JP2015024982A - Advanced glycation end products formation inhibitor - Google Patents
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本発明は、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物から選ばれる１種以上の植物抽出物を有効成分として含む終末糖化産物生成抑制剤に関するものである。 The present invention relates to a terminal glycation product production inhibitor containing, as an active ingredient, one or more plant extracts selected from a lemon myrtle extract, a cassava extract and a cassava extract.
アミノ酸と還元糖の混合物を加熱すると褐変する現象は、一般にメイラード反応と呼ばれ、食品分野では、食品の加熱処理や貯蔵中に生じる現象として知られている。メイラード反応は、生体内においても発生しており、１９６８年にはグリコシルヘモグロビン（ＨｂＡ１ｃ）が生体内で同定されたことにより、糖尿病や老化の進行に伴い蛋白質の糖化反応が進行することが明らかにされた。そして、近年では、蛋白質の糖化反応における終末糖化産物（Ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ ｐｒｏｄｕｃｔｓ、以下「ＡＧＥｓ」とも称する）が糖尿病合併症や動脈硬化といった生活習慣病の発症や老化の進行に関与することが報告されている（非特許文献１〜３）。 The phenomenon of browning when a mixture of amino acid and reducing sugar is heated is generally called the Maillard reaction, and is known in the food field as a phenomenon that occurs during heat treatment and storage of food. The Maillard reaction also occurs in vivo, and it was clear that glycosylation hemoglobin (HbA1c) was identified in vivo in 1968, leading to the progress of protein glycation with the progress of diabetes and aging. It was done. In recent years, advanced glycation end products (hereinafter also referred to as “AGEs”) in protein glycation reactions have been reported to be involved in the onset of lifestyle-related diseases such as diabetic complications and arteriosclerosis and the progression of aging. (Non-Patent Documents 1 to 3).
生体内における蛋白質糖化反応の詳細は明らかとなっていないが、蛋白質に存在するアミノ基と還元糖に存在するアルデヒド基が反応し、シッフ塩基を形成した後、安定なアマドリ化合物を経由し、さらに長期の反応を経て、ＡＧＥｓに移行すると考えられている。 The details of the protein saccharification reaction in vivo are not clear, but the amino group present in the protein reacts with the aldehyde group present in the reducing sugar to form a Schiff base, which then passes through a stable Amadori compound. It is believed that the transition to AGEs will occur after a long-term reaction.
ＡＧＥｓは特定の物質を指すものではなく、その全容は未だ解明されていないが、蛍光性や架橋構造の有無によって、ペントシジン、ピロピリジン、ピラリン等、様々な物質の存在が確認されている。ＡＧＥｓの物理化学的な特徴としては、黄褐色で蛍光性（主に、Ｅｘ:３７０ｎｍ、Ｅｍ:４４０ｎｍ）を持ち、タンパク質間に架橋を形成することが挙げられる。しかし、ピラリンのように蛍光性を示さず、架橋構造を有しないＡＧＥｓも確認されている。 AGEs do not refer to specific substances, and the whole of them has not yet been elucidated, but the presence of various substances such as pentosidine, pyropyridine, and pyralin has been confirmed depending on the presence of fluorescence and cross-linking structure. The physicochemical characteristics of AGEs include yellowish brown and fluorescence (mainly Ex: 370 nm, Em: 440 nm) and the formation of crosslinks between proteins. However, AGEs that do not show fluorescence like pyralin and do not have a crosslinked structure have also been confirmed.
これらＡＧＥｓの体内蓄積は、腎糸球体組織硬化症や腎動脈硬化症などの糖尿病合併症だけでなく、アルツハイマー病などの神経変性疾患、皮膚の弾力性の低下や黄色化といった皮膚老化、骨の老化、眼球の老化、アルブミン蛋白の老化に深く関与することが報告されている（非特許文献４および５） The accumulation of these AGEs is not only due to diabetic complications such as glomerular tissue sclerosis and renal arteriosclerosis, but also neurodegenerative diseases such as Alzheimer's disease, skin aging such as reduced skin elasticity and yellowing, It is reported to be deeply involved in aging, ocular aging, and albumin protein aging (Non-patent Documents 4 and 5).
したがって、生体内の糖化反応を阻止することは、糖尿病合併症、動脈硬化、骨粗鬆症、変形性関節症などの疾患の発症・進展を防ぐ効果が期待される。また、糖化反応の阻止は、皮膚の弾力性の低下や黄色化等の予防・改善にも効果があるものと期待される。このため、さまざまな糖化反応阻害剤やＡＧＥｓ生成阻害剤が開発・検討されてきている。 Therefore, blocking the glycation reaction in the living body is expected to have an effect of preventing the onset and progression of diseases such as diabetic complications, arteriosclerosis, osteoporosis, and osteoarthritis. In addition, inhibition of the saccharification reaction is expected to be effective in preventing and improving skin elasticity and yellowing. For this reason, various saccharification reaction inhibitors and AGEs production inhibitors have been developed and studied.
特許文献１には、アミノグアニジン等の化合物を含むメイラード反応阻害剤が開示されている。アミノグアニジンは当該技術分野における開発において最も研究されている、抗糖化作用を示す物質であるが、肝障害等の副作用を有することが確認されており、実用化には至っていない。 Patent Document 1 discloses a Maillard reaction inhibitor containing a compound such as aminoguanidine. Aminoguanidine is a substance exhibiting an anti-glycation effect that has been most studied in development in the technical field, but has been confirmed to have side effects such as liver damage and has not yet been put into practical use.
その他にも、特許文献２に記載のガンビールノキやシラカバ等の植物の抽出物によるメイラード反応阻害剤などが知られているが、有効性や安全性の点に課題を残すものであった。 In addition, the Maillard reaction inhibitor by the extract of plants, such as a ganbei tree and a birch described in patent document 2, etc. are known, but problems remain in terms of effectiveness and safety.
そこで、種々の疾患の発症や悪化ならびに皮膚老化等に関与するＡＧＥｓの生成を抑制することができ、かつ、重篤な副作用を有さない安全性の高い薬剤が求められている。 Therefore, there is a demand for highly safe drugs that can suppress the generation of AGEs involved in the onset and worsening of various diseases and skin aging, and that have no serious side effects.
本発明は、副作用の少ないＡＧＥｓ生成抑制剤を提供することを目的とする。本発明はまた、生活習慣病、特に糖尿病もしくは糖尿病合併症の予防および／または治療に有用であり、且つ副作用の少ない医薬組成物、ならびに皮膚老化、特に皮膚の弾力性低下や黄色化の予防および／または改善に有用であり、且つ副作用の少ない皮膚外用剤を提供することを目的とする。 An object of this invention is to provide the AGEs production inhibitor with few side effects. The present invention is also useful for the prevention and / or treatment of lifestyle-related diseases, particularly diabetes or diabetic complications, and has few side effects, as well as prevention of skin aging, particularly skin elasticity loss and yellowing. An object is to provide an external preparation for skin that is useful for improvement and / or has few side effects.
本発明者らは、抗糖化作用に優れ、且つ副作用が少ない物質について、天然物であり、食経験のある植物からのスクリーニングを試みた結果、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物が強いＡＧＥｓ生成抑制効果を発揮することを見出し、本発明を完成させた。 As a result of the screening of plants that are natural products and have dietary experiences with respect to substances that have excellent anti-glycation effects and few side effects, the present inventors have a strong lemon myrtle extract, hamboufu extract, and cassava extract. The present invention has been completed by finding out that it exhibits the effect of suppressing the generation of AGEs.
すなわち本発明は、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を有効成分として含有する終末糖化産物生成抑制剤（以下、「本発明のＡＧＥｓ生成抑制剤」とも称する）を提供する。 That is, the present invention provides a terminal glycation product production inhibitor (hereinafter referred to as “the present invention”) containing, as an active ingredient, one or more anti-glycated plant extracts selected from the group consisting of a lemon myrtle extract, a hamboufu extract and a cassava extract. AGEs production inhibitor ").
本発明はまた、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を有効成分として含有する、糖尿病もしくは糖尿病合併症の予防および／または治療のための医薬組成物（以下、「本発明の糖尿病予防／治療用医薬組成物」とも称する）を提供する。 The present invention also includes one or more anti-glycated plant extracts selected from the group consisting of a lemon myrtle extract, a sunflower extract and a cassava extract as an active ingredient, for prevention and / or treatment of diabetes or diabetic complications. (Hereinafter also referred to as “the pharmaceutical composition for the prevention / treatment of diabetes of the present invention”).
本発明はさらに、非ヒト動物における糖尿病もしくは糖尿病合併症の予防および／または治療のための、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を有効成分として含む非ヒト動物用医薬組成物、該医薬組成物または本発明のＡＧＥｓ生成抑制剤を非ヒト動物に投与することを含む、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療する方法、該抗糖化植物抽出物を飲食品に配合または添加することを特徴とする機能性飲食品の製造方法、ならびにサプリメントの製造のための該抗糖化植物抽出物の使用を提供する。 The present invention further provides one or more anti-glycated plant extracts selected from the group consisting of lemon myrtle extract, hamboufu extract and cassava extract for the prevention and / or treatment of diabetes or diabetic complications in non-human animals. And / or preventing and / or preventing diabetes or diabetic complications in a non-human animal, comprising administering to the non-human animal the pharmaceutical composition for a non-human animal comprising the active ingredient as an active ingredient, the pharmaceutical composition or the AGEs production inhibitor of the present invention. Provided are a method for treating, a method for producing a functional food or drink characterized by adding or adding the anti-glycated plant extract to a food or drink, and use of the anti-glycated plant extract for the production of a supplement.
本発明はさらに、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を有効成分として含有する、皮膚老化の予防および／または改善のための皮膚外用剤（以下、「本発明の皮膚老化予防／改善用皮膚外用剤」とも称する）を提供する。 The present invention further includes a skin for preventing and / or improving skin aging, which contains, as an active ingredient, one or more anti-glycated plant extracts selected from the group consisting of a lemon myrtle extract, a sunflower extract and a cassava extract. An external preparation (hereinafter also referred to as “skin external preparation for skin aging prevention / improvement of the present invention”) is provided.
ＡＧＥｓは、種々の生活習慣病や皮膚老化との関連が示唆されている物質であり、特に、糖尿病およびその合併症の発症および／または進行、ならびに皮膚の弾力性低下および皮膚の黄色化の発生および／または進行に深く関与するものである。したがって、本発明者らによりＡＧＥｓの生成を抑制する作用が見出されたレモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物は、糖尿病もしくは糖尿病合併症の予防および／または治療のための医薬組成物、または皮膚の弾力性低下もしくは黄色化等の皮膚老化の予防および／または改善のための皮膚外用剤における、副作用の少ない有効成分として有用である。 AGEs are substances that have been suggested to be associated with various lifestyle-related diseases and skin aging, and in particular, the onset and / or progression of diabetes and its complications, as well as the occurrence of reduced skin elasticity and skin yellowing. And / or are deeply involved in progression. Therefore, the lemon myrtle extract, hamboufu extract and cassava extract, which have been found to have an action of suppressing the production of AGEs by the present inventors, are pharmaceutical compositions for the prevention and / or treatment of diabetes or diabetic complications, Or it is useful as an active ingredient with few side effects in the skin external preparation for prevention and / or improvement of skin aging, such as skin elasticity fall or yellowing.
本明細書において「抗糖化植物抽出物」とは、アミノ酸、ペプチドもしくはタンパク質の糖化反応（メイラード反応）を阻害する作用を有する植物抽出物をいう。 In the present specification, the “anti-glycated plant extract” refers to a plant extract having an action of inhibiting a saccharification reaction (Maillard reaction) of amino acids, peptides or proteins.
本発明のＡＧＥｓ生成抑制剤は、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を有効成分として含有するものである。 The AGEs production inhibitor of the present invention contains, as an active ingredient, one or more anti-glycated plant extracts selected from the group consisting of a lemon myrtle extract, a sunflower extract and a cassava extract.
レモンマートル（Ｂａｃｋｈｏｕｓｉａ ｃｉｔｒｉｏｄｏｒａ）は、オーストラリア原住民であるアボリジニが料理や薬草に使用している他、リラックス効果を有することが知られており、世界中でハーブティーの茶葉として用いられている。ハマボウフウ（Ｇｌｅｈｎｉａ ｌｉｔｔｏｒａｌｉｓ）は、おとその材料として用いられている他、天ぷらや酢の物として食されている。キャッサバ（Ｍａｎｉｈｏｔ ｅｓｃｕｌｅｎｔａ）は、その塊根部がデンプンの原料として用いられているほか、ビタミン類を豊富に含む葉部が食材として利用されている。 Lemon Myrtle (Backhouse citriodora) is known to have a relaxing effect, as well as being used for cooking and medicinal herbs by Australian aboriginals, and is used throughout the world as a tea leaf for herbal tea. In addition to being used as a material for hamabofu (Glehnia littoralis), it is eaten as a product of tempura and vinegar. In cassava (Manihot esculenta), the tuberous root portion is used as a raw material for starch, and the leaf portion rich in vitamins is used as a food material.
抗糖化植物抽出物の原料として用いるレモンマートル、ハマボウフウおよびキャッサバの植物体は、開花期のものであっても栄養生長期のものであってもよく、生の状態であっても乾燥させたものであってもよい。また、抽出効率を高めるため、植物体を適宜細断ないし粉砕等したもの、例えば粉末状のものを抽出原料として用いてもよい。原料として用いる植物体の部位に関しては、花、葉、茎、根、またはこれらを含む全草のいずれを抽出原料として用いてもよいが、特に葉部からの抽出物に強い効果が認められるため、かかる部位を用いることが好ましい。 Lemon myrtle, hamboufu and cassava plants used as raw materials for anti-glycated plant extracts may be in the flowering stage or in the vegetative long-term, and dried in the raw state. There may be. Further, in order to increase the extraction efficiency, a plant body that has been appropriately shredded or pulverized, for example, a powdered one, may be used as an extraction raw material. As for the plant part used as a raw material, any of flowers, leaves, stems, roots, or whole plants containing these may be used as an extraction raw material. It is preferable to use such a site.
以下、抗糖化植物抽出物を得るための抽出方法および条件について記載する。レモンマートル、ハマボウフウおよびキャッサバのいずれを原料として用いる場合であっても、本明細書に記載の抽出方法および条件を同等に適用することができる。 Hereinafter, the extraction method and conditions for obtaining the anti-glycated plant extract will be described. The extraction method and conditions described in the present specification can be equally applied to any of lemon lemon, hamboufu and cassava as raw materials.
本発明のＡＧＥｓ生成抑制剤、糖尿病予防／治療用医薬組成物および皮膚老化予防／改善用皮膚外用剤の有効成分である抗糖化植物抽出物は、一般的な植物成分抽出方法を用いて得ることができる。かかる抽出方法としては、例えば、熱水抽出法、煮沸抽出法、浸漬抽出法、振とう抽出法、ソックスレー抽出法、水蒸気蒸留法などが挙げられる。 The anti-glycated plant extract which is an active ingredient of the AGEs production inhibitor, the pharmaceutical composition for preventing / treating diabetes and the external preparation for skin aging prevention / amelioration of the present invention is obtained by using a general plant component extraction method. Can do. Examples of the extraction method include a hot water extraction method, a boiling extraction method, a soaking extraction method, a shaking extraction method, a Soxhlet extraction method, and a steam distillation method.
抽出溶媒としては、水、メタノール、エタノール等の低級アルコール類、プロピレングリコール、１，３‐ブチレングリコールなどの多価アルコール類、アセトン等のケトン類、ジエチルエーテル、アセトニトリル、酢酸エチル等のエステル類等を用いることができ、これらの中から抽出原料とする植物の種類、後に行う加工処理、抽出物の使用目的などを考慮した上で選択すれば良い。抽出物を食品用途等へ使用するため有機溶媒の含有が好ましくない場合においては、水のみを使用すれば良く、抽出後に除去しやすいエタノールを単独で、又は水との任意の割合の混液として使用しても良い。一つの態様において、好ましい抽出溶媒は、水またはエタノールである。 Extraction solvents include water, lower alcohols such as methanol and ethanol, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, ketones such as acetone, esters such as diethyl ether, acetonitrile, and ethyl acetate. These may be selected in consideration of the type of plant used as the raw material for extraction, the subsequent processing, the purpose of use of the extract, and the like. If the organic solvent is not preferred because the extract is used for food applications, etc., only water should be used. Ethanol that can be easily removed after extraction is used alone or as a mixture with water in any proportion. You may do it. In one embodiment, the preferred extraction solvent is water or ethanol.
抽出溶媒の使用量は、重量比で、１：２〜１：３０（植物原料：抽出溶媒）が好ましく、１：３〜１：２５がより好ましく、１：４〜１：２０がさらに好ましい。 The use amount of the extraction solvent is preferably from 1: 2 to 1:30 (plant raw material: extraction solvent), more preferably from 1: 3 to 1:25, and even more preferably from 1: 4 to 1:20 by weight.
抽出温度は各溶媒によって適宜決定すれば良いが、水抽出の場合は４〜１３０℃が好ましく、２５〜１１０℃がより好ましく、４０〜１００℃がさらに好ましい。抽出温度が４℃未満の場合、活性成分が抽出されにくい傾向があり、１３０℃を越える場合、活性成分が分解されやすい傾向がある。エタノール抽出の場合、抽出温度は４〜１００℃が好ましく、１０〜８０℃がより好ましく、２０〜６０℃がさらに好ましい。抽出温度が４℃未満の場合、活性成分が抽出されにくい傾向があり、１００℃を越える場合、活性成分が分解されやすい傾向がある。 The extraction temperature may be appropriately determined depending on each solvent, but in the case of water extraction, it is preferably 4 to 130 ° C, more preferably 25 to 110 ° C, and further preferably 40 to 100 ° C. When the extraction temperature is less than 4 ° C, the active ingredient tends to be difficult to extract, and when it exceeds 130 ° C, the active ingredient tends to be easily decomposed. In the case of ethanol extraction, the extraction temperature is preferably 4 to 100 ° C, more preferably 10 to 80 ° C, further preferably 20 to 60 ° C. When the extraction temperature is less than 4 ° C., the active ingredient tends to be difficult to extract, and when it exceeds 100 ° C., the active ingredient tends to be easily decomposed.
また、抽出時間は、水抽出の場合は１分〜１５日間が好ましく、３分〜１日間がより好ましく、５分〜１時間がさらに好ましい。抽出時間が１分間未満の場合、活性成分が抽出されにくい傾向があり、１５日間を越える場合、活性成分が分解されやすい傾向がある。エタノール抽出の場合は１時間〜１５日間が好ましく、１２時間〜１０日間がより好ましく、１〜８日間がさらに好ましい。抽出時間が１時間未満の場合、活性成分が抽出されにくい傾向があり、１５日間を越える場合、活性成分が分解されやすい傾向がある。 In the case of water extraction, the extraction time is preferably 1 minute to 15 days, more preferably 3 minutes to 1 day, and further preferably 5 minutes to 1 hour. When the extraction time is less than 1 minute, the active ingredient tends to be difficult to extract, and when it exceeds 15 days, the active ingredient tends to be easily decomposed. In the case of ethanol extraction, 1 hour to 15 days is preferable, 12 hours to 10 days is more preferable, and 1 to 8 days is more preferable. When the extraction time is less than 1 hour, the active ingredient tends to be difficult to extract, and when it exceeds 15 days, the active ingredient tends to be easily decomposed.
本明細書において、「抽出物」の用語には、溶媒を用いる各種抽出方法によって得られる抽出液、該抽出液を濃縮した濃縮液、該抽出液を適当な溶媒で希釈した希釈液、該抽出液またはその濃縮液もしくは希釈液から加熱乾燥、減圧乾燥、凍結乾燥などの種々の方法によって溶媒を除くことにより得られる固形の抽出物（例えば粉末状や粒状のもの）、ならびに固形化剤やゲル化剤の添加等によって得られる固形もしくは半固形の抽出物が含まれるものとする。一つの好ましい態様において、本発明のＡＧＥｓ生成抑制剤、糖尿病予防／治療用医薬組成物および皮膚老化予防／改善用皮膚外用剤の有効成分として用いる抗糖化植物抽出物は、抽出液から溶媒を除去した固形抽出物の形態である。 In the present specification, the term “extract” includes an extract obtained by various extraction methods using a solvent, a concentrated solution obtained by concentrating the extract, a diluted solution obtained by diluting the extract with an appropriate solvent, and the extraction. Solid extract (for example, powdered or granular) obtained by removing the solvent from the liquid or its concentrated or diluted liquid by various methods such as heat drying, vacuum drying, freeze drying, etc., and solidifying agent or gel A solid or semi-solid extract obtained by addition of an agent is included. In one preferred embodiment, the anti-glycated plant extract used as an active ingredient of the AGEs production inhibitor of the present invention, the pharmaceutical composition for preventing / treating diabetes and the external preparation for skin aging prevention / amelioration removes the solvent from the extract. In the form of a solid extract.
抗糖化植物抽出物の抽出方法の例としては、（１）原料の植物体１重量部に対して２〜３０重量部、好ましくは３〜２５重量部の水を加え、４０〜９０℃で１０〜１２０分間静置、あるいは１００℃で５〜１５分間煮沸する方法や、（２）原料の植物体１重量部に対して２〜３０重量部、好ましくは３〜２５重量部のエタノールを加え、１０〜８０℃で１２時間〜１０日間静置、好ましくは２０〜６０℃で１〜８日間静置する方法等が挙げられる。 As an example of the extraction method of an anti-glycation plant extract, (1) 2 to 30 parts by weight, preferably 3 to 25 parts by weight of water is added to 1 part by weight of the raw material plant and 10 to 40 to 90 ° C. The method of boiling for 120 minutes or boiling at 100 ° C. for 5 to 15 minutes, (2) 2 to 30 parts by weight, preferably 3 to 25 parts by weight of ethanol is added to 1 part by weight of the plant body, The method of leaving still at 10-80 degreeC for 12 hours-10 days, Preferably leaving still at 20-60 degreeC for 1-8 days etc. are mentioned.
本発明において用い得る抗糖化植物抽出物の具体例としては、レモンマートル水抽出物、レモンマートルエタノール抽出物、ハマボウフウ水抽出物、ハマボウフウエタノール抽出物、キャッサバ水抽出物およびキャッサバエタノール抽出物が挙げられる。 Specific examples of the anti-glycated plant extract that can be used in the present invention include a lemon myrtle water extract, a lemon myrtle ethanol extract, a hamabofu water extract, a hamabofu ethanol extract, a cassava water extract, and a cassava ethanol extract.
本発明のＡＧＥｓ生成抑制剤は、抗糖化植物抽出物によるＡＧＥｓ生成抑制効果が妨げられない限り、賦形剤等を含むものであってもよい。 The AGEs production inhibitor of the present invention may contain excipients and the like as long as the AGEs production inhibitory effect of the anti-glycated plant extract is not hindered.
本発明のＡＧＥｓ生成抑制剤中の抗糖化植物抽出物の割合は特に限定されない。例えば、固形抽出物を用いる場合、本発明のＡＧＥｓ生成抑制剤は、抗糖化植物抽出物を４０重量％以上、４５重量％以上、５０重量％以上、６０重量％以上、７０重量％以上、８０重量％以上、８５重量％以上、９０重量％以上、９５重量％以上または９８重量％以上含むものであり得る。あるいは、本発明のＡＧＥｓ生成抑制剤は、抗糖化植物抽出物のみからなるものであってもよい。 The proportion of the anti-glycated plant extract in the AGEs production inhibitor of the present invention is not particularly limited. For example, when a solid extract is used, the AGEs production inhibitor of the present invention contains 40% by weight, 45% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight of anti-glycated plant extract. It may contain at least 85%, 85%, 90%, 95% or 98% by weight. Or the AGEs production | generation inhibitor of this invention may consist only of an anti-glycation plant extract.
本発明のＡＧＥｓ生成抑制剤は、ペントシジン、クロスリン、ピロピリジン、グリオキサール由来リジンダイマー（ＧＯＬＤ）、メチルグリオキサール由来リジンダイマー（ＭＯＬＤ）等の蛍光性ＡＧＥｓおよびピラリン、カルボキシメチルリジン（ＣＭＬ）、３−デオキシグルコソン由来リジンダイマー（ＤＯＬＤ）、イミダゾロン化合物等の非蛍光性ＡＧＥｓを含む種々のＡＧＥｓの生成を抑制することができる。一つの態様において、本発明のＡＧＥｓ生成抑制剤は、蛍光性ＡＧＥｓであるペントシジンの生成抑制のために用いられるものである。別の態様において、本発明のＡＧＥｓ生成抑制剤は、非蛍光性ＡＧＥｓであるカルボキシメチルリジン（ＣＭＬ）の生成抑制のために用いられるものである。 The AGEs production inhibitor of the present invention includes fluorescent AGEs such as pentosidine, croslin, pyropyridine, glyoxal-derived lysine dimer (GOLD), methylglyoxal-derived lysine dimer (MOLD), pyralin, carboxymethyllysine (CML), 3-deoxyglucose. Generation of various AGEs including non-fluorescent AGEs such as son-derived lysine dimer (DOLD) and imidazolone compounds can be suppressed. In one embodiment, the AGEs production inhibitor of the present invention is used for inhibiting the production of pentosidine, which is a fluorescent AGE. In another embodiment, the AGEs production inhibitor of the present invention is used for inhibiting the production of carboxymethyllysine (CML), which is a non-fluorescent AGE.
本発明の糖尿病予防／治療用医薬組成物および皮膚老化予防／改善用皮膚外用剤は、本発明のＡＧＥｓ生成抑制剤と同様、有効成分としてレモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を含有するものである。 The pharmaceutical composition for preventing / treating diabetes and the skin external preparation for preventing / ameliorating skin aging according to the present invention are the group consisting of lemon myrtle extract, hamboufu extract and cassava extract as active ingredients, like the AGEs production inhibitor of the present invention. It contains one or more anti-glycated plant extracts selected from the above.
本発明の糖尿病予防／治療用医薬組成物中に有効成分として含有させる抗糖化植物抽出物の合計量は、目的とする剤形等に応じて適宜設定することができるが、固形抽出物を用いる場合、通常、１〜９８重量％程度であればよく、２〜９５重量％程度であることが好ましく、３〜９０重量％程度であることがより好ましい。 The total amount of the anti-glycated plant extract to be contained as an active ingredient in the pharmaceutical composition for prevention / treatment of diabetes of the present invention can be appropriately set according to the target dosage form, etc., but a solid extract is used. In this case, it may be about 1 to 98% by weight, preferably about 2 to 95% by weight, and more preferably about 3 to 90% by weight.
本明細書において、糖尿病もしくは糖尿病合併症の「治療」には、既に糖尿病もしくは糖尿病合併症に罹患している患者における症状の進行を阻止することも含まれる。 As used herein, “treatment” of diabetes or diabetic complications includes preventing the progression of symptoms in a patient already suffering from diabetes or diabetic complications.
本発明の糖尿病予防／治療用医薬組成物の投与方法は、経口投与または非経口投与のいずれであってもよい。また、投与時期は特に限定されず、食前、食中、食後、食間のいずれであってもよいが、例えば、糖類含有量が５ｇ以上である食事、またはアミノ酸および／または蛋白質含有量が２０ｇ以上である食事の食前、食中、食後および食間のいずれかに投与するのが好ましく、糖類ならびにアミノ酸および／または蛋白質を前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに投与するのがより好ましい。前記アミノ酸としては、リジン、アルギニン等が挙げられ、その中でもＡＧＥｓ化し易いリジンおよび／またはアルギニンを前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに投与するのが好ましい。また、前記蛋白質としては、卵白アルブミン、乳アルブミン、グリアジン、乳カゼイン、大豆カゼイン等が挙げられ、その中でもＡＧＥｓ化し易い卵白アルブミン、乳アルブミンおよび乳カゼインから選ばれる１種以上を前記含有量以上含有する食事の食前、食中、食後および食間のいずれかに投与するのが好ましい。ここで、一般的に「食事」とは、生存に必要な栄養分をとるために毎日の習慣として物を食べること（飲食行為）あるいはその飲食物を意味するが、本明細書において用いる「食事」の用語は、一回の飲食行為において摂取する一まとまりの飲食物を意味するものとする。 The method for administering the pharmaceutical composition for preventing / treating diabetes of the present invention may be either oral administration or parenteral administration. The administration time is not particularly limited and may be any of before, during, after, and between meals. For example, a meal having a saccharide content of 5 g or more, or an amino acid and / or protein content of 20 g or more. It is preferably administered before meal, during meal, after meal and between meals, and before meal, during meal, after meal and between meals of meal containing sugar and amino acid and / or protein above the above content. More preferably it is administered. Examples of the amino acid include lysine, arginine, and the like. Among them, lysine and / or arginine, which is easily converted to AGEs, is preferably administered before, during, after, and between meals. Examples of the protein include ovalbumin, milk albumin, gliadin, milk casein, soybean casein, and the like. Among them, one or more selected from ovalbumin, milk albumin, and milk casein which are easily converted to AGEs are contained in the above content. It is preferably administered before, during, after and between meals. Here, “meal” generally means eating (drinking action) or eating or drinking food as a daily habit for taking the nutrients necessary for survival, but “meal” as used herein. The term "" means a set of food and drink taken in a single eating and drinking act.
本明細書において用いる場合、「糖類」とは、単糖および／または二糖を意味するものとする。これに対し「糖質」とは、炭水化物のうち食物繊維以外のものをいい、糖類の他に、オリゴ糖、多糖、糖アルコール等が含まれる。 As used herein, “saccharides” shall mean monosaccharides and / or disaccharides. On the other hand, “sugar” refers to carbohydrates other than dietary fiber, and includes oligosaccharides, polysaccharides, sugar alcohols and the like in addition to sugars.
本発明の糖尿病予防／治療用医薬組成物の投与量は、糖尿病またはその合併症の程度、その他の疾病の程度、年齢、性別等の条件に応じて適宜選択されるが、ＡＧＥｓの生成を抑制することができる量（以下、「ＡＧＥｓ生成抑制有効量」とも称する）を投与すればよい。ＡＧＥｓ生成抑制有効量は、当業者に周知の方法（各種の非臨床および／または臨床試験を含む）を用いて適宜決定することができる。 The dosage of the pharmaceutical composition for preventing / treating diabetes according to the present invention is appropriately selected according to conditions such as the degree of diabetes or its complications, the degree of other diseases, age, and sex, but suppresses the generation of AGEs. An amount that can be administered (hereinafter also referred to as “AGEs suppression effective amount”) may be administered. An effective amount for inhibiting the generation of AGEs can be appropriately determined using methods well known to those skilled in the art (including various non-clinical and / or clinical trials).
本発明の糖尿病予防／治療用医薬組成物は、ＡＧＥｓ生成抑制有効量の抗糖化植物抽出物を一度に投与するものであっても良く、間隔を置いて複数回に分けて投与するものであっても良い。複数回に分けて投与する場合は、一日に投与される抗糖化植物抽出物の合計量がＡＧＥｓ生成抑制有効量となればよく、食事の回数に合わせて投与するのが好ましい。 The pharmaceutical composition for preventing / treating diabetes of the present invention may be one in which an anti-glycated plant extract in an effective amount for inhibiting the generation of AGEs is administered at once, and is administered in multiple portions at intervals. May be. When administered in multiple doses, the total amount of the anti-glycated plant extract administered per day may be an effective amount for inhibiting the generation of AGEs, and is preferably administered according to the number of meals.
本発明の糖尿病予防／治療用医薬組成物は、糖尿病もしくは糖尿病合併症に罹患した者、または健常者のいずれに対しても投与することができるが、ＡＧＥｓとの関連性が高い糖尿病に罹患した者に対して投与するのが好ましい。糖尿病に罹患した者とは、ヘモグロビンＡ１ｃが６．１％（ＪＤＳ値）以上で、且つ、以下の（１）〜（３）のいずれかに該当する者を指す：（１）空腹時血糖値が１２６ｍｇ／ｄＬ以上、（２）随時血糖値（空腹か食後かにかかわらず）が２００ｍｇ／ｄＬ以上、および（３）ブドウ糖負荷後２時間値が２００ｍｇ／ｄＬ以上。糖尿病診断基準の詳細は、日本糖尿病学会による「糖尿病の分類と診断基準に関する委員会報告」（同学会のホームページ等から入手可能）に記載されている。 The pharmaceutical composition for preventing / treating diabetes according to the present invention can be administered to either a person suffering from diabetes or diabetic complications or a healthy person, but suffering from diabetes having a high association with AGEs. It is preferably administered to a person. A person suffering from diabetes refers to a person whose hemoglobin A1c is 6.1% (JDS value) or more and falls under any of the following (1) to (3): (1) Fasting blood glucose level 126 mg / dL or more, (2) Adequate blood glucose level (whether fasting or after eating) is 200 mg / dL or more, and (3) 2 hours after glucose loading is 200 mg / dL or more. Details of the diagnostic criteria for diabetes are described in the “Report of the Committee on Diabetes Classification and Diagnostic Criteria” by the Japanese Diabetes Association (available from the association's website).
また、本発明の糖尿病予防／治療用医薬組成物は、糖尿病に伴って発症する糖尿病網膜症、糖尿病腎症、糖尿病神経障害、糖尿病血管合併症、動脈硬化症、腎不全、アルツハイマー病、神経変性疾患、がん等の糖尿病合併症の予防および／または治療のために用いることができる。これらの糖尿病合併症の中でも、発症率の高い糖尿病網膜症、糖尿病腎症および／または糖尿病神経障害の予防および／または治療のために用いることが好ましい。 Further, the pharmaceutical composition for preventing / treating diabetes of the present invention comprises diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, arteriosclerosis, renal failure, Alzheimer's disease, neurodegeneration, which develops with diabetes. It can be used for the prevention and / or treatment of diabetic complications such as diseases and cancer. Among these diabetic complications, it is preferably used for the prevention and / or treatment of diabetic retinopathy, diabetic nephropathy and / or diabetic neuropathy with a high incidence.
さらに、本発明の糖尿病予防／治療用医薬組成物は、健常者に投与することにより、糖尿病およびその合併症を効果的に予防することができる。 Furthermore, diabetes and its complications can be effectively prevented by administering the pharmaceutical composition for preventing / treating diabetes of the present invention to healthy individuals.
本発明の糖尿病予防／治療用医薬組成物は、抗糖化植物抽出物による糖尿病もしくは糖尿病合併症の予防および／または治療効果を妨げない範囲であれば、抗糖化植物抽出物の他にさらに賦形剤、安定剤、保存剤、緩衝剤、矯味剤、懸濁化剤、乳化剤、着香剤、溶解補助剤、着色剤、粘稠剤等の成分を添加して各種の剤形とすることができる。 The pharmaceutical composition for preventing / treating diabetes according to the present invention is further shaped in addition to the anti-glycated plant extract as long as it does not interfere with the prevention and / or treatment effect of diabetes or diabetic complications by the anti-glycated plant extract. It is possible to add various components such as additives, stabilizers, preservatives, buffering agents, flavoring agents, suspending agents, emulsifiers, flavoring agents, solubilizing agents, coloring agents, and thickeners. it can.
本発明の糖尿病予防／治療用医薬組成物の剤形としては、錠剤（口腔内崩壊錠、チュアブル錠、発泡錠、分散錠、溶解錠）、カプセル剤、顆粒剤（発泡顆粒剤）、散剤、経口液剤（エリキシル剤、懸濁剤、乳剤、リモナーデ剤）、シロップ剤（シロップ用剤）、経口ゼリー剤、口腔用錠剤（トローチ剤、舌下錠、バッカル錠、付着錠、ガム剤）、口腔用スプレー剤、口腔用半固形剤、含嗽剤、注射剤（輸液剤、埋め込み注射剤、持続性注射剤）、透析用剤（腹膜透析用剤、血液透析用剤）、吸入剤（吸入粉末剤、吸入液剤、吸入エアゾール剤）、坐剤、直腸用半固形剤、注腸剤、点眼剤、眼軟膏剤、点耳剤、点鼻剤（点鼻粉末剤、点鼻液剤）、膣錠、膣用坐剤、外用固形剤（外用散剤）、外用液剤（リニメント剤、ローション剤）、スプレー剤（外用エアゾール剤、ポンプスプレー剤）、軟膏剤、クリーム剤、ゲル剤、貼付剤（テープ剤、パップ剤）等が挙げられる。これらの中でも経口投与のし易さの点で、錠剤、カプセル剤、顆粒剤、散剤、経口液剤、シロップ剤、経口ゼリー剤、口腔用錠剤、口腔用スプレー剤、口腔用半固形剤および含嗽剤が好ましく、生体内に吸収され易い点で、錠剤、カプセル剤、顆粒剤、散剤がより好ましい。 Examples of the dosage form of the pharmaceutical composition for prevention / treatment of diabetes of the present invention include tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets), capsules, granules (expanded granules), powders, Oral solution (elixir, suspension, emulsion, limonade), syrup (syrup), oral jelly, oral tablet (troche, sublingual, buccal, adhesive tablet, gum), oral Spray, oral semi-solid preparation, mouthwash, injection (infusion solution, implantable injection, continuous injection), dialysis agent (peritoneal dialysis agent, hemodialysis agent), inhalant (inhalation powder) , Inhalants, aerosols), suppositories, rectal semisolids, enemas, eye drops, eye ointments, ear drops, nasal drops (nasal powders, nasal drops), vaginal tablets, Vaginal suppository, external solid preparation (external powder), external liquid (liniment, lotion), spray (External aerosols, pump sprays), ointments, creams, gels, patches (tape, cataplasms), and the like. Among these, tablets, capsules, granules, powders, oral solutions, syrups, oral jelly, oral tablets, oral sprays, oral semisolids and mouthwashes are easy to administer. From the viewpoint of being easily absorbed in the living body, tablets, capsules, granules, and powders are more preferable.
また、本発明の糖尿病予防／治療用医薬組成物は、エキス剤、丸剤、酒精剤、浸剤・煎剤、茶剤、チンキ剤、芳香水剤、流エキス剤等の生薬関連製剤に本発明のＡＧＥｓ生成抑制剤を添加した剤形で使用することもできる。これら剤形は、糖尿病またはその合併症の程度、その他の疾病の程度、年齢、性別等の条件に応じて適宜選択される。 Further, the pharmaceutical composition for preventing / treating diabetes of the present invention can be applied to herbal medicine-related preparations such as extracts, pills, spirits, soaking agents, decoction, teas, tinctures, fragrances, and flow extracts. It can also be used in a dosage form to which an AGEs production inhibitor is added. These dosage forms are appropriately selected according to conditions such as the degree of diabetes or its complications, the degree of other diseases, age, and sex.
更に、本発明の糖尿病予防／治療用医薬組成物は、ＡＧＥｓ生成抑制効果を有する他の薬剤をさらに含むものとすることができる。これらの薬剤としては、例えば、インスリン抵抗性改善薬、脂質異常症治療薬、アンジオテンシンII１型受容体拮抗薬、アンジオテンシン変換酵素阻害剤、メトホルミン等が例示される。 Furthermore, the pharmaceutical composition for preventing / treating diabetes according to the present invention may further contain other drugs having an AGEs production inhibitory effect. Examples of these drugs include insulin resistance improving drugs, dyslipidemic drugs, angiotensin II type 1 receptor antagonists, angiotensin converting enzyme inhibitors, metformin, and the like.
本発明のＡＧＥｓ生成抑制剤またはその有効成分である抗糖化植物抽出物は、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療するために用いることもできる。したがって、本発明の一態様において、非ヒト動物における糖尿病もしくは糖尿病合併症の予防および／または治療のための、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を含む非ヒト動物用医薬組成物（以下、本発明の非ヒト動物用医薬組成物とも称する）が提供される。また、該非ヒト動物用医薬組成物または本発明のＡＧＥｓ生成抑制剤を非ヒト動物に投与することを含む、非ヒト動物における糖尿病もしくは糖尿病合併症を予防および／または治療する方法も提供される。非ヒト動物としては、ウシ、ウマ、ブタ、ヒツジ、ヤギ、ニワトリ等の家畜類や、イヌ、ネコ等のペットとして飼育される動物等が例示される。 The AGEs production inhibitor of the present invention or an anti-glycated plant extract that is an active ingredient thereof can also be used to prevent and / or treat diabetes or diabetic complications in non-human animals. Accordingly, in one aspect of the present invention, one or more anti-antibodies selected from the group consisting of a lemon myrtle extract, a sunflower extract, and a cassava extract for the prevention and / or treatment of diabetes or diabetic complications in a non-human animal. There is provided a pharmaceutical composition for non-human animals comprising a saccharified plant extract (hereinafter also referred to as a pharmaceutical composition for non-human animals of the present invention). In addition, a method for preventing and / or treating diabetes or diabetic complications in a non-human animal, comprising administering the non-human animal pharmaceutical composition or the AGEs production inhibitor of the present invention to a non-human animal is also provided. Examples of non-human animals include livestock such as cows, horses, pigs, sheep, goats and chickens, and animals raised as pets such as dogs and cats.
本発明の非ヒト動物用医薬組成物に含有させる抗糖化植物抽出物の量、該医薬組成物の投与方法、投与時期、投与量および剤形、該医薬組成物において抗糖化植物抽出物と併用可能な他の薬剤、天然物および天然由来物質、ならびに本発明のＡＧＥｓ生成抑制剤を非ヒト動物に投与する場合における投与方法、投与時期、投与量等については、「本発明の糖尿病予防／治療用医薬組成物」について上述した内容と同様である。 Amount of anti-glycated plant extract to be contained in the non-human animal pharmaceutical composition of the present invention, administration method, timing, dosage and dosage form of the pharmaceutical composition, combined use with the anti-glycated plant extract in the pharmaceutical composition For other possible drugs, natural products and naturally-derived substances, and the administration method, administration time, dosage, etc. when administering the AGEs production inhibitor of the present invention to non-human animals, refer to “Prevention / Treatment of Diabetes of the Present Invention”. The same as described above for the “pharmaceutical composition”.
上記の抗糖化植物抽出物は、食品添加剤として用いることもできる。したがって、レモンマートル抽出物、ハマボウフウ抽出物およびキャッサバ抽出物からなる群より選ばれる１種以上の抗糖化植物抽出物を飲食品に配合または添加することにより、ＡＧＥｓの生成を抑制する機能性飲食品を提供することができる。また、該抗糖化植物抽出物を成分として使用することにより、ＡＧＥｓ生成を抑制するサプリメントを製造することもできる。この場合、製造するサプリメントの剤形に応じて適宜、賦形剤等を配合することができる。 Said anti-glycation plant extract can also be used as a food additive. Therefore, the functional food / beverage products which suppress the production | generation of AGEs by mix | blending or adding 1 or more types of anti-glycation plant extracts selected from the group which consists of a lemon myrtle extract, a hamboufu extract, and a cassava extract to food / beverage products Can be provided. Moreover, the supplement which suppresses AGE production | generation can also be manufactured by using this anti-glycation plant extract as a component. In this case, excipients and the like can be appropriately blended according to the dosage form of the supplement to be produced.
さらに、上記の抗糖化植物抽出物は、皮膚老化の予防／改善のための皮膚外用剤の有効成分として用いることもできる。本発明の皮膚老化予防／改善用皮膚外用剤中に含有させる抗糖化植物抽出物の合計量は、目的とする剤形等に応じて適宜設定することができるが、固形抽出物を用いる場合、通常、０．０１〜２０重量％程度であればよく、０．０５〜１５重量％程度であることが好ましく、０．１〜１０重量％程度であることがより好ましい。 Furthermore, the above-mentioned anti-glycated plant extract can also be used as an active ingredient of a skin external preparation for preventing / improving skin aging. The total amount of the anti-glycated plant extract to be contained in the skin external preparation for skin aging prevention / improvement of the present invention can be appropriately set according to the intended dosage form, etc., but when using a solid extract, Usually, it may be about 0.01 to 20% by weight, preferably about 0.05 to 15% by weight, and more preferably about 0.1 to 10% by weight.
本発明の皮膚老化予防／改善用皮膚外用剤の皮膚への適用量は、皮膚老化の程度、その他の皮膚トラブルの程度、年齢、性別等の条件に応じて適宜選択されるが、ＡＧＥｓの生成を抑制することができる量（ＡＧＥｓ生成抑制有効量）を適用すればよい。ＡＧＥｓ生成抑制有効量は、当業者に周知の方法（各種の非臨床および／または臨床試験を含む）を用いて適宜決定することができる。 The applied amount of the external preparation for skin aging prevention / improvement of the present invention to the skin is appropriately selected according to conditions such as the degree of skin aging, the degree of other skin troubles, age, and sex, but the generation of AGEs It is sufficient to apply an amount that can suppress the amount (AGEs generation suppression effective amount). An effective amount for inhibiting the generation of AGEs can be appropriately determined using methods well known to those skilled in the art (including various non-clinical and / or clinical trials).
本発明の皮膚老化予防／改善用皮膚外用剤は、ＡＧＥｓ生成抑制有効量の抗糖化植物抽出物を一度に適用するものであっても良く、間隔を置いて複数回に分けて適用するものであっても良い。複数回に分けて適用する場合は、一日に適用される抗糖化植物抽出物の合計量がＡＧＥｓ生成抑制有効量となればよい。 The skin external preparation for skin aging prevention / improvement of the present invention may be one in which an anti-glycated plant extract in an effective amount for inhibiting AGE generation is applied at once, and is applied in multiple times at intervals. There may be. When the application is divided into a plurality of times, the total amount of the anti-glycated plant extract to be applied per day may be an effective amount for inhibiting the generation of AGEs.
本発明の皮膚老化予防／改善用皮膚外用剤は、塗布等の方法で皮膚に適用することにより、皮膚老化、例えば皮膚の弾力性低下や黄色化を効果的に予防することができるほか、これら皮膚老化を改善し、または皮膚老化の進行を阻止することができる。 The skin external preparation for skin aging prevention / improvement of the present invention can effectively prevent skin aging, for example, skin elasticity reduction or yellowing, by applying to the skin by a method such as application. It can improve skin aging or prevent the progression of skin aging.
本発明の皮膚老化予防／改善用皮膚外用剤は、抗糖化植物抽出物による皮膚老化の予防および／または改善効果を妨げない範囲であれば、抗糖化植物抽出物の他にさらに安定剤、防腐剤、緩衝剤、酸化防止剤、懸濁化剤、界面活性剤、溶解補助剤、着色剤、粘稠剤、香料等の成分を添加して各種の剤形とすることができる。 The skin external preparation for skin aging prevention / improvement according to the present invention may further contain a stabilizer, antiseptic, in addition to the anti-glycated plant extract, as long as the effect of preventing and / or improving skin aging by the anti-glycated plant extract is not disturbed Components such as an agent, a buffer, an antioxidant, a suspending agent, a surfactant, a solubilizing agent, a colorant, a thickener, and a fragrance can be added to form various dosage forms.
本発明の皮膚老化予防／改善用皮膚外用剤は、化粧料または医薬組成物であり得る。該化粧料は、通常、化粧品もしくは医薬部外品として提供され得るものである。 The skin external preparation for preventing / ameliorating skin aging according to the present invention may be a cosmetic or a pharmaceutical composition. The cosmetics can be usually provided as cosmetics or quasi drugs.
本発明の皮膚老化予防／改善用皮膚外用剤の形態としては、乳液、美容液、ローション、パック、クリーム、ハンドクリームなどの基礎化粧料、ファンデーション、化粧下地、リップクリーム、口紅などのメークアップ化粧料、洗顔料、石鹸、メイク落とし、ボディーシャンプーなどの洗浄用化粧料、ヘアートニック、ヘアーローションなどの頭髪用化粧料、入浴剤などが挙げられる。 The form of the external preparation for skin aging prevention / improvement of the present invention includes basic cosmetics such as milky lotion, cosmetic liquid, lotion, pack, cream and hand cream, makeup makeup such as foundation, makeup base, lip balm and lipstick. Cosmetics, facial cleansers, soaps, makeup removers, body cosmetics such as body shampoos, hair cosmetics such as hair nicks and hair lotions, and bath preparations.
また、本発明の糖尿病予防／治療用医薬組成物、非ヒト動物用医薬組成物、食品添加剤および皮膚老化予防／改善用皮膚外用剤は、ＡＧＥｓ生成抑制効果を有する天然物または天然由来物質をさらに含むものとすることもできる。これらの天然物または天然由来物質としては、例えば、ドクダミ、セイヨウサンザシ、カモミール、ブドウ葉等のハーブ類、桜の花、トウモロコシの花柱および柱頭、セイヨウオオバコ種子、マロニエ、シャクヤク、バラの花、梅果実、山ぶどう、紫菊花、小麦胚芽、小麦胚芽由来のポリアミン、マンゴスチン等が例示される。 In addition, the pharmaceutical composition for preventing / treating diabetes, the pharmaceutical composition for non-human animals, the food additive, and the external preparation for skin aging prevention / amelioration of the present invention are natural products or naturally derived substances having an AGEs production inhibitory effect. It can also be included. Examples of these natural products or naturally-derived substances include herbs such as dokudami, hawthorn, chamomile, grape leaves, cherry blossoms, corn stigma and stigmas, plantain seeds, maronier, peonies, rose flowers, plum fruit , Mountain grape, purple chrysanthemum flower, wheat germ, wheat germ-derived polyamine, mangosteen and the like.
以下、実施例を挙げて、本発明を具体的に説明するが、本発明はこれら記載に限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these description.
実施例１
レモンマートル抽出物の製造（エタノールによる抽出）
レモンマートルの乾燥させた葉（５００．５ｇ）を９９．５％エタノール（８０９１．８ｇ）に浸漬し、２５℃で７日間静置して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：１６）。抽出液を減圧乾燥にて乾燥し、固形抽出物（４１．８ｇ）を得た。以下、かかるエタノール抽出物をレモンマートル抽出物（Ｅ）と表記する。
Example 1
Manufacture of lemon myrtle extract (extraction with ethanol)
Lemon Myrtle's dried leaves (500.5 g) were immersed in 99.5% ethanol (8091.8 g), left to stand at 25 ° C. for 7 days, extracted, and separated into an extract and a residue by filtration. A liquid was obtained (weight ratio of raw material to extraction solvent was 1:16). The extract was dried under reduced pressure to obtain a solid extract (41.8 g). Hereinafter, this ethanol extract is described as a lemon myrtle extract (E).
レモンマートル抽出物の製造（水による抽出）
レモンマートルの乾燥させた葉（１１１ｇ）に水（２２３２．２ｇ）を加え、１００℃で５分間煮沸して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：２０）。抽出液を凍結乾燥にて乾燥し、固形抽出物（２６．８ｇ）を得た。以下、かかる熱水抽出物をレモンマートル抽出物（Ｗ）と表記する。
Manufacture of lemon myrtle extract (extraction with water)
Water (2232.2 g) was added to the dried leaves (111 g) of Lemon Myrtle, and the mixture was boiled at 100 ° C. for 5 minutes for extraction, and separated into an extract and a residue by filtration to obtain an extract (material and extraction The solvent weight ratio is 1:20). The extract was dried by lyophilization to obtain a solid extract (26.8 g). Hereinafter, this hot water extract is referred to as a lemon myrtle extract (W).
蛍光性ＡＧＥｓ強度の測定
レモンマートル抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法で蛍光性ＡＧＥｓ強度を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表１に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液の蛍光強度（励起波長３８０ｎｍ、蛍光波長４００−６００ｎｍ）を９６ウエルマイクロプレートリーダーを用いて測定し、この値を以下に示す蛍光性ＡＧＥｓ強度計算式中のａ１とした。該計算式中のｂ１〜ｄ１についても同様の条件で反応させて蛍光強度を測定し、蛍光性ＡＧＥｓ強度を算出した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
蛍光性ＡＧＥｓ強度＝ａ１−ｂ１−ｃ１−ｄ１
ａ１：各試験区のサンプル溶液０．２５ｍＬ＋アミノ酸液１．０ｍＬ＋糖液０．２５ｍｌ
ｂ１：各試験区のサンプル溶液０．２５ｍＬ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
ｃ１：アミノ酸液１．０ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）０．５ｍｌ
ｄ１：糖液０．２５ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
Measurement of Fluorescence AGE Intensity Using a lemon myrtle extract, the fluorescence AGE intensity was measured by the following method in order to confirm the AGE suppression effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 1 were mixed and allowed to stand at 37 ° C. for 7 days. The fluorescence intensity (excitation wavelength: 380 nm, fluorescence wavelength: 400-600 nm) of the reaction solution was measured using a 96-well microplate reader, and this value was defined as a1 in the fluorescent AGEs intensity calculation formula shown below. B1 to d1 in the calculation formula were also reacted under the same conditions to measure the fluorescence intensity, and the fluorescent AGEs intensity was calculated. The blank was measured by adding a solvent instead of adding the sample solution.
Fluorescent AGE intensity = a1-b1-c1-d1
a1: 0.25 mL of sample solution in each test group + 1.0 mL of amino acid solution + 0.25 mL of sugar solution
b1: 0.25 ml of sample solution of each test group 0.25 ml + 0.5 M phosphate buffer (pH 7.2)
c1: 1.0 ml of amino acid solution + 0.5 ml of 0.5 M phosphate buffer (pH 7.2)
d1: 1.25 ml of sugar solution 0.25 ml + 0.5 M phosphate buffer (pH 7.2)
本発明のＡＧＥｓ生成抑制剤の有効成分であるレモンマートル抽出物を用いた試験区では、ブランクに比べ蛍光性ＡＧＥｓ強度が低下しており、該抽出物が蛍光性ＡＧＥｓの生成を抑制していることが確認された。結果を表２に示す。 In the test section using the lemon myrtle extract which is an active ingredient of the AGEs production inhibitor of the present invention, the intensity of the fluorescent AGEs is lower than that of the blank, and the extract inhibits the production of the fluorescent AGEs. Was confirmed. The results are shown in Table 2.
ＣＭＬ生成量の測定
レモンマートル抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でＣＭＬ生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表１に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のＣＭＬ生成量をＣｉｒｃｕＬｅｘ ＣＭＬ／Ｎε−（ｃａｒｂｏｘｙｍｅｔｈｙｌ）ｌｙｓｉｎｅ ＥＬＩＳＡ ｋｉｔ（株式会社サイクレックス製）を用いて測定した。まず、反応液６０μＬに第一次抗体液６０μＬを加え混合した。次にＣＭＬ固層化プレートに混合液１００μＬを添加し、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１０分間振とうした。その後、プレートに反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるＣＭＬ量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of CML Production Amount Using the lemon myrtle extract, the CML production amount was measured by the following method in order to confirm the AGEs inhibitory effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 1 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of CML produced in the reaction solution was measured using CircuLex CML / Nε- (carbomethyl) lysine ELISA kit (manufactured by Cyclex Co., Ltd.). First, 60 μL of the primary antibody solution was added to 60 μL of the reaction solution and mixed. Next, 100 μL of the mixed solution was added to the CML solidified plate and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a secondary antibody solution was added and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added and shaken at 25 ° C. and 300 rpm for 10 minutes. Thereafter, 100 μL of a reaction stop solution was added to the plate, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of CML contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるレモンマートル抽出物を用いた試験区では、ブランクに比べＣＭＬ生成量が減少しており、該抽出物がＣＭＬの生成を抑制していることが確認された。結果を表３に示す。 In the test section using the lemon myrtle extract, which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of CML produced decreased compared to the blank, and it was confirmed that the extract inhibits the production of CML. It was. The results are shown in Table 3.
ペントシジン生成量の測定
レモンマートル抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でペントシジン生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表１に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のペントシジン生成量を、ペントシジン測定キット「ＦＳＫペントシジン(登録商標)」（株式会社伏見製薬所）を用いて測定した。まず、反応液５０μＬを酵素剤チューブに添加し、５５℃で９０分間反応させた。つぎに、１００℃で１５分間処理し酵素を失活させた。室温まで冷却した後、補助剤５０μＬを添加し混合した。次に抗原固層プレートに混合液５０μＬおよび第一次抗体液５０μＬを添加した後、２５℃、３００ｒｐｍで１分間振とうし、３７℃で１時間静置した。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で１時間静置した。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で９分間静置した。その後、反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるペントシジン量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of Pentosidine Production Amount Using a lemon myrtle extract, the amount of pentosidine production was measured by the following method in order to confirm the AGEs inhibitory effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 1 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of pentosidine produced in the reaction solution was measured using a pentosidine measurement kit “FSK Pentosidine (registered trademark) ” (Fushimi Pharmaceutical Co., Ltd.). First, 50 μL of the reaction solution was added to the enzyme agent tube and reacted at 55 ° C. for 90 minutes. Next, the enzyme was inactivated by treatment at 100 ° C. for 15 minutes. After cooling to room temperature, 50 μL of adjuvant was added and mixed. Next, 50 μL of the mixed solution and 50 μL of the primary antibody solution were added to the antigen solid layer plate, and then shaken at 25 ° C. and 300 rpm for 1 minute and allowed to stand at 37 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of the secondary antibody solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 9 minutes. Thereafter, 100 μL of the reaction stop solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of pentosidine contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるレモンマートル抽出物を用いた試験区では、ブランクに比べペントシジン生成量が減少しており、該抽出物がペントシジンの生成を抑制していることが確認された。結果を表４に示す。 In the test section using the lemon myrtle extract, which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of pentosidine produced was reduced compared to the blank, and it was confirmed that the extract inhibited the production of pentosidine. It was. The results are shown in Table 4.
実施例２
ハマボウフウ抽出物の製造（エタノールによる抽出）
２〜１０ｍｍ四方の大きさに細断したハマボウフウの葉（６０１．６ｇ）を９９．５％エタノール（３００４．９ｇ）に浸漬し、２５℃で７日間静置して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：５）。抽出液を減圧乾燥にて乾燥し、固形抽出物（１７．３ｇ）を得た。以下、かかるエタノール抽出物をハマボウフウ抽出物（Ｅ）と表記する。
Example 2
Mangrove extract production (extraction with ethanol)
2-10 mm squared chopped leaves (601.6 g) were immersed in 99.5% ethanol (3004.9 g), left to stand at 25 ° C. for 7 days, extracted, and filtered. The extract was divided into an extract and a residue to obtain an extract (the weight ratio of the raw material to the extraction solvent was 1: 5). The extract was dried under reduced pressure to obtain a solid extract (17.3 g). Hereinafter, such an ethanol extract will be referred to as a hamboufu extract (E).
ハマボウフウ抽出物の製造（水による抽出）
２〜１０ｍｍ四方の大きさに細断したハマボウフウの葉（３４１．９ｇ）に水（２７３５．９ｇ）を加え、１００℃で５分間煮沸して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：８）。抽出液を凍結乾燥にて乾燥し、固形抽出物（１５．７ｇ）を得た。以下、かかる熱水抽出物をハマボウフウ抽出物（Ｗ）と表記する。
Mangrove extract production (water extraction)
Water (2735.9 g) is added to the leaf of kingfisher (341.9 g) shredded to a size of 2 to 10 mm square, boiled at 100 ° C. for 5 minutes, extracted, and separated into an extract and a residue by filtration. An extract was obtained (weight ratio of material to extraction solvent was 1: 8). The extract was lyophilized to obtain a solid extract (15.7 g). Hereinafter, such a hot water extract will be referred to as a hamboufu extract (W).
蛍光性ＡＧＥｓ強度の測定
ハマボウフウ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法で蛍光性ＡＧＥｓ強度を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表５に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液の蛍光強度（励起波長３８０ｎｍ、蛍光波長４００−６００ｎｍ）を９６ウエルマイクロプレートリーダーを用いて測定し、この値を以下に示す蛍光性ＡＧＥｓ強度計算式中のａ２とした。該計算式中のｂ２〜ｄ２についても同様の条件で反応させて蛍光強度を測定し、蛍光性ＡＧＥｓ強度を算出した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
蛍光性ＡＧＥｓ強度＝ａ２−ｂ２−ｃ２−ｄ２
ａ２：各試験区のサンプル溶液０．２５ｍＬ＋アミノ酸液１．０ｍＬ＋糖液０．２５ｍｌ
ｂ２：各試験区のサンプル溶液０．２５ｍＬ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
ｃ２：アミノ酸液１．０ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）０．５ｍｌ
ｄ２：糖液０．２５ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
Measurement of fluorescent AGE intensity In order to confirm the AGE suppression effect, the fluorescent AGE intensity was measured by the method described below using the extract of Hamabou fu. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 5 were mixed and allowed to stand at 37 ° C. for 7 days. The fluorescence intensity (excitation wavelength: 380 nm, fluorescence wavelength: 400-600 nm) of the reaction solution was measured using a 96-well microplate reader, and this value was defined as a2 in the fluorescent AGEs intensity calculation formula shown below. Regarding b2 to d2 in the calculation formula, the fluorescence intensity was measured by reacting under the same conditions, and the fluorescence AGEs intensity was calculated. The blank was measured by adding a solvent instead of adding the sample solution.
Fluorescence AGE intensity = a2-b2-c2-d2
a2: 0.25 mL of sample solution in each test group + 1.0 mL of amino acid solution + 0.25 mL of sugar solution
b2: 0.25 ml of sample solution of each test group + 1.25 ml of 0.5 M phosphate buffer (pH 7.2)
c2: Amino acid solution 1.0 ml + 0.5 M phosphate buffer (pH 7.2) 0.5 ml
d2: 1.25 ml of sugar solution 0.25 ml + 0.5 M phosphate buffer (pH 7.2)
本発明のＡＧＥｓ生成抑制剤の有効成分であるハマボウフウ抽出物を用いた試験区では、ブランクに比べ蛍光性ＡＧＥｓ強度が低下しており、該抽出物が蛍光性ＡＧＥｓの生成を抑制していることが確認された。結果を表６に示す。 In the test section using the hamboufu extract, which is an active ingredient of the AGEs production inhibitor of the present invention, the fluorescence AGEs intensity is lower than that of the blank, and the extract inhibits the production of fluorescent AGEs. Was confirmed. The results are shown in Table 6.
ＣＭＬ生成量の測定
ハマボウフウ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でＣＭＬ生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表５に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のＣＭＬ生成量をＣｉｒｃｕＬｅｘ ＣＭＬ／Ｎε−（ｃａｒｂｏｘｙｍｅｔｈｙｌ）ｌｙｓｉｎｅ ＥＬＩＳＡ ｋｉｔ（株式会社サイクレックス製）を用いて測定した。まず、反応液６０μＬに第一次抗体液６０μＬを加え混合した。次にＣＭＬ固層化プレートに混合液１００μＬを添加し、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１０分間振とうした。その後、プレートに反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるＣＭＬ量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of CML production amount The CML production amount was measured by the following method in order to confirm the AGEs inhibitory effect using the extract of Hamabou fuu. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 5 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of CML produced in the reaction solution was measured using CircuLex CML / Nε- (carbomethyl) lysine ELISA kit (manufactured by Cyclex Co., Ltd.). First, 60 μL of the primary antibody solution was added to 60 μL of the reaction solution and mixed. Next, 100 μL of the mixed solution was added to the CML solidified plate and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a secondary antibody solution was added and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added and shaken at 25 ° C. and 300 rpm for 10 minutes. Thereafter, 100 μL of a reaction stop solution was added to the plate, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of CML contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるハマボウフウ抽出物を用いた試験区では、ブランクに比べＣＭＬ生成量が減少しており、該抽出物がＣＭＬの生成を抑制していることが確認された。結果を表７に示す。 In the test section using the hamboufu extract, which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of CML produced was reduced compared to the blank, and it was confirmed that the extract inhibits the production of CML. It was. The results are shown in Table 7.
ペントシジン生成量の測定
ハマボウフウ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でペントシジン生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表５に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のペントシジン生成量を、ペントシジン測定キット「ＦＳＫペントシジン(登録商標)」（株式会社伏見製薬所）を用いて測定した。まず、反応液５０μＬを酵素剤チューブに添加し、５５℃で９０分間反応させた。つぎに、１００℃で１５分間処理し酵素を失活させた。室温まで冷却した後、補助剤５０μＬを添加し混合した。次に抗原固層プレートに混合液５０μＬおよび第一次抗体液５０μＬを添加した後、２５℃、３００ｒｐｍで１分間振とうし、３７℃で１時間静置した。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で１時間静置した。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で９分間静置した。その後、反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるペントシジン量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of Pentosidine Production Amount In order to confirm the AGEs inhibitory effect, the amount of pentosidine production was measured by the following method. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 5 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of pentosidine produced in the reaction solution was measured using a pentosidine measurement kit “FSK Pentosidine (registered trademark) ” (Fushimi Pharmaceutical Co., Ltd.). First, 50 μL of the reaction solution was added to the enzyme agent tube and reacted at 55 ° C. for 90 minutes. Next, the enzyme was inactivated by treatment at 100 ° C. for 15 minutes. After cooling to room temperature, 50 μL of adjuvant was added and mixed. Next, 50 μL of the mixed solution and 50 μL of the primary antibody solution were added to the antigen solid layer plate, and then shaken at 25 ° C. and 300 rpm for 1 minute and allowed to stand at 37 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of the secondary antibody solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 9 minutes. Thereafter, 100 μL of the reaction stop solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of pentosidine contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるハマボウフウ抽出物を用いた試験区では、ブランクに比べペントシジン生成量が減少しており、該抽出物がペントシジンの生成を抑制していることが確認された。結果を表８に示す。 In the test section using the hamboufu extract, which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of pentosidine produced was reduced compared to the blank, and it was confirmed that the extract inhibited the production of pentosidine. It was. The results are shown in Table 8.
実施例３
キャッサバ抽出物の製造（エタノールによる抽出）
２〜１０ｍｍ四方の大きさに細断したキャッサバの乾燥させた葉（９９．９ｇ）を９９．５％エタノール（１９９８．９ｇ）に浸漬し、２５℃で７日間静置して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：２０）。抽出液を減圧乾燥にて乾燥し、固形抽出物（６．５ｇ）を得た。以下、かかるエタノール抽出物をキャッサバ抽出物（Ｅ）と表記する。
Example 3
Manufacture of cassava extract (extraction with ethanol)
Cassava dried leaves (99.9 g) shredded to a size of 2 to 10 mm square were soaked in 99.5% ethanol (19988.9 g), left at 25 ° C. for 7 days for extraction, The extract was separated into an extract and a residue by filtration to obtain an extract (the weight ratio of the raw material to the extraction solvent was 1:20). The extract was dried under reduced pressure to obtain a solid extract (6.5 g). Hereinafter, such an ethanol extract is referred to as a cassava extract (E).
キャッサバ抽出物の製造（水による抽出）
２〜１０ｍｍ四方の大きさに細断したキャッサバの乾燥させた葉（１００．０ｇ）に水（４００．０ｇ）を加え、１００℃で５分間煮沸して抽出を行い、濾過にて抽出液と残渣に分け、抽出液を得た（素材と抽出溶媒の重量比は１：４）。抽出液を凍結乾燥にて乾燥し、固形抽出物（４．２ｇ）を得た。以下、かかる熱水抽出物をキャッサバ抽出物（Ｗ）と表記する。
Manufacture of cassava extract (extraction with water)
Water (400.0 g) is added to dried cassava leaves (100.0 g) shredded to a size of 2 to 10 mm square, boiled at 100 ° C. for 5 minutes, extracted, and filtered with the extract. Divided into residues, an extract was obtained (weight ratio of material to extraction solvent is 1: 4). The extract was lyophilized to obtain a solid extract (4.2 g). Hereinafter, such hot water extract is referred to as cassava extract (W).
蛍光性ＡＧＥｓ強度の測定
キャッサバ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法で蛍光性ＡＧＥｓ強度を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表９に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液の蛍光強度（励起波長３８０ｎｍ、蛍光波長４００−６００ｎｍ）を９６ウエルマイクロプレートリーダーを用いて測定し、この値を以下に示す蛍光性ＡＧＥｓ強度計算式中のａ３とした。該計算式中のｂ３〜ｄ３についても同様の条件で反応させて蛍光強度を測定し、蛍光性ＡＧＥｓ強度を算出した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
蛍光性ＡＧＥｓ強度＝ａ３−ｂ３−ｃ３−ｄ３
ａ３：各試験区のサンプル溶液０．２５ｍＬ＋アミノ酸液１．０ｍＬ＋糖液０．２５ｍｌ
ｂ３：各試験区のサンプル溶液０．２５ｍＬ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
ｃ３：アミノ酸液１．０ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）０．５ｍｌ
ｄ３：糖液０．２５ｍｌ＋０．５Ｍリン酸バッファー（ｐＨ７．２）１．２５ｍｌ
Measurement of Fluorescence AGE Intensity Using the cassava extract, the fluorescence AGE intensity was measured by the following method in order to confirm the AGE suppression effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 9 were mixed and allowed to stand at 37 ° C. for 7 days. The fluorescence intensity (excitation wavelength: 380 nm, fluorescence wavelength: 400-600 nm) of the reaction solution was measured using a 96-well microplate reader, and this value was defined as a3 in the fluorescent AGEs intensity calculation formula shown below. The b3 to d3 in the calculation formula were also reacted under the same conditions to measure the fluorescence intensity, and the fluorescence AGEs intensity was calculated. The blank was measured by adding a solvent instead of adding the sample solution.
Fluorescence AGE intensity = a3-b3-c3-d3
a3: 0.25 mL of sample solution of each test group + 1.0 mL of amino acid solution + 0.25 mL of sugar solution
b3: 0.25 ml of sample solution of each test group 0.25 ml + 0.5 M phosphate buffer (pH 7.2)
c3: Amino acid solution 1.0 ml + 0.5 M phosphate buffer (pH 7.2) 0.5 ml
d3: 1.25 ml of sugar solution 0.25 ml + 0.5 M phosphate buffer (pH 7.2)
本発明のＡＧＥｓ生成抑制剤の有効成分であるキャッサバ抽出物を用いた試験区では、ブランクに比べ蛍光性ＡＧＥｓ強度が低下しており、該抽出物が蛍光性ＡＧＥｓの生成を抑制していることが確認された。結果を表１０に示す。 In the test section using the cassava extract which is an active ingredient of the AGEs production inhibitor of the present invention, the intensity of the fluorescent AGEs is lower than that of the blank, and the extract suppresses the production of the fluorescent AGEs. Was confirmed. The results are shown in Table 10.
ＣＭＬ生成量の測定
キャッサバ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でＣＭＬ生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表９に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のＣＭＬ生成量をＣｉｒｃｕＬｅｘ ＣＭＬ／Ｎε−（ｃａｒｂｏｘｙｍｅｔｈｙｌ）ｌｙｓｉｎｅ ＥＬＩＳＡ ｋｉｔ（株式会社サイクレックス製）を用いて測定した。まず、反応液６０μＬに第一次抗体液６０μＬを加え混合した。次にＣＭＬ固層化プレートに混合液１００μＬを添加し、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１時間振とうした。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１０分間振とうした。その後、プレートに反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるＣＭＬ量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of CML Production Amount Using the cassava extract, the CML production amount was measured by the following method in order to confirm the AGEs inhibitory effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 9 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of CML produced in the reaction solution was measured using CircuLex CML / Nε- (carbomethyl) lysine ELISA kit (manufactured by Cyclex Co., Ltd.). First, 60 μL of the primary antibody solution was added to 60 μL of the reaction solution and mixed. Next, 100 μL of the mixed solution was added to the CML solidified plate and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a secondary antibody solution was added and shaken at 25 ° C. and 300 rpm for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added and shaken at 25 ° C. and 300 rpm for 10 minutes. Thereafter, 100 μL of a reaction stop solution was added to the plate, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of CML contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるキャッサバ抽出物を用いた試験区では、ブランクに比べＣＭＬ生成量が減少しており、該抽出物がＣＭＬの生成を抑制していることが確認された。結果を表１１に示す。 In the test section using the cassava extract which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of CML produced was reduced compared to the blank, and it was confirmed that the extract inhibits the production of CML. It was. The results are shown in Table 11.
ペントシジン生成量の測定
キャッサバ抽出物を用いて、ＡＧＥｓ抑制効果を確認するため下記方法でペントシジン生成量を測定した。７．５ｍｇ／ｍｌＢｏｃ−リジンおよび７．５ｍｇ／ｍｌＢｏｃ−アルギニンを含有する０．５Ｍリン酸バッファー液（ｐＨ７．２）（以下、アミノ酸液とする）１．０ｍｌ、５４ｍｇ／ｍｌＤＬ−グリセルアルデヒド液（以下、糖液とする）０．２５ｍｌおよび表９に示す組成のサンプル溶液０．２５ｍｌを混合し、３７℃で７日間静置した。反応液のペントシジン生成量を、ペントシジン測定キット「ＦＳＫペントシジン(登録商標)」（株式会社伏見製薬所）を用いて測定した。まず、反応液５０μＬを酵素剤チューブに添加し、５５℃で９０分間反応させた。つぎに、１００℃で１５分間処理し酵素を失活させた。室温まで冷却した後、補助剤５０μＬを添加し混合した。次に抗原固層プレートに混合液５０μＬおよび第一次抗体液５０μＬを添加した後、２５℃、３００ｒｐｍで１分間振とうし、３７℃で１時間静置した。プレートを洗浄液でよく洗浄した後、第二次抗体液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で１時間静置した。プレートを洗浄液でよく洗浄した後、発色液１００μＬを添加し、２５℃、３００ｒｐｍで１分間振とうした後、２５℃で９分間静置した。その後、反応停止液１００μＬを加え、２５℃、３００ｒｐｍで１分間振とうし、２５℃で４分間静置した。９６ウエルマイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定し、各反応液に含まれるペントシジン量を検量線を用いて計算した。ブランクは、サンプル溶液を加える代わりに、溶媒を加え測定した。
Measurement of Pentosidine Production Amount Using the cassava extract, the amount of pentosidine production was measured by the following method in order to confirm the AGEs inhibitory effect. 0.5 ml phosphate buffer solution (pH 7.2) (hereinafter referred to as amino acid solution) containing 7.5 mg / ml Boc-lysine and 7.5 mg / ml Boc-arginine 1.0 ml, 54 mg / ml DL-glyceraldehyde solution 0.25 ml (hereinafter referred to as a sugar solution) and 0.25 ml of a sample solution having the composition shown in Table 9 were mixed and allowed to stand at 37 ° C. for 7 days. The amount of pentosidine produced in the reaction solution was measured using a pentosidine measurement kit “FSK Pentosidine (registered trademark) ” (Fushimi Pharmaceutical Co., Ltd.). First, 50 μL of the reaction solution was added to the enzyme agent tube and reacted at 55 ° C. for 90 minutes. Next, the enzyme was inactivated by treatment at 100 ° C. for 15 minutes. After cooling to room temperature, 50 μL of adjuvant was added and mixed. Next, 50 μL of the mixed solution and 50 μL of the primary antibody solution were added to the antigen solid layer plate, and then shaken at 25 ° C. and 300 rpm for 1 minute and allowed to stand at 37 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of the secondary antibody solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 1 hour. After thoroughly washing the plate with a washing solution, 100 μL of a coloring solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and then allowed to stand at 25 ° C. for 9 minutes. Thereafter, 100 μL of the reaction stop solution was added, shaken at 25 ° C. and 300 rpm for 1 minute, and allowed to stand at 25 ° C. for 4 minutes. Absorbance at 450 nm was measured using a 96-well microplate reader, and the amount of pentosidine contained in each reaction solution was calculated using a calibration curve. The blank was measured by adding a solvent instead of adding the sample solution.
本発明のＡＧＥｓ生成抑制剤の有効成分であるキャッサバ抽出物を用いた試験区では、ブランクに比べペントシジン生成量が減少しており、該抽出物がペントシジンの生成を抑制していることが確認された。結果を表１２に示す。 In the test section using the cassava extract which is an active ingredient of the AGEs production inhibitor of the present invention, the amount of pentosidine produced was reduced compared to the blank, and it was confirmed that the extract inhibits the production of pentosidine. It was. The results are shown in Table 12.
実施例４
散剤の製造
表１３に示す割合で原材料を混合し、散剤を製造した。該散剤は、本発明のＡＧＥｓ生成抑制剤、または本発明の糖尿病予防／治療用医薬組成物の一形態である。
Example 4
Production of powder The raw materials were mixed in the ratio shown in Table 13 to produce a powder. The powder is one form of the AGE production inhibitor of the present invention or the pharmaceutical composition for preventing / treating diabetes of the present invention.
錠剤の製造
表１４に示す割合で原材料を混合した後、連続式打錠機（Ｐｉｃｃｏｌａ Ｂ−１０／ＲＩＶＡ社製）を用い、杵金型（φ８ｍｍ、Ｒ１２ｍｍ）、１錠あたりの重量１５０〜２００ｍｇ、回転盤の回転数１２ｒｐｍ、打錠圧４ｋＮの打錠条件で直接打錠し、錠剤を製造した。該錠剤は、本発明のＡＧＥｓ生成抑制剤、または本発明の糖尿病予防／治療用医薬組成物の一形態である。
Preparation of tablets After mixing the raw materials in the proportions shown in Table 14, using a continuous tableting machine (Piccola B-10 / RIVA), a persimmon mold (φ8 mm, R12 mm), a weight per tablet of 150 to 200 mg Tablets were manufactured directly by tableting under the tableting conditions of 12 rpm and a tableting pressure of 4 kN. The tablet is one form of the AGE production inhibitor of the present invention or the pharmaceutical composition for preventing / treating diabetes of the present invention.
経口液剤の製造
表１５に示す割合で原材料を蒸留水に溶解し、経口液剤を製造した。該経口液剤は、本発明の糖尿病予防／治療用医薬組成物の一形態である。
Production of Oral Solution The raw material was dissolved in distilled water at the ratio shown in Table 15 to produce an oral solution. The oral solution is one form of the pharmaceutical composition for preventing / treating diabetes of the present invention.
化粧水の製造
表１６に示す割合で原材料を蒸留水に溶解し、化粧水を製造した。該化粧水は、本発明の皮膚老化予防／改善用皮膚外用剤の一形態である。
Production of lotion The raw materials were dissolved in distilled water at the ratio shown in Table 16 to produce lotion. The lotion is one form of the external preparation for skin aging prevention / amelioration of the present invention.
乳液の製造
表１７に示す割合で原材料を混合し、乳液を製造した。該乳液は、本発明の皮膚老化予防／改善用皮膚外用剤の一形態である。
Production of emulsion The raw materials were mixed in the proportions shown in Table 17 to produce an emulsion. The emulsion is one form of the external preparation for skin aging prevention / amelioration of the present invention.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170037230A (en) * | 2015-09-25 | 2017-04-04 | 주식회사 엘지생활건강 | Oral composition comprising cassava extract |
| CN108135955A (en) * | 2015-10-16 | 2018-06-08 | 株式会社钟化 | muscle-building agent |
| JP2021104950A (en) * | 2019-12-26 | 2021-07-26 | 小林製薬株式会社 | FGF21 production promoter |
| CN113349233A (en) * | 2021-06-19 | 2021-09-07 | 武汉轻工大学 | Nutrition-enriched cookie and preparation method thereof |
| CN114767600A (en) * | 2021-06-02 | 2022-07-22 | 吴哲贤 | Antioxidant, anti-inflammatory and anti-pollution composition containing mixed extract of myrtle and sparassis crispa |
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2013
- 2013-07-29 JP JP2013156989A patent/JP2015024982A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170037230A (en) * | 2015-09-25 | 2017-04-04 | 주식회사 엘지생활건강 | Oral composition comprising cassava extract |
| KR102509579B1 (en) * | 2015-09-25 | 2023-03-10 | 주식회사 엘지생활건강 | Oral composition comprising cassava extract |
| CN108135955A (en) * | 2015-10-16 | 2018-06-08 | 株式会社钟化 | muscle-building agent |
| EP3363449A4 (en) * | 2015-10-16 | 2019-06-26 | Kaneka Corporation | Muscle-enhancing agent |
| US10729737B2 (en) | 2015-10-16 | 2020-08-04 | Kaneka Corporation | Muscle-enhancing agent |
| JP2021104950A (en) * | 2019-12-26 | 2021-07-26 | 小林製薬株式会社 | FGF21 production promoter |
| CN114767600A (en) * | 2021-06-02 | 2022-07-22 | 吴哲贤 | Antioxidant, anti-inflammatory and anti-pollution composition containing mixed extract of myrtle and sparassis crispa |
| CN113349233A (en) * | 2021-06-19 | 2021-09-07 | 武汉轻工大学 | Nutrition-enriched cookie and preparation method thereof |
| CN113349233B (en) * | 2021-06-19 | 2023-04-25 | 武汉轻工大学 | Nutrient enrichment cookie and preparation method thereof |
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