glucosyl-2,4,4',6-tetrahydroxybenzophenone （特に3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenone）又はchlorophyllは、いずれもマンゴー（特にマンゴー葉）から取得することが可能である。3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneが特に有効であることから、類似した生理活性（J. Agric. Food Chem. 2011, 59, 11526-11533）を持つベンゾフェノンのC-配糖体にも同様の効果が想定される。また、3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneは、別名として、(3-β-D-glucopyranosyl-2,4,6-trihydroxyphenyl)(4-hydroxyphenyl)methanone、又はiriflophenone 3-C-β-glycosideと呼ばれることがある。 Furthermore, benzophenone C-glycoside and 3-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone (especially 3-C-β-D-glucosyl-2,4,4', 6-tetrahydroxybenzophenone) or chlorophyll are both It can be obtained from mango (especially mango leaves). Since 3-C-β-D-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone is particularly effective, it has similar physiological activity (J. Agric. Food Chem. 2011, 59, 11526-11533) Similar effects are expected for C-glycosides of benzophenone. In addition, 3-C-β-D-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone is also known as (3-β-D-glucopyranosyl-2,4,6-trihydroxyphenyl) (4-hydroxyphenyl) methanone Or iriflophenone 3-C-β-glycoside.
Chlorophyll（クロロフィル）は、葉緑素としてマンゴー葉に含まれる成分であるが、他の植物にも含まれる成分であり、chlorophyll単体としても市販されている。 Chlorophyll (chlorophyll) is a component contained in mango leaves as chlorophyll, but is also a component contained in other plants, and is also commercially available as chlorophyll alone.
本発明によれば、新規かつ安全性の高いAGE産生抑制剤（最終糖化産物産生抑制剤）を提供することが出来る。 According to the present invention, a novel and highly safe AGE production inhibitor (final glycation product production inhibitor) can be provided.
アーウィン種マンゴーの葉（緑色葉）、果皮、種子、果肉および花エキス（抽出物）の取得
実験材料および被検体：実験材料は近畿大学附属農場湯浅農場にて栽培されているマンゴー（採取対象；250本、樹高；2 m、樹齢；18-26年）から採取したものを実験に供した。乾燥した実験材料をそれぞれクラッシュミルを用いて細切後、20倍量のメタノール（花のみ10倍量）に浸漬し、72時間室温抽出した。抽出溶媒は抽出を均一にさせるため24時間に1回数分程度撹拌し、合計72時間抽出を行った。抽出後、No.5 A濾紙（110 mm ADVANTEC）にて濾過し、残渣を除去した。得られたろ液を減圧下で溶媒を留去してエキスを得た。
なお、葉（緑色葉）、果皮、種子、果肉および花のエキス収率は、それぞれ23、23、6、63、23%であった。
Acquisition of Irwin mango leaves (green leaves), pericarp, seeds, pulp and flower extracts (extracts) Experimental materials and specimens: Mangos grown at Yuasa Farm, Kinki University Attached Farm (collection target; Samples from 250 trees, tree height; 2 m, tree age; 18-26 years) were used for the experiment. Each dried experimental material was cut into pieces using a crush mill, immersed in 20 times the amount of methanol (10 times the amount of flowers only), and extracted at room temperature for 72 hours. The extraction solvent was stirred about once every 24 hours to make the extraction uniform, and extracted for a total of 72 hours. After extraction, the mixture was filtered through No. 5 A filter paper (110 mm ADVANTEC) to remove the residue. The obtained filtrate was distilled off the solvent under reduced pressure to obtain an extract.
The extract yields of leaves (green leaves), pericarp, seeds, pulp and flowers were 23, 23, 6, 63 and 23%, respectively.
アーウィン種以外の品種（アルフォンソ種，エドワード種，トミーアトキンス種）のマンゴー葉（緑色葉）エキスの取得
いずれの葉（緑色葉）も近畿大学附属農場湯浅農場にて栽培されているマンゴーから採取したものを実験に供した。エキス収率は乾燥葉50 gに対して、上記アーウィン種葉と同じ抽出方法で取得した。エキス収率は、アルフォンソ種：16%、エドワード種：22%、トミーアトキンス種：20%であった。
Acquisition of mango leaf (green leaf) extract of varieties other than Irwin (Alfonso, Edward, Tommy Atkins) All leaves (green leaves) were collected from mango cultivated at Kinsai University Farm Yuasa Farm Things were subjected to experiments. The extract yield was obtained by the same extraction method as that for the above Irwin seed leaves with respect to 50 g of dried leaves. The extract yield was 16% for Alfonso species, 22% for Edward species, and 20% for Tommy Atkins species.
AGE産生抑制試験
AGE産生抑制試験はShimodaらの方法に準じて行った。すなわち、被検体はDMSOに溶解した（DMSO最終濃度；10%）。2 mlチューブに被検液100μlおよび1/5M のリン酸緩衝液（PBS，pH 7.2）に溶解した10% D-glucoseと1% BSAの混合液900μlをそれぞれ加え，60℃，48時間インキュベートした溶液を黒色96wellプレートに移し，蛍光強度（励起波長 370nm/蛍光波長 460nm）をマルチラベルリーダー（PerkinElmer 2030 ARVO X4，PerkinElmer Life and Analytical Sciences）により測定した。混合液非添加群は混合液の代わりにPBSを用いた。AGE産生抑制率（%）は、以下の式により算出した。陽性対照薬としては、aminoguanidine hydrochloride（アミノグアニジン塩酸塩）を用いた。下記表１は、AGE産生を50%阻害するために必要な濃度（IC50値）である。
AGE production inhibition test
The AGE production inhibition test was performed according to the method of Shimoda et al. That is, the specimen was dissolved in DMSO (DMSO final concentration; 10%). Add 100 μl of the test solution and 900 μl of 10% D-glucose and 1% BSA dissolved in 1/5 M phosphate buffer (PBS, pH 7.2) to each 2 ml tube and incubate at 60 ° C for 48 hours. The solution was transferred to a black 96-well plate, and the fluorescence intensity (excitation wavelength: 370 nm / fluorescence wavelength: 460 nm) was measured with a multi-label reader (PerkinElmer 2030 ARVO X4, PerkinElmer Life and Analytical Sciences). PBS was used instead of the mixed solution in the mixed solution non-added group. The AGE production inhibition rate (%) was calculated by the following formula. As a positive control, aminoguanidine hydrochloride was used. Table 1 below shows the concentration (IC 50 value) required to inhibit AGE production by 50%.
抑制率（%）＝1−（C−D）／（A−B）×100
A：混合液（glucoseとBSA）添加、DMSO添加群（60℃，48時間インキュベート）の測定値、
B：混合液添加、DMSO添加群（4℃，48時間インキュベート）の測定値、
C：混合液添加および被検液添加群の測定値、
D；混合液非添加，被検液添加群の測定値。
Inhibition rate (%) = 1- (C-D) / (A-B) x 100
A: Measured value of mixed solution (glucose and BSA) addition, DMSO addition group (60 ° C, 48 hours incubation),
B: Measured value of mixed solution addition, DMSO addition group (4 ° C, incubation for 48 hours),
C: Measured value of mixed solution addition and test solution addition group,
D: Measured value of the group not added with the mixed solution and the test solution added group.
以上の結果から、種・部位の相違にもかかわらず、マンゴーの果皮、種子、果肉、花、及び葉のいずれについても、顕著なAGE産生抑制作用を示した。これらのうち、種子や果皮も好適だが、採取量・採取効率や商用利用の優先度などを総合的に考慮すると、一般的に食用にされていない葉が最も実用的であると考えられる。そこで、葉について見ると、若い暗赤褐色葉エキス、若い黄色葉エキス、成熟した緑色葉のいずれも顕著なAGE産生抑制作用を示した。マンゴーは常緑果樹植物のため、同時期にこれら３種類の葉を収穫することができる利点がある。ただし、マンゴー葉は萌芽してからおよそ１ヶ月程度で成熟した緑色葉となるために、成熟した葉を使用することが実用的であると想定される。 From the above results, all of mango peels, seeds, flesh, flowers, and leaves showed a remarkable AGE production inhibitory action, regardless of the species and site. Of these, seeds and peels are also suitable, but considering the amount collected, efficiency, and priority for commercial use, leaves that are generally not edible are considered the most practical. Therefore, when looking at the leaves, all of the young dark red-brown leaf extract, young yellow leaf extract, and mature green leaf showed significant AGE production inhibitory action. Since mango is an evergreen fruit tree plant, there is an advantage that these three kinds of leaves can be harvested at the same time. However, since mango leaves become mature green leaves in about one month after budding, it is assumed that it is practical to use mature leaves.
そこで、活性を有し、かつ素材確保が容易な成熟した緑色の葉がAGE産生抑制作用を有する素材として有望であると考え、成熟した緑色の葉からAGE産生抑制作用を指標に有効成分を探索した。その結果、3-C-β-D-glucosyl- 2,4,4',6-tetrahydroxybenzophenoneおよびchlorophyllを有効成分のひとつとして同定した。 Therefore, it is considered that mature green leaves that are active and easy to secure materials are promising as materials that have AGE production inhibitory action, and search for active ingredients from mature green leaves using AGE production inhibitory action as an indicator. did. As a result, 3-C-β-D-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone and chlorophyll were identified as one of the active ingredients.
3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneの単離とクロロフィルの同定、およびAGE産生抑制作用
葉エキス（緑色葉）を粗分画するため、葉エキスをオープンカラムクロマトグラフィーに供した。充填剤にはシリカゲル（Wakogel C-100, Wako）を使用した。試料（葉エキス；5.4g）に対して20倍量の充填剤を用意し、クロロホルムを用いて湿式充填法によりオープンカラムクロマト管（φφの充填剤を用意し）に充填した。次に、メタノール抽出物を10倍量の充填剤に吸着させるため、少量のメタノールに溶解し吸着用充填剤と混合した。メタノールを除くため、ロータリーエバポレーターを用い減圧留去した。乾燥後の充填剤をクロロホルムに懸濁しカラムに供した。溶出溶媒はオープンカラムクロマト管に充填した充填剤の体積に対して5倍量の溶媒を用い、クロロホルム：メタノール＝10：0 , 30：1, 10：1, 5：1, 0：10の順に溶出を行った。
Isolation of 3-C-β-D-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone, identification of chlorophyll, and AGE production-inhibiting leaf extract (green leaf) for rough fractionation Open leaf extract It used for column chromatography. Silica gel (Wakogel C-100, Wako) was used as the filler. A 20-fold amount of filler was prepared with respect to the sample (leaf extract; 5.4 g), and was packed in an open column chromatograph tube (prepared with φφ filler) using chloroform with a wet packing method. Next, the methanol extract was dissolved in a small amount of methanol and mixed with the adsorption filler in order to adsorb the methanol extract to a 10-fold amount of the filler. In order to remove methanol, it was distilled off under reduced pressure using a rotary evaporator. The dried packing material was suspended in chloroform and applied to a column. The elution solvent is 5 times the volume of the packing material packed in the open column chromatography tube, and chloroform: methanol = 10: 0, 30: 1, 10: 1, 5: 1, 0:10 in this order. Elution was performed.
はじめに溶出されたクロロホルム：メタノール＝10：0溶出画分（フラクションＩ）にAGE産生抑制作用が認められた。フラクションＩは深緑色を呈していたことから、葉の緑色素成分が含有することが推察された。葉の緑色素成分として知られるクロロフィルをPorraらの方法（1989）により測定した結果、葉エキス中に4.34 mg/g含有することが明らかとなったことから、下記の市販試薬（東京化成工業株式会社にて購入）を用いて評価したところ、クロロフィルにAGE産生抑制作用が認められた（IC50；41μg/ml）。このことから、葉エキスの有効成分のひとつであることが明らかとなった。 In the first eluted chloroform: methanol = 10: 0 eluted fraction (fraction I), an AGE production inhibitory action was observed. Since fraction I was dark green, it was speculated that the green pigment component of the leaf contained it. As a result of measuring chlorophyll, which is known as a green pigment component of leaves, by Porra et al.'S method (1989), it was revealed that it contained 4.34 mg / g in leaf extract. Chlorophyll was found to inhibit AGE production (IC 50 ; 41 μg / ml). From this, it became clear that it is one of the active ingredients of a leaf extract.
クロロホルム：メタノール＝30：1, 10：1, 5：1, 0：10溶出画分（フラクションII）にもAGE産生抑制作用が認められたことから、本画分を単離するため分取HPLCに供した。分取HPLC分析にはシステムにLC-10Aシステム、分取カラムには逆相系カラム(Inertsil ODS-3 JET Column 20×50mm, GL Sciences)を用い、カラムオーブンを40℃に設定し分析した。溶媒は15 %アセトニトリル水溶液を用い、流量は9.0ml/minで溶出した。検出はSPD-10A(SHIMADZU)を用いてUV 254nmの吸光度により行った。この条件において、3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneは保持時間17分に溶出した。
3-C-β-D-Glucosyl-2,4,4',6-tetrahydroxybenzophenoneの同定は、Severiらの報告（2009）にある1Hおよび13C-NMR並びにマススペクトルに一致したことにより支持された。また、3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenone含量は、205.9±7.6mg/gであった。
Chloroform: methanol = 30: 1, 10: 1, 5: 1, 0:10 Since the AGE production inhibitory action was also observed in the fraction eluted (fraction II), preparative HPLC was used to isolate this fraction. It was used for. For preparative HPLC analysis, an LC-10A system was used as the system, a reverse phase column (Inertsil ODS-3 JET Column 20 × 50 mm, GL Sciences) was used as the preparative column, and the column oven was set at 40 ° C. for analysis. The solvent was a 15% acetonitrile aqueous solution, and the flow rate was eluted at 9.0 ml / min. Detection was carried out by absorbance at UV 254 nm using SPD-10A (SHIMADZU). Under these conditions, 3-C-β-D-glucosyl-2,4,4 ′, 6-tetrahydroxybenzophenone was eluted at a retention time of 17 minutes.
The identification of 3-C-β-D-Glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone was supported by agreement with 1 H and 13 C-NMR and mass spectra in the report of Severi et al. (2009). It was. In addition, the content of 3-C-β-D-glucosyl-2,4,4 ′, 6-tetrahydroxybenzophenone was 205.9 ± 7.6 mg / g.
この3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneを上記と同様の方法で試験したところ、顕著なAGE産生抑制作用が認められた（表２：IC50；85 μg/ml）。3-C-β-D-glucosyl-2,4,4',6-tetrahydroxybenzophenoneに強い活性が認められることから、benzophenone C-glycosideのβ-グルコシド及び3-Glucosyl-2,4,4',6-tetrahydroxybenzophenoneのC-グルコシドだけでなく、類似する構成及び活性を有する3-glucosyl-2,4,4',6-tetrahydroxybenzophenoneのα-グルコシド及びO-グルコシド、benzophenone C-glycosideのα-グルコシドにも十分な活性があると想定される。また、植物中の採取可能収量と収集率、効果を考慮すれば、市場においてAGE産生抑制用組成物の十分な供給を行うことが可能である。 When this 3-C-β-D-glucosyl-2,4,4 ′, 6-tetrahydroxybenzophenone was tested in the same manner as above, a remarkable inhibitory effect on AGE production was observed (Table 2: IC 50 ; 85 μg / ml). Since 3-C-β-D-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone has strong activity, β-glucoside of benzophenone C-glycoside and 3-Glucosyl-2,4,4', 6 Not only C-glucoside of -tetrahydroxybenzophenone but also α-glucoside and O-glucoside of 3-glucosyl-2,4,4 ', 6-tetrahydroxybenzophenone and benzophenone C-glycoside α-glucoside having similar structure and activity It is assumed that there is sufficient activity. In addition, considering the yield, collection rate, and effects that can be collected in plants, it is possible to sufficiently supply the AGE production-suppressing composition on the market.
以上により、自然由来で新規かつ安全性が高く、実用性の高いAGE産生抑制剤を提供することが可能である。
As described above, it is possible to provide a natural, novel, highly safe, and practical AGE production inhibitor.
Claims (14)
The food for inhibiting AGEs production according to claim 12 or 13, wherein the effect of inhibiting AGEs production is displayed.
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