JP7286109B2 - Novel pharmacological applications of fermented plant extracts - Google Patents
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Description
本発明は、植物発酵エキスの新規な薬理用途に関する。 The present invention relates to novel pharmacological uses of fermented plant extracts.
近年、植物素材を微生物の力によって発酵熟成させて得られるエキス（植物発酵エキス）が、健康の維持や増進に有用であるとして愛好する人が増えていることは周知の通りである。植物発酵エキスの人気の高まりに伴い、その機能性についての研究も行われており、植物発酵エキスが有する薬理作用として、例えば特許文献１では老化抑制作用が報告されている。しかしながら、植物発酵エキスが有する薬理作用についての報告はこれまでのところわずかである。 In recent years, it is well known that an increasing number of people love extracts (fermented plant extracts) obtained by fermenting and maturing plant materials with the power of microorganisms, as they are useful for maintaining and improving health. With the increasing popularity of fermented plant extracts, research has also been conducted on their functionality, and Patent Document 1 reports, for example, an anti-aging effect as a pharmacological action of fermented plant extracts. However, there are only a few reports so far on the pharmacological actions of fermented plant extracts.
そこで本発明は、植物発酵エキスの新規な薬理用途を提供することを目的とする。 Accordingly, an object of the present invention is to provide a novel pharmacological use of a fermented plant extract.
本発明者らは上記の点に鑑みて鋭意検討を行った結果、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスが、皮膚障害抑制剤およびダイエット剤としての効果が期待できる各種の作用を有することを見出した。 In view of the above points, the present inventors conducted intensive studies and found that a fermented plant extract obtained by fermenting and maturing a plant material with three kinds of yeast, koji mold, and lactic acid bacteria can be used as a skin disorder inhibitor and a diet agent. It was found that it has various actions that can be expected from the effect of
上記の知見に基づいてなされた本発明の皮膚障害抑制剤は、請求項１記載の通り、トマト、ニンジン、タマネギ、パセリ、キャベツ、ゴボウ、モヤシ、ニンニク、ピーマン、ホウレン草、レンコン、山芋、インゲン豆、ユリ根、アスパラガス、ニラ、ネギ及び大豆を含む野菜、ミカン、パインアップル、リンゴ、グレープ、メロン、レモン、グレープフルーツ、イチゴ、バナナ、パッションフルーツ、ブルーベリー、ブラックベリー、カシス及びラズベリーを含む果物、ヨモギ、ウコン、ドクダミ、ハスの葉、高麗人参、センシンレン、オトギリソウ、クマザサ、タンポポの根、霊芝、アマチャヅル、トチュウ葉、オオバコ、カンゾウ、マツ葉、ナンテンの葉、アマドコロ、ツユ草、ツルナ、マカ、トンカットアリ、ハブソウ、ハト麦、スギナ、ビワ葉、ラカンカ、クコの実及びレンセンソウを含む和漢植物、コンブ、フノリ、わかめ、海苔及びヒバマタを含む海藻を、植物素材として、煎じることで得られるエキスに、ワイン酵母菌、麹菌、乳酸菌を接種し、糖蜜、黒砂糖及びオリゴ糖を加えて１２～１８ヶ月間発酵熟成させた後、濾過して得られる液体エキスである植物発酵エキスを有効成分とし、ラジカル消去作用、ＴＮＦ－α産生抑制作用、ＮＯ産生抑制作用、ＡＧＥ生成抑制作用、チロシナーゼ阻害作用の少なくとも１つの作用に基づく（但し、肌荒れ、吹き出物に用いる態様を除く）。 The skin disorder inhibitor of the present invention, which has been made based on the above findings, is, as described in claim 1, tomatoes, carrots, onions, parsley, cabbage, burdock, sprouts, garlic, green peppers, spinach, lotus roots, yam, and kidney beans. , vegetables including lily root, asparagus, chives, green onions and soybeans, mandarin oranges, pineapples, apples, grapes, melons, lemons, grapefruits, strawberries, bananas, passion fruit, blueberries, blackberries, blackcurrants and raspberries, Mugwort, turmeric, Houttuynia cordata, lotus leaf, ginseng, ginseng, hypericum perforatum, kumazasa, dandelion root, ganoderma lucidum, Gynostemma pentaphyllum, eucommia leaf, plantain, licorice, pine leaf, nanten leaf, Polygonatum, daywort grass, tuna, maca , tongkat ali, herb sow, pigeon barley, horsetail, loquat leaf, lanquat, goji berry and lotus root, and seaweed including kelp, funori, wakame seaweed, seaweed and fucus as plant materials. The extract is inoculated with wine yeast, koji mold, and lactic acid bacteria, molasses, brown sugar, and oligosaccharides are added, fermented and matured for 12 to 18 months, and then filtered to obtain a fermented plant extract as an active ingredient. and is based on at least one of radical scavenging action, TNF-α production inhibitory action, NO production inhibitory action, AGE production inhibitory action, and tyrosinase inhibitory action (except for rough skin and pimples).
本発明によれば、植物発酵エキスの新規な薬理用途として、皮膚障害抑制剤およびダイエット剤を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide skin disorder inhibitors and dieting agents as novel pharmacological uses of fermented plant extracts.
本発明の皮膚障害抑制剤およびダイエット剤は、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスを有効成分とするものである。 The skin disorder inhibitor and diet agent of the present invention contain, as an active ingredient, a fermented plant extract obtained by fermenting and maturing a plant material with yeast, koji mold, and lactic acid bacteria.
本発明の皮膚障害抑制剤およびダイエット剤の有効成分とする、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、公知のものであってよい。公知の植物発酵エキスとしては、例えばインターナショナルフーズ株式会社から「植物発酵エキスＩＬ」の商品名で販売されているものが挙げられる。「植物発酵エキスＩＬ」は、植物素材として、野菜（トマト、ニンジン、タマネギ、パセリ、キャベツ、ゴボウ、モヤシ、ニンニク、ピーマン、ホウレン草、レンコン、山芋、インゲン豆、ユリ根、アスパラガス、ニラ、ネギ、大豆など）、果物（ミカン、パインアップル、リンゴ、グレープ、メロン、レモン、グレープフルーツ、イチゴ、バナナ、パッションフルーツ、ブルーベリー、ブラックベリー、カシス、ラズベリーなど）、和漢植物（ヨモギ、ウコン、ドクダミ、ハスの葉、高麗人参、センシンレン、オトギリソウ、クマザサ、タンポポの根、霊芝、アマチャヅル、トチュウ葉、オオバコ、カンゾウ、マツ葉、ナンテンの葉、アマドコロ、ツユ草、ツルナ、マカ、トンカットアリ、ハブソウ、ハト麦、スギナ、ビワ葉、ラカンカ、クコの実、レンセンソウなど）を用い、これらを煎じる（煮出す）ことで得られるエキスに、ワイン酵母菌、麹菌、乳酸菌を接種し、糖（糖蜜、黒砂糖、オリゴ糖）を加えて一定期間（例えば１２～１８ヶ月間）発酵熟成させた後、濾過して得られる液体エキスである。植物素材としてさらに海藻（コンブ、フノリ、わかめ、海苔、ヒバマタなど）を用いてもよく、野菜、果物、和漢植物に、海藻を加えた植物素材を、ワイン酵母菌、麹菌、乳酸菌の３種類で発酵熟成させて得られる植物発酵エキスは、例えばインターナショナルフーズ株式会社から「植物発酵エキスパウダーＺ」の商品名で販売されている（液体エキスをスプレードライした粉体エキス）。また、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、特許文献１に記載されているものでもよい。 The fermented plant extract obtained by fermenting and maturing a plant material with three kinds of yeast, koji mold, and lactic acid bacteria, which is used as an active ingredient of the skin disorder inhibitor and diet agent of the present invention, may be a known one. Examples of known fermented plant extracts include those sold by International Foods Co., Ltd. under the trade name of "fermented plant extract IL". "Fermented plant extract IL" contains vegetables (tomato, carrot, onion, parsley, cabbage, burdock, bean sprout, garlic, green pepper, spinach, lotus root, yam, kidney bean, lily root, asparagus, chive, green onion) as plant materials. , soybeans, etc.), fruits (mandarin oranges, pineapples, apples, grapes, melons, lemons, grapefruits, strawberries, bananas, passion fruits, blueberries, blackberries, cassis, raspberries, etc.), oriental plants (wormwood, turmeric, houttuynia, lotus, etc.) Leaves, Ginseng, Sinensis, Hypericum perforatum, Kumazasa, Dandelion root, Ganoderma lucidum, Jiaogulan, Eucommia leaf, Plantain, Glycyrrhiza, Pine leaf, Nanten leaf, Polygonatum, Tsuyu grass, Tsuna, Maca, Tongkat ali, Habso, Pigeon barley, horsetail, loquat leaf, lanquat, goji berry, lotus root, etc.) are used, and the extract obtained by boiling (boiling) these is inoculated with wine yeast, koji mold, and lactic acid bacteria, and sugar (molasses, black It is a liquid extract obtained by adding sugar, oligosaccharide), fermenting and maturing for a certain period (for example, 12 to 18 months), and then filtering. Seaweed (kelp, funori, wakame seaweed, seaweed, fucus, etc.) may also be used as a plant material. Vegetables, fruits, Japanese and Chinese plants, and seaweed are added to the three types of wine yeast, koji mold, and lactic acid bacteria. A fermented plant extract obtained by fermentation and maturation is sold, for example, by International Foods Co., Ltd. under the trade name of "fermented plant extract powder Z" (powder extract obtained by spray-drying a liquid extract). Also, the fermented plant extract obtained by fermenting and maturing a plant material with three types of yeast, koji mold, and lactic acid bacteria may be the one described in Patent Document 1.
酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、皮膚障害の因子として知られている各種の物質に対して効果的に作用する。具体的には、ラジカルに対する消去作用、炎症物質であるＴＮＦ（腫瘍壊死因子）－αおよびＮＯ（一酸化窒素）に対する産生抑制作用、皮膚に沈着したり炎症性変化を惹起したりするＡＧＥ（終末糖化産物）に対する生成抑制作用を有する。さらに、シミの原因として知られているメラニンの生成酵素であるチロシナーゼに対する阻害作用を有する。従って、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、これらの作用が相まって皮膚障害抑制剤としての効果が期待できる。 A fermented plant extract obtained by fermenting and maturing a plant material with three types of yeast, koji mold, and lactic acid bacteria effectively acts on various substances known to cause skin disorders. Specifically, it has a scavenging effect on radicals, an inhibitory effect on the production of inflammatory substances TNF (tumor necrosis factor)-α and NO (nitric oxide), and an AGE (terminal saccharification products). Furthermore, it has an inhibitory effect on tyrosinase, a melanin-producing enzyme known to cause dark spots. Therefore, a fermented plant extract obtained by fermenting and maturing a plant material with yeast, koji mold, and lactic acid bacteria is expected to be effective as an agent for suppressing skin disorders in combination with these actions.
また、酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、糖質の分解酵素であるαアミラーゼおよびαグルコシダーゼに対する阻害作用を有するとともに、脂質の分解酵素であるリパーゼに対する阻害作用を有し、これらの作用によって糖質と脂質の吸収を抑制することでダイエット剤としての効果が期待できる。 In addition, the fermented plant extract, which is obtained by fermenting and maturing plant materials with three types of yeast, koji mold, and lactic acid bacteria, has an inhibitory effect on α-amylase and α-glucosidase, which are enzymes that decompose carbohydrates, and is a lipid-decomposing enzyme. It has an inhibitory effect on certain lipases, and is expected to be effective as a dieting agent by suppressing the absorption of carbohydrates and lipids by these effects.
本発明において用いる酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスは、液体エキスの場合にはそのまま飲用したり水で希釈して飲用したりすればよく、粉体エキスの場合には水で服用したりすればよい。また、各種の飲食品に配合や調合して飲食したりしてもよい。いずれの摂取形態においても、皮膚障害抑制剤およびダイエット剤としての効果が期待できる。その摂取量は、摂取者の年齢や体重、症状の程度、摂取形態などによって異なり得るが、特に制限されるものではない。 The fermented plant extract obtained by fermenting and maturing plant materials with three types of yeast, koji mold, and lactic acid bacteria used in the present invention may be drunk as it is or after being diluted with water in the case of a liquid extract. In the case of a powder extract, it may be taken with water. Moreover, you may mix|blend or formulate in various foods and drinks, and may eat and drink. In any form of ingestion, it can be expected to be effective as a skin disorder suppressing agent and a dieting agent. The amount to be ingested may vary depending on the age and weight of the ingestor, severity of symptoms, form of ingestion, etc., but is not particularly limited.
以下、本発明を実施例によって詳細に説明するが、本発明は以下の記載に限定して解釈されるものではない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to the following description.
試験例１：
酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスとして、インターナショナルフーズ株式会社から販売されている商品名「植物発酵エキスパウダーＺ」を用い、その皮膚障害抑制剤としての効果が期待できる作用を以下の方法で確認した。
Test Example 1:
As a fermented plant extract obtained by fermenting and maturing plant materials with three types of yeast, koji mold, and lactic acid bacteria, the product name "Fermented plant extract powder Z" sold by International Foods Co., Ltd. is used. The effect that can be expected as an effect was confirmed by the following method.
１．ラジカルの消去作用（抗酸化作用）
（試験方法）
ＤＰＰＨ（２，２－ジフェニル－１－ピクリルヒドラジル）をエタノールに溶解し、濃度が８０μｇ／ｍＬの溶液を調製した。こうして調製したＤＰＰＨのエタノール溶液５００μＬと、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液５００μＬを混合し、室温で３０分間静置した後、吸光度（ＯＤ：４９５ｎｍ）を測定し、検体溶液のラジカル消去率を、コントロール（純水）に対する抑制％として算出した。
（試験結果）
図１に示す（図中の濃度は最終濃度）。図１から明らかなように、「植物発酵エキスパウダーＺ」は、濃度依存的にＤＰＰＨラジカルの消去作用を示した。
1. Radical scavenging action (antioxidant action)
(Test method)
DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to prepare a solution with a concentration of 80 μg/mL. 500 μL of ethanol solution of DPPH thus prepared and 500 μL of specimen solution of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water were mixed and allowed to stand at room temperature for 30 minutes. ) was measured, and the radical scavenging rate of the sample solution was calculated as % inhibition relative to the control (pure water).
(Test results)
It is shown in FIG. 1 (the concentration in the figure is the final concentration). As is clear from FIG. 1, "Fermented plant extract powder Z" exhibited a DPPH radical scavenging effect in a concentration-dependent manner.
２．ＴＮＦ－αおよびＮＯの産生抑制作用（抗炎症作用）
（試験方法）
マウス由来マクロファージ細胞であるＲＡＷ２６４細胞を１０％ＦＣＳ含有ＲＰＭＩ－１６４０培地に懸濁してフラスコに播種し、３７℃、５％ＣＯ２のインキュベーター内で継代培養した。コンフルエントになったＲＡＷ２６４細胞をフラスコから回収し、培地に懸濁して６．２５×１０５ｃｅｌｌｓ／ｍＬの濃度に調整した。こうして調製した細胞懸濁液を２４ｗｅｌｌプレートに８００μＬ／ｗｅｌｌで播種した後、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液１００μＬを添加し、３７℃、５％ＣＯ２のインキュベーター内で２時間培養した。培養後、濃度を１０μｇ／ｍＬに調整したＬＰＳ（リポポリサッカライド）溶液１００μＬを添加してさらに２４時間培養してから、０．４％トリパンブルー溶液を用いた染色により細胞毒性試験を行い、検体溶液の細胞障害性の有無を調べた。また、細胞培養上清に含まれるＴＮＦ－αおよびＮＯを定量した。ＴＮＦ－αの定量は、ＥＬＩＳＡキット（レビスＴＮＦ－αマウスＥＬＩＳＡキット：レビス社）を用い、プロトコルに従って行った。ＮＯの定量は、グリース法によって行った。具体的には、細胞培養上清５００μＬに同量のグリース試薬（１％スルファニルアミド）を添加し、１０分間反応させた後、吸光度（ＯＤ：４９５ｎｍ）を測定し、ＮＯの酸化によって生成するＮＯ２
－の濃度を測定することで行った。
（試験結果）
図２にＴＮＦ－αについての結果を示し、図３にＮＯについての結果を示す（図中の濃度は最終濃度）。図２および図３から明らかなように、ＲＡＷ２６４細胞は、ＬＰＳ刺激によってＴＮＦ－αおよびＮＯを産生して細胞培養上清に分泌するが、「植物発酵エキスパウダーＺ」は、濃度依存的にこれらの炎症物質の産生を抑制した。なお、いずれの濃度の検体溶液についても、トリパンブルー染色による細胞毒性試験において、細胞障害性は認められなかった（検体溶液のかわりに純水を添加したコントロールと比較して生細胞数の減少は認められなかった）。
2. TNF-α and NO production inhibitory action (anti-inflammatory action)
(Test method)
RAW264 cells, which are mouse-derived macrophage cells, were suspended in RPMI-1640 medium containing 10% FCS, seeded in flasks, and subcultured in an incubator at 37° C. and 5% CO 2 . Confluent RAW264 cells were recovered from the flask and suspended in medium to adjust the concentration to 6.25×10 5 cells/mL. After inoculating the cell suspension thus prepared at 800 μL/well in a 24-well plate, 100 μL of sample solutions of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water were added, and 37° C., 5% Cultured for 2 hours in a CO2 incubator. After culturing, 100 μL of LPS (lipopolysaccharide) solution adjusted to a concentration of 10 μg/mL was added and further cultured for 24 hours. The solution was examined for cytotoxicity. Also, TNF-α and NO contained in the cell culture supernatant were quantified. TNF-α was quantified using an ELISA kit (Revis TNF-α mouse ELISA kit: Revis) according to the protocol. Quantification of NO was performed by the Griess method. Specifically, the same amount of Griess reagent (1% sulfanilamide) was added to 500 μL of the cell culture supernatant, and after reacting for 10 minutes, the absorbance (OD: 495 nm) was measured, and NO produced by oxidation of NO This was done by measuring the concentration of 2- .
(Test results)
FIG. 2 shows the results for TNF-α, and FIG. 3 shows the results for NO (concentrations in the figure are final concentrations). As is clear from FIGS. 2 and 3, RAW264 cells produce TNF-α and NO by LPS stimulation and secrete them into the cell culture supernatant. suppresses the production of inflammatory substances in In the cytotoxicity test by trypan blue staining, no cytotoxicity was observed for any concentration of sample solution (compared to the control in which pure water was added instead of the sample solution, the number of viable cells decreased. I was not able to admit).
３．ＡＧＥの生成抑制作用（抗糖化作用）
（試験方法）
グリシンおよびＤ－グルコースをそれぞれリン酸緩衝液（１００ｍＭ、ｐＨ７．４）に溶解し、濃度が１０％の溶液を調製した。こうして調製したグリシン溶液４５０μＬとＤ－グルコース溶液４５０μＬを混合した後、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液１００μＬを添加し、６０℃で４８時間反応させた後、４５０ｎｍで吸光度を測定し、検体溶液のＡＧＥ生成抑制率を、コントロール（純水）に対する抑制％として算出した。
（試験結果）
図４に示す（図中の濃度は最終濃度）。図４から明らかなように、「植物発酵エキスパウダーＺ」は、グリシン溶液とＤ－グルコース溶液を混合して６０℃で４８時間反応させることで生成するＡＧＥの生成抑制作用を、濃度依存的に示した。
3. AGE production inhibitory action (anti-glycation action)
(Test method)
Glycine and D-glucose were each dissolved in a phosphate buffer (100 mM, pH 7.4) to prepare a 10% solution. After mixing 450 μL of the glycine solution and 450 μL of the D-glucose solution prepared in this way, 100 μL of sample solutions of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water are added and reacted at 60 ° C. for 48 hours. After that, the absorbance was measured at 450 nm, and the AGE production inhibition rate of the sample solution was calculated as % inhibition relative to the control (pure water).
(Test results)
It is shown in FIG. 4 (the concentration in the figure is the final concentration). As is clear from FIG. 4, "Fermented Plant Extract Powder Z" suppresses the production of AGE produced by mixing the glycine solution and the D-glucose solution and reacting them at 60° C. for 48 hours in a concentration-dependent manner. Indicated.
４．チロシナーゼの阻害作用
（試験方法）
リン酸緩衝液（１００ｍＭ、ｐＨ７．４）で濃度を１００ｍＵ／ｍＬに調整したチロシナーゼ（ｆｒｏｍ ｍｕｓｈｒｏｏｍ：シグマ社）溶液５μＬと、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液１００μＬと、リン酸緩衝液４５μＬを、９６ｗｅｌｌプレートの各ｗｅｌｌに加え、室温で１０分間静置してから、基質として濃度が２ｍｇ／ｍＬのＬ－ＤＯＰＡ（３－（３，４－ジヒドロキシフェニル）－Ｌ－アラニン）溶液５０μＬを各ｗｅｌｌに添加し、室温で３０分間反応させた後、吸光度（ＯＤ：４９２ｎｍ）を測定し、検体溶液のチロシナーゼ阻害率を、コントロール（純水）に対する阻害％として算出した。
（試験結果）
図５に示す（図中の濃度は最終濃度）。図５から明らかなように、「植物発酵エキスパウダーＺ」は、Ｌ－ＤＯＰＡからメラニンを生成させるチロシナーゼの阻害作用を、濃度依存的に示した。
4. Tyrosinase inhibitory action (test method)
Various concentrations prepared by dissolving 5 μL of tyrosinase (from mushroom: Sigma) solution adjusted to 100 mU / mL with phosphate buffer (100 mM, pH 7.4) and “fermented plant extract powder Z” in
(Test results)
It is shown in FIG. 5 (the concentration in the figure is the final concentration). As is clear from FIG. 5, "Fermented Plant Extract Powder Z" exhibited concentration-dependent inhibition of tyrosinase, which produces melanin from L-DOPA.
試験例２：
酵母菌、麹菌、乳酸菌の３種類で植物素材を発酵熟成させて得られる植物発酵エキスとして、インターナショナルフーズ株式会社から販売されている商品名「植物発酵エキスパウダーＺ」を用い、そのダイエット剤としての効果が期待できる作用を以下の方法で確認した。
Test example 2:
As a plant fermented extract obtained by fermenting and maturing plant materials with three types of yeast, koji mold, and lactic acid bacteria, the product name "Plant Fermented Extract Powder Z" sold by International Foods Co., Ltd. is used. Actions that can be expected to be effective were confirmed by the following methods.
１．αアミラーゼの阻害作用
（試験方法）
αアミラーゼ（Ｔｙｐｅ ＶＩ－Ｂ ｆｒｏｍ ｐｏｒｃｉｎｅ ｐａｎｃｒｅａｓ：シグマ社）を２０ｍＭ塩化カルシウム溶液に溶解し、濃度が１２５Ｕの溶液を調製した。こうして調製したαアミラーゼ溶液１０μＬと、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液５０μＬを、９６ｗｅｌｌプレートの各ｗｅｌｌに加え、室温で５分間静置してから、基質として濃度が１％のデンプン溶液１２５μＬを各ｗｅｌｌに添加し、室温で７．５分間反応させた。反応後、濃度が０．０２Ｎのヨウ素溶液１２５μＬを各ｗｅｌｌに添加し、αアミラーゼによって分解されていないデンプンを発色させた反応液を純水で５倍希釈し、吸光度（ＯＤ：６２０ｎｍ）を測定することで、残存するデンプンを定量し、検体溶液のαアミラーゼ阻害率を、コントロール（純水）に対する抑制％として算出した。
（試験結果）
図６に示す（図中の濃度は最終濃度）。図６から明らかなように、「植物発酵エキスパウダーＺ」は、デンプンを分解するαアミラーゼの阻害作用を、濃度依存的に示した。
1. α-amylase inhibitory action (test method)
α-amylase (Type VI-B from porcine pancreas: Sigma) was dissolved in a 20 mM calcium chloride solution to prepare a solution with a concentration of 125 U. 10 μL of the α-amylase solution thus prepared and 50 μL of sample solutions of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water are added to each well of a 96-well plate, and allowed to stand at room temperature for 5 minutes. , 125 μL of a 1% starch solution as a substrate was added to each well and allowed to react at room temperature for 7.5 minutes. After the reaction, 125 μL of an iodine solution with a concentration of 0.02 N was added to each well, and the reaction solution in which starch that had not been decomposed by α-amylase was colored was diluted 5-fold with pure water, and the absorbance (OD: 620 nm) was measured. By doing so, the remaining starch was quantified, and the α-amylase inhibition rate of the sample solution was calculated as % inhibition relative to the control (pure water).
(Test results)
It is shown in FIG. 6 (the concentration in the figure is the final concentration). As is clear from FIG. 6, "Fermented Plant Extract Powder Z" showed concentration-dependent inhibition of α-amylase, which decomposes starch.
２．αグルコシダーゼの阻害作用
（試験方法）
αグルコシダーゼ（シグマ社）をリン酸緩衝液（５０ｍＭ、ｐＨ７．４）に溶解し、濃度が１４０Ｕの溶液を調製した。こうして調製したαグルコシダーゼ溶液２５μＬと、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液２５μＬを、９６ｗｅｌｌプレートの各ｗｅｌｌに加え、室温で５分間静置してから、基質としてリン酸緩衝液で濃度を１．５ｍｇ／ｍＬに調整したＰＮＰＧ（ｐ－ニトロフェニル－α－Ｄ－グルコピラノシド）溶液５０μＬを各ｗｅｌｌに添加し、室温で１０分間反応させた後、吸光度（ＯＤ：４０５ｎｍ）を測定し、検体溶液のαグルコシダーゼ阻害率を、コントロール（純水）に対する抑制％として算出した。
（試験結果）
図７に示す（図中の濃度は最終濃度）。図７から明らかなように、「植物発酵エキスパウダーＺ」は、ＰＮＰＧを分解するαグルコシダーゼの阻害作用を、濃度依存的に示した。
2. Inhibitory action of α-glucosidase (test method)
α-Glucosidase (Sigma) was dissolved in a phosphate buffer (50 mM, pH 7.4) to prepare a solution with a concentration of 140 U. 25 μL of the thus prepared α-glucosidase solution and 25 μL of sample solutions of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water are added to each well of a 96-well plate and allowed to stand at room temperature for 5 minutes. As a substrate, 50 μL of a PNPG (p-nitrophenyl-α-D-glucopyranoside) solution adjusted to a concentration of 1.5 mg/mL with a phosphate buffer was added to each well and allowed to react at room temperature for 10 minutes. (OD: 405 nm) was measured, and the α-glucosidase inhibition rate of the sample solution was calculated as % inhibition relative to the control (pure water).
(Test results)
It is shown in FIG. 7 (the concentration in the figure is the final concentration). As is clear from FIG. 7, "Fermented Plant Extract Powder Z" exhibited concentration-dependent inhibitory action on α-glucosidase that decomposes PNPG.
３．リパーゼの阻害作用
（試験方法）
Ｌｉｐａｓｅ Ａｃｔｉｖｉｔｙ Ａｓｓａｙ Ｋｉｔ（ケイマン社）を用いて調べた。具体的には、リパーゼ（ｆｒｏｍ ｐｏｒｃｉｎｅ ｐａｎｃｒｅａｓ：シグマ社）をリン酸緩衝液（５０ｍＭ、ｐＨ７．４）に溶解し、濃度が１ｍｇ／ｍＬの溶液を調製した。こうして調製したリパーゼ溶液１０μＬと、「植物発酵エキスパウダーＺ」を純水に溶解して調製した各種濃度の検体溶液１００μＬと、リン酸緩衝液７０μＬを、９６ｗｅｌｌプレートの各ｗｅｌｌに加え、室温で１０分間静置してから、Ｆｌｕｏｒｏｍｅｔｒｉｃ ｔｈｉｏｌ ｄｅｔｅｃｔｏｒ１０μＬとＬｉｐａｓｅ ｓｕｂｓｔｒａｔｅ１０μＬを各ｗｅｌｌに添加し、室温で１５分間反応させた後、励起波長／測定波長：４３０ｎｍ／５３５ｎｍの蛍光強度を測定し、検体溶液のリパーゼ阻害率を、コントロール（純水）に対する抑制％として算出した。
（試験結果）
図８に示す（図中の濃度は最終濃度）。図８から明らかなように、「植物発酵エキスパウダーＺ」は、基質を分解するリパーゼの阻害作用を、濃度依存的に示した。
3. Inhibitory action of lipase (test method)
It was investigated using Lipase Activity Assay Kit (Cayman). Specifically, lipase (from porcine pancreas: Sigma) was dissolved in a phosphate buffer (50 mM, pH 7.4) to prepare a solution with a concentration of 1 mg/mL. 10 μL of the lipase solution prepared in this way, 100 μL of sample solutions of various concentrations prepared by dissolving “fermented plant extract powder Z” in pure water, and 70 μL of phosphate buffer are added to each well of a 96-well plate. After standing still for 10 minutes, 10 μL of fluorometric thiol detector and 10 μL of Lipase substrate were added to each well and allowed to react at room temperature for 15 minutes. Percentage was calculated as % inhibition relative to control (pure water).
(Test results)
It is shown in FIG. 8 (the concentration in the figure is the final concentration). As is clear from FIG. 8, "Fermented Plant Extract Powder Z" exhibited concentration-dependent inhibition of lipase that decomposes substrates.
本発明は、植物発酵エキスの新規な薬理用途として、皮膚障害抑制剤およびダイエット剤を提供することができる点において、産業上の利用可能性を有する。 INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide skin disorder inhibitors and dieting agents as novel pharmacological uses of fermented plant extracts.
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