JP2016020338A - Novel compound - Google Patents
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- JP2016020338A JP2016020338A JP2015120890A JP2015120890A JP2016020338A JP 2016020338 A JP2016020338 A JP 2016020338A JP 2015120890 A JP2015120890 A JP 2015120890A JP 2015120890 A JP2015120890 A JP 2015120890A JP 2016020338 A JP2016020338 A JP 2016020338A
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Abstract
Description
本発明は、ゲットウ抽出物由来の新規化合物に関し、更に詳細には、糖尿病モジュレーターの抑制、ラジカル生成阻害作用、低比重リポタンパク（ＬＤＬ、一般に悪玉コレステロールとも言われる）の抗酸化作用及びメラニン生成抑制作用を有する新規化合物に関する。 The present invention relates to a novel compound derived from a ghetto extract, and more specifically, suppression of diabetes modulator, radical generation inhibitory activity, low density lipoprotein (LDL, also commonly referred to as bad cholesterol) antioxidant activity and melanin production suppression. The present invention relates to a novel compound having an action.
今日、先進国全体では糖尿疾患の発生率の上昇にともない、糖尿病を抑制する物質の研究が加速している。糖尿病モジュレーター（修飾因子）には、食後の高血糖を低下させるα‐グルコシダーゼ、消化酵素の一つであるトリプシン及び血管拡張物質である一酸化窒素などがあり、これらの因子に対して抑制効果を示す物質が望まれていた。 Today, in developed countries as a whole, research on substances that suppress diabetes is accelerating as the incidence of diabetes mellitus increases. Diabetes modulators (modifiers) include α-glucosidase, which reduces postprandial hyperglycemia, trypsin, one of the digestive enzymes, and nitric oxide, a vasodilator, which have an inhibitory effect on these factors. The substance to be shown was desired.
また、糖尿病とともに生活習慣病として知られる脂質異常症（高脂血症）は、血液中に含まれる脂質の量が過剰となる状態であり、特に、ＬＤＬの酸化型（酸化ＬＤＬ）が血管壁に溜まると、動脈硬化の原因となる。したがって、ＬＤＬの酸化を抑制したり、活性酸素種の生成を抑制する物質が望まれていた。 In addition, dyslipidemia (hyperlipidemia) known as a lifestyle-related disease together with diabetes is a state in which the amount of lipid contained in blood is excessive, and in particular, the oxidized form of LDL (oxidized LDL) is a blood vessel wall. If it accumulates in the water, it will cause arteriosclerosis. Therefore, a substance that suppresses the oxidation of LDL or suppresses the generation of reactive oxygen species has been desired.
ところで、天然物中に生活習慣病の予防、治療等に対し有効な化合物が存在することが知られており、例えば、特許文献１には、糖尿病治療に効果を有するものとして、アブラヤシ属の果実から得られる抽出物がグルコース濃度やトリグリセリド濃度を調整することが記載されている。しかしながら、同抽出物はα‐グルコシダーゼやトリプシン等を抑制するものではなかった。
By the way, it is known that compounds effective for the prevention, treatment, etc. of lifestyle-related diseases exist in natural products. For example,
また、特許文献２には、マメ科植物であるカッシア アウリクラタ（Cassia auriculata）の葉部から水‐アセトン混合液を用いて抽出したフラバンダイマー化合物が、ラジカル消去作用を有することが記載されている。しかしながら、同フラバンダイマー化合物は、ＬＤＬの酸化を抑制するものではなかった。
糖尿病の主要な危険因子として肥満が知られているが、肥満はその他にも、心臓病、高血圧、脳卒中などにおける発症リスクとなる。世界保健機関は、少なくとも１０億人の大人が体重過剰で、このうち３億人が肥満体であるとし、そしてこれらの数は、医療介入なしでは更に上昇すると予想されることを報告している。昨今では、肥満の蔓延は子供にも影響しており、小児肥満の普及は過去３０年間で３倍になり、この影響されやすい人口における健康上の問題を引き起こすことが予想される。 Obesity is known as a major risk factor for diabetes, but obesity is also an onset risk in heart disease, hypertension, and stroke. The World Health Organization reports that at least 1 billion adults are overweight, of which 300 million are obese, and these numbers are expected to rise further without medical intervention . In recent years, the prevalence of obesity has also affected children, and the prevalence of childhood obesity has tripled over the past 30 years and is expected to cause health problems in this susceptible population.
現在、肥満症の治療方法には、食事療法、運動療法、行動療法、薬物療法等があるが、基本となるのは食事療法と運動療法で、これを同時に進めることが一般的である。この食事療法と運動療法の実施には、行動療法という食事と運動の生活指導が具体的に行なわれるが、一般に肥満患者では、強い意志をもってこれに堪えられる人が少なく、結局失敗に終わることが多いとされている。この行動療法では、まれに薬物療法が補助的に使われることがあるとされており、これらの方法で効果がない場合にだけ、胃を小さくする外科療法(手術)が行われることがある。 Currently, there are diet therapy, exercise therapy, behavioral therapy, drug therapy, and the like as methods for treating obesity, but the basics are diet therapy and exercise therapy, which are generally advanced simultaneously. This dietary therapy and exercise therapy are specifically provided with behavioral dietary and exercise lifestyle guidance, but generally there are few obese patients who can withstand this with strong will and eventually fail. It is said that there are many. In this behavioral therapy, it is said that drug therapy is sometimes used as ancillary, and surgical treatment (surgery) to make the stomach small may be performed only when these methods are not effective.
このように、現在の肥満症の治療においては、薬物療法は限られた範囲でしか使用されていないが、現在の肥満の蔓延を抑制するには、患者の意志に関わらず肥満を有効に抑制する医薬の開発が強く求められている。 Thus, in the current treatment of obesity, pharmacotherapy is only used to a limited extent, but to suppress the current prevalence of obesity, effectively suppress obesity regardless of the patient's will There is a strong demand for the development of pharmaceuticals.
また、皮膚が紫外線に曝露されると、皮膚内で発生する活性酸素、過酸化脂質等は、炎症を引き起こし、皮膚組織に大きな損傷を与える。皮膚や毛髪等に存在する色素であるメラニンは、このような紫外線による損傷から皮膚を保護する役割を有する。しかし、メラニンが過剰産生されると、低色素沈着あるいは色素沈着過剰などの皮膚異常を引き起こす他、シミ、ソバカスなどが生じるため、メラニンの生成を抑制することを目的として種々の美白剤が開発されている。メラニンは、メラノサイトにおいてチロシンがチロシナーゼによって酸化されることにより産生されるため、美白剤の多くはメラニン産生における鍵酵素であるチロシナーゼ活性阻害物質である。 Further, when the skin is exposed to ultraviolet rays, active oxygen, lipid peroxide, and the like generated in the skin cause inflammation and seriously damage the skin tissue. Melanin, which is a pigment present in skin and hair, has a role of protecting the skin from damage caused by such ultraviolet rays. However, excessive production of melanin causes skin abnormalities such as hypopigmentation or hyperpigmentation, as well as spots and freckles. Various whitening agents have been developed with the aim of suppressing the production of melanin. ing. Since melanin is produced by oxidation of tyrosine by tyrosinase in melanocytes, many whitening agents are tyrosinase activity inhibitors that are key enzymes in melanin production.
チロシナーゼ活性阻害物質の代表的なものとして、コウジ酸やアルブチン、アスコルビン酸などがよく知られており、メラニンの生成および沈着を抑制する美白剤として利用されている（特許文献３〜５）。しかし、これらの中には活性が十分でないものもあり、天然物由来で強力かつ安全性の高いチロシナーゼ阻害剤がなお強く求められている。 As typical tyrosinase activity inhibitors, kojic acid, arbutin, ascorbic acid, and the like are well known, and are used as whitening agents that suppress the production and deposition of melanin (Patent Documents 3 to 5). However, some of these are not sufficiently active, and there is still a strong demand for tyrosinase inhibitors that are derived from natural products and have high safety and safety.
従って、本発明の課題は、天然物中から従来知られていなかったα‐グルコシダーゼ等の糖尿病モジュレーターの抑制作用、ラジカル生成阻害作用やＬＤＬの抗酸化作用、チロシナーゼ活性阻害作用等の薬理活性を有する新規化合物を見出し、これを提供することである。 Accordingly, the object of the present invention is to have pharmacological activities such as an inhibitory action of diabetes modulators such as α-glucosidase, a radical generation inhibitory action, an LDL antioxidant action, and a tyrosinase activity inhibitory action, which have not been known from natural products. To find and provide new compounds.
本発明者らは、従来から、沖縄県に自生する植物であるゲットウ（月桃）に着目し、この植物中に含まれる成分の薬理活性について研究を行っていたが、今回新たにゲットウ抽出物中から、次の式（Ｉ）で表される２，５−ビス（１Ｅ，３Ｅ，５Ｅ）−６−メトキシヘキサ−１，３，５−トリエン−１−イル−２，５−ジヒドロフラン(2,5-bis(1E,3E,5E)-6-methoxyhexa-1,3,5-trien-1-yl-2,5-dihydrofuran；以下「ＭＴＤ」という）及び次の式（II）で表される（Ｅ）−２，２，３，３−テトラメチル−８−メチレン−７−（オクト−６−エン−１−イル）オクタヒドロ−１Ｈ―キノリジン((E)-2,2,3,3-tetramethyl-8-methylene-7-(oct-6-en-1-yl)octahydro-1H-quinolizine；以下「ＴＭＯＱ」という）を分離し、これらが糖尿病モジュレーターの抑制、ラジカル生成阻害作用、ＬＤＬ酸化阻害活性、チロシナーゼ活性阻害作用等を有することを見出し、本発明を完成した。 The inventors of the present invention have conventionally focused on ghetto (moon peach), a plant that grows naturally in Okinawa Prefecture, and researched the pharmacological activity of the components contained in this plant. Among them, 2,5-bis (1E, 3E, 5E) -6-methoxyhexa-1,3,5-trien-1-yl-2,5-dihydrofuran represented by the following formula (I) ( 2,5-bis (1E, 3E, 5E) -6-methoxyhexa-1,3,5-trien-1-yl-2,5-dihydrofuran (hereinafter referred to as “MTD”) and the following formula (II) (E) -2,2,3,3-tetramethyl-8-methylene-7- (oct-6-en-1-yl) octahydro-1H-quinolidine ((E) -2,2,3, 3-tetramethyl-8-methylene-7- (oct-6-en-1-yl) octahydro-1H-quinolizine (hereinafter referred to as “TMOQ”), which inhibits diabetic modulators, inhibits radical production, LDL Oxidation Harm activity, found to have tyrosinase activity-inhibiting action and the like, and completed the present invention.
すなわち本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物である。 That is, the present invention is a novel compound represented by the above formula (I) or formula (II).
また、本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有するα‐グルコシダーゼ、トリプシン、キサンチンオキシダーゼ及びチロシナーゼからなる群より選ばれる酵素活性阻害剤である。 In addition, the present invention is an enzyme activity inhibitor selected from the group consisting of α-glucosidase, trypsin, xanthine oxidase and tyrosinase containing the novel compound represented by the above formula (I) or formula (II) as an active ingredient.
更に、本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有する一酸化窒素生成阻害剤である。 Furthermore, this invention is a nitric oxide production inhibitor containing the novel compound represented by the above formula (I) or formula (II) as an active ingredient.
また更に、本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有するラジカル生成阻害剤である。 Furthermore, the present invention is a radical production inhibitor containing a novel compound represented by the above formula (I) or formula (II) as an active ingredient.
そして更に、本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有するＬＤＬ酸化阻害剤である。 Furthermore, the present invention is an LDL oxidation inhibitor containing a novel compound represented by the above formula (I) or formula (II) as an active ingredient.
また本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有する抗肥満剤である。 The present invention also provides an antiobesity agent containing a novel compound represented by the above formula (I) or formula (II) as an active ingredient.
また本発明は、上記式（Ｉ）または式（ＩＩ）で表わされる新規化合物を有効成分として含有するメラニン生成抑制剤または美白剤である。 Moreover, this invention is a melanin production inhibitor or whitening agent which contains the novel compound represented by the said Formula (I) or Formula (II) as an active ingredient.
本発明の式（Ｉ）および（ＩＩ）の化合物は、α‐グルコシダーゼ、トリプシン及びキサンチンオキシダーゼの活性を抑制し、また、一酸化窒素生成を抑制する。さらに、これらの化合物は、ラジカル生成を阻害し、またＬＤＬの酸化を抑制する作用を有するものである。 The compounds of the formulas (I) and (II) of the present invention suppress the activities of α-glucosidase, trypsin and xanthine oxidase, and suppress nitric oxide production. Furthermore, these compounds have an action of inhibiting radical generation and suppressing oxidation of LDL.
従ってこれら化合物は、糖尿病モジュレーター（修飾因子）を抑制するものであり、これを含有する医薬品あるいは飲食品は、糖尿病の予防や治療に有用である。 Therefore, these compounds suppress diabetes modulators (modifiers), and pharmaceuticals or foods and drinks containing them are useful for the prevention and treatment of diabetes.
また肥満では、脂肪組織において、例えばスーパーオキシドアニオンラジカル、ヒドロキシラジカル等のラジカルや過酸化水素、一重項酸素等の活性酸素種（ＲＯＳ）生成が促進し、酸化ストレスが亢進する。ＲＯＳは脂肪細胞の分化に重要な役割を果たしており、酸化ストレス下で脂肪組織が増加すると考えられる。本発明の式（Ｉ）および（ＩＩ）の化合物は、脂肪細胞におけるＲＯＳ及び一酸化窒素（ＮＯ）の生成を有効に抑制し、さらに活性酸素産生酵素であるキサンチンオキシダーゼ阻害活性を有するため、肥満の治療・予防に有効である。 In obesity, for example, radicals such as superoxide anion radicals and hydroxy radicals, and reactive oxygen species (ROS) such as singlet oxygen are promoted in adipose tissue, and oxidative stress is increased. ROS plays an important role in adipocyte differentiation, and it is considered that adipose tissue increases under oxidative stress. The compounds of the formulas (I) and (II) of the present invention effectively suppress the production of ROS and nitric oxide (NO) in adipocytes, and further have xanthine oxidase inhibitory activity which is an active oxygen producing enzyme. It is effective for treatment and prevention.
さらに本発明の式（Ｉ）および（ＩＩ）の化合物は、優れたチロシナーゼ阻害活性を有し、メラニン生成を有効に抑制するため、低色素沈着あるいは色素沈着過剰などの皮膚異常疾患に対する治療・予防に有効であり、またシミ、ソバカス等を防ぎ、美白効果に優れ、さらに安全性も高いものである。 Furthermore, the compounds of the formulas (I) and (II) of the present invention have excellent tyrosinase inhibitory activity and effectively suppress melanogenesis, so that the treatment / prevention for abnormal skin diseases such as hypopigmentation or hyperpigmentation In addition, it is effective in preventing stains, freckles, etc., has an excellent whitening effect, and has high safety.
本発明の式（Ｉ）および（ＩＩ）で表されるＭＴＤおよびびＴＭＯＱは、ゲットウ（月桃）（学名：Alpinia zerumbet）を水、低級アルコールなどの溶媒で抽出したゲットウ抽出物中から、単離、精製することにより取得することができる。 The MTD and TMOQ represented by the formulas (I) and (II) of the present invention are obtained from a ghetto extract obtained by extracting ghetto (moon peach) (scientific name: Alpinia zerumbet ) with a solvent such as water or a lower alcohol. It can be obtained by separation and purification.
このゲットウは、ショウガ科ハナミョウガ属（アルピニア属）の多年草で、熱帯から亜熱帯アジアに分布し、日本では沖縄県から九州南部に分布する。ゲットウの葉から取った精油が甘い香を放つので、アロマオイルや香料として使用されており、また、その葉や茎は抗菌剤や防虫剤としても知られているものである。 This ghetto is a perennial plant belonging to the genus Glyceraceae (Alpinia), distributed from the tropics to subtropical Asia, and in Japan from Okinawa Prefecture to the southern part of Kyushu. The essential oil taken from the leaves of ghetto gives off a sweet incense and is used as an aroma oil or fragrance, and its leaves and stems are also known as antibacterial and insect repellents.
式（Ｉ）および（ＩＩ）をゲットウから得るには、抽出物の原料として、ゲットウの根茎や種子を用いる。このゲットウの根茎や種子は、収穫したものをそのまま使用しても良いし、乾燥させたものを使用しても良い。この抽出原料は、好ましくは、風乾した後、適切な大きさに細断ないし粉砕し、次の抽出行程において使用する。 In order to obtain the formulas (I) and (II) from the ghetto, ghetto rhizomes and seeds are used as the raw material of the extract. As for the rhizomes and seeds of this ghetto, harvested ones may be used as they are, or dried ones may be used. This extraction raw material is preferably air-dried, then chopped or pulverized to an appropriate size, and used in the next extraction step.
次いで、上記のように準備した抽出原料に対し、その５ないし１００重量倍の抽出溶媒を加えた後、２０分ないし４８時間程度抽出を行う。抽出に用いる抽出溶媒としては、水や、エタノール等の低級アルコール、アセトン、酢酸エチル等の溶媒、あるいはこれらの混液等の溶媒（以下、「水性溶媒」という）が好ましい。上記水性溶媒のうち、混液としては、例えば、１０ないし９６％程度の、任意の割合のエタノール−水混液のような混合溶媒であっても良い。 Next, 5 to 100 times by weight of the extraction solvent is added to the extraction raw material prepared as described above, followed by extraction for about 20 minutes to 48 hours. The extraction solvent used for extraction is preferably water, a lower alcohol such as ethanol, a solvent such as acetone or ethyl acetate, or a solvent such as a mixture thereof (hereinafter referred to as “aqueous solvent”). Among the aqueous solvents, the mixed solution may be a mixed solvent such as an ethanol-water mixed solution having an arbitrary ratio of about 10 to 96%.
上記の抽出に当たっての抽出温度は室温が好ましく、抽出中、必要により連続あるいは間欠的に攪拌すればよい。 The extraction temperature for the above extraction is preferably room temperature, and may be stirred continuously or intermittently as necessary during extraction.
ＭＴＤおよびＴＭＯＱの単離、精製は、周知の単離、精製方法により行うことができる。 Isolation and purification of MTD and TMOQ can be performed by well-known isolation and purification methods.
すなわち、例えば、前記したゲットウ抽出物からカラムクロマトグラフィーを用い、例えば、勾配溶離を行うことにより、ＭＴＤおよびＴＭＯＱを単離精製することができる。 That is, for example, MTD and TMOQ can be isolated and purified by using column chromatography from the above-mentioned ghetto extract, for example, by performing gradient elution.
このようにして得られた、ＭＴＤおよびＴＭＯＱは、α−グルコシダーゼ、トリプシン、キサンチンオキシダーゼ及びチロシナーゼの活性阻害作用を有し、また、一酸化窒素生成の阻害作用も有する。さらに、ＭＴＤおよびＴＭＯＱは、ラジカル生成阻害作用及びＬＤＬ酸化阻害作用を有するものであり、特に脂肪細胞における活性酸素種（ＲＯＳ）及び一酸化窒素の生成を抑制し得る。 The MTD and TMOQ thus obtained have an activity inhibiting activity of α-glucosidase, trypsin, xanthine oxidase and tyrosinase, and also have an inhibitory effect on nitric oxide production. Furthermore, MTD and TMOQ have a radical production inhibitory action and an LDL oxidation inhibitory action, and can particularly suppress the production of reactive oxygen species (ROS) and nitric oxide in adipocytes.
以上のようにして得られる本発明のＭＴＤやＴＭＯＱは、上記のような効果を有するため、飲食品、医薬品及び医薬部外品等に利用可能である。特に、糖尿病及び動脈硬化を治療又は予防するための飲食品、医薬品、医薬部外品として用いることができる。また肥満やメタボリックシンドロームを治療又は予防するための飲食品、医薬品、医薬部外品として利用し得る。さらに低色素沈着あるいは色素沈着過剰などの皮膚異常疾患用の医薬品や美白用の医薬部外品、化粧料等とすることが可能である。 Since the MTD and TMOQ of the present invention obtained as described above have the effects as described above, they can be used for foods and drinks, pharmaceuticals, quasi drugs and the like. In particular, it can be used as a food / beverage product, a pharmaceutical product, or a quasi-drug for treating or preventing diabetes and arteriosclerosis. It can also be used as a food, drink, pharmaceutical, or quasi-drug for treating or preventing obesity and metabolic syndrome. Furthermore, it can be used as a pharmaceutical for skin abnormalities such as hypopigmentation or hyperpigmentation, a quasi-drug for whitening, and a cosmetic.
本発明の化合物を配合成分とした医薬の製造は、これを薬学的に許容される担体や添加剤と組み合わせ、軟膏剤、クリーム剤、乳剤、ゲル剤、ローション剤、貼付剤等の形態に調製すればよい。医薬品、医薬部外品の形態で使用する場合の投与形態は、経口であっても非経口であっても良い。経口投与による場合は、通常の経口投与製剤、例えば、錠剤、散剤、顆粒剤、カプセル剤等の固形剤；水剤；油性懸濁剤；又はシロップ剤もしくはエリキシル剤等の液剤のいずれかの剤形としても用いることができる。非経口投与による場合には、水性又は油性懸濁注射剤として用いることができる。 The manufacture of a pharmaceutical comprising the compound of the present invention as a compounding ingredient is combined with a pharmaceutically acceptable carrier or additive to prepare an ointment, cream, emulsion, gel, lotion, patch, etc. do it. When used in the form of a pharmaceutical or quasi drug, the dosage form may be oral or parenteral. In the case of oral administration, it is a usual oral preparation, for example, any solid agent such as tablets, powders, granules, capsules, etc .; a liquid agent; an oily suspension; or a liquid agent such as a syrup or elixir. It can also be used as a shape. In the case of parenteral administration, it can be used as an aqueous or oily suspension injection.
なお、本発明のＭＴＤやＴＭＯＱを飲食品に配合する形態や、経口医薬品などとして使用する形態とする場合の、その配合量は、特に制約はないが成人１日当たりの投与量として５〜２００ｍｇ程度、好ましくは５０〜１００ｍｇ程度が適当である。 The amount of the MTD or TMOQ of the present invention in a form to be blended in a food or drink or a form to be used as an oral medicine is not particularly limited, but is about 5 to 200 mg as a daily dose for an adult. Preferably, about 50 to 100 mg is appropriate.
一方、本発明のＭＴＤやＴＭＯＱを配合して美白化粧料とする場合には、例えば、公知の化粧料基剤にＭＴＤやＴＭＯＱを０．０００１〜１０質量％程度配合し、常法に従って、溶液状、可溶化状、乳化状、粉末状、ペースト状、ムース状、ジェル状の形態とすることにより製造され、化粧水、乳液、クリーム、パック、軟膏等として提供される。 On the other hand, in the case of blending the MTD or TMOQ of the present invention into a whitening cosmetic, for example, about 0.0001 to 10% by mass of MTD or TMOQ is blended with a known cosmetic base, , Solubilized, emulsified, powdered, pasty, mousse, and gel-like forms and provided as a lotion, emulsion, cream, pack, ointment, and the like.
また、上記美白化粧料の製造にあたっては、必要に応じて本発明の効果を損なわない範囲で、通常、化粧料に使用される成分、すなわち、精製水、アルコール類、水溶性高分子、油剤、界面活性剤、ゲル化剤、保湿剤、ビタミン類、抗菌剤、香料、塩類、ｐＨ調整剤等を加えることができる。 Further, in the production of the above-mentioned whitening cosmetics, components that are usually used in cosmetics, that is, purified water, alcohols, water-soluble polymers, oils, as long as the effects of the present invention are not impaired as necessary. Surfactants, gelling agents, humectants, vitamins, antibacterial agents, fragrances, salts, pH adjusters and the like can be added.
以下、実施例により本発明を更に詳細に説明するが、本発明はこれら実施例に何ら制約されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples at all.
実 施 例 １
ＭＴＤの抽出と単離：
琉球大学（沖縄県中頭郡西原町千原１）のキャンパスからゲットウ（Alpinia zerumbet）を採取し、ゲットウの根茎を抽出試料とした。
乾燥重量４００ｇの根茎をカンナで削り、これにメタノール２Ｌを加え、２日間、室温で抽出した。
Example 1
Extraction and isolation of MTD:
The ghetto ( Alpinia zerumbet ) was collected from the campus of the University of the Ryukyus (1 Chihara, Nishihara-cho, Nakagami-gun, Okinawa Prefecture), and the rhizome of the ghetto was used as an extraction sample.
A rhizome having a dry weight of 400 g was shaved with a canna, and 2 L of methanol was added thereto, followed by extraction at room temperature for 2 days.
抽出溶媒を留去させた後、蒸留水３００ｍＬに乾燥抽出物５．７gを溶かし、ヘキサン３００ｍＬで脱脂した。脱脂後の水性抽出物２．９ｇをクロロホルム２００ｍＬとそれに続くエチルアセテート２００ｍＬで分画し、エチルアセテート画分１．６ｇをシリカゲルが入ったガラスクロマトグラフィーカラム（Ｓｉｌｉｃａ ｇｅｌ ６０Ｎ, ｐａｒｔｉｃｌｅ ｓｉｚｅ ６３−１２０μｍ，７０−２３０ ｍｅｓｈ ＡＳＴＭ)に付し、石油エーテル：クロロホルム（０−１００％）の直線勾配で溶出し、３つの画分を得た。２番目の画分をＨＰＬＣを使用して更に精製した。 After the extraction solvent was distilled off, 5.7 g of the dried extract was dissolved in 300 mL of distilled water and degreased with 300 mL of hexane. 2.9 g of the degreased aqueous extract was fractionated with 200 mL of chloroform and then with 200 mL of ethyl acetate, and 1.6 g of the ethyl acetate fraction was fractionated with a glass chromatography column containing silica gel (Silica gel 60N, particle size 63-120 μm, 70-230 mesh ASTM) and eluted with a linear gradient of petroleum ether: chloroform (0-100%) to give three fractions. The second fraction was further purified using HPLC.
次にＳｙｎｅｒｇｉ ４ｕ ＭＡＸ−ＲＰ ８０Ａ ｃｏｌｕｍｎ（１５０ｘ４．６０ｍｍ,４ｍｉｃｒｏｎ；Ｐｈｅｎｏｍｅｎｅｘ, Ｔｏｒｒａｎｃｅ, ＣＡ)を用い、２８０ｎｍで単離化合物を収集した。移動相は、０．１％酢酸溶液（ｖ／ｖ）(溶液Ａ)と０．１％酢酸を含むアセトニトリル(溶液Ｂ)を用い、流速を１．００ｍＬ/ｍｉｎとした。グラジエント溶離の条件は、０〜７分間：溶液Ｂの４０〜７０％勾配、７〜２０分間：溶液Ｂの７０〜１００％直線勾配、２０〜３０分間：溶液Ｂの１００％定組成溶離とした。分画１ｍｇ/ｍＬのメタノール溶液５μＬを化合物単離に使用した。ＭＴＤを含む画分を図１に示す。
The isolated compounds were then collected at 280 nm using Synergi 4u MAX-RP 80A column (150 × 4.60 mm, 4 micron; Phenomenex, Torrance, Calif.). As the mobile phase, 0.1% acetic acid solution (v / v) (solution A) and acetonitrile containing 0.1% acetic acid (solution B) were used, and the flow rate was 1.00 mL / min. Gradient elution conditions were 0-7 minutes: 40-70% gradient of solution B, 7-20 minutes: 70-100% linear gradient of solution B, 20-30 minutes: 100% isocratic elution of solution B. .
また、ＭＴＤの物理化学的性質を以下に示す。
HREIMS m/z 285.1 [M+] (calcd for C18H22O3, 286.16). IR v (KBr) cm-1: 669, 1646, 2341, 2359. 1H-NMR (500 MHz, MeOD-d4): 3.83 (q, 3H, OCH3, J = 5.5 Hz, 9), 5.62 (d, 1H, OCH, 2), 6.24 (d, 1H, CH, 7), 6.86 (d, 1H, CH, 6), 7.58 (s, 1H, CH, 5), 7.59 (d, 1H, CH, 4), 7.14 (q, 1H, CH, J = 5.0 Hz, 1), 7.41 (t, 1H, CH, J = 7.0 Hz, 3), 7.36 (t, 1H, CH, J = 4.0 Hz, 8). 13C-NMR (500 MHz, MeOD-d4): 57.01 (C-9), 89.42 (C-2), 102.78 (C-7), 120.05 (C-6), 128.63 (C-5), 129.14 (C-4), 129.99 (C-3), 130.55 (C-1), 136.66 (C-8)
The physicochemical properties of MTD are shown below.
HREIMS m / z 285.1 [M + ] (calcd for C 18 H 22 O 3 , 286.16). IR v (KBr) cm -1 : 669, 1646, 2341, 2359. 1 H-NMR (500 MHz, MeOD-d 4 ): 3.83 (q, 3H, OCH 3 , J = 5.5 Hz, 9), 5.62 (d, 1H, OCH, 2), 6.24 (d, 1H, CH, 7), 6.86 (d, 1H, CH, 6), 7.58 (s, 1H, CH, 5), 7.59 (d, 1H, CH, 4), 7.14 (q, 1H, CH, J = 5.0 Hz, 1), 7.41 (t, 1H, CH, J = 7.0 Hz, 3), 7.36 (t, 1H, CH, J = 4.0 Hz, 8) 13 C-NMR (500 MHz, MeOD-d 4):. 57.01 (C-9), 89.42 (C-2) , 102.78 (C-7), 120.05 (C-6), 128.63 (C-5), 129.14 (C-4), 129.99 (C-3), 130.55 (C-1), 136.66 (C-8)
実 施 例 ２
ＴＭＯＱの抽出と単離：
ゲットウの種子１００ｇを乳鉢で粉砕して、二日間、室温でメタノール５００ｍＬに浸漬し抽出した。濾過後、その濾液からメタノールを留去させ、濃いシロップ状の抽出物２１．６ｇ得た。この抽出物を蒸留水５００ｍＬに懸濁し、ヘキサン５００ｍＬとエチルアセテート５００ｍＬで分画した。エチルアセテート抽出画分１１．０７ｇを、シリカゲルを含むガラスクロマトグラフィーカラム（Ｓｉｌｉｃａ ｇｅｌ ６０Ｎ, ｐａｒｔｉｃｌｅ ｓｉｚｅ ６３−１２０μｍ, ７０−２３０ ｍｅｓｈ ＡＳＴＭ）に付し、１％から５０％へ段階勾配的にクロロホルムにメタノールを混合し溶出させた。４画分が溶出したが、このうち画分４を、実施例１のＨＰＬＣと同じカラム及び同じ条件で更に精製した。得られたＴＭＯＱ画分を図２に示す。
Example 2
Extraction and isolation of TMOQ:
100 g of ghetto seeds were pulverized in a mortar and immersed in 500 mL of methanol at room temperature for 2 days for extraction. After filtration, methanol was distilled off from the filtrate to obtain 21.6 g of a thick syrupy extract. This extract was suspended in 500 mL of distilled water and fractionated with 500 mL of hexane and 500 mL of ethyl acetate. The ethyl acetate extract fraction (11.07 g) was applied to a glass chromatography column containing silica gel (Silica gel 60N, particle size 63-120 μm, 70-230 mesh ASTM), and gradually changed from 1% to 50% to chloroform. Methanol was mixed and eluted. Four fractions eluted, among which
また、ＴＭＯＱの物理化学的性質を以下に示す。
HREIMS m/z 317.55 [M+] (calcd for C22H39N, 317.2). IR v (KBr) cm-1: 1024, 1121, 1456, 1507, 1541, 1558, 1646, 1698, 1748, 2360, 2927, 3448. 1H-NMR (500 MHz, MeOD-d4): 0.75 (q, 3H, CH3, J = 10.5 Hz, 20), 0.85 (t, 3H, CH3, J = 1.5 Hz, 18), 0.89 (q, 3H, CH3, J = 4.0 Hz, 21), 0.94 (t, 3H, CH3, J = 2.5 Hz, 1), 1.09 (t, 2H, CH2, J = 2.5, 8), 1.17 (q, 2H, CH2, 11), 1.22 (q, 2H, CH2, J = 1.5 Hz, 14), 1.28 (s, 2H, CH2, 14), 1.39 (q, 1H, NCH, J = 2.5 Hz, 12), 1.43 (q, 2H, CH2, J = 2.2 Hz, 4), 1.58 (t, 3H, CH3, J = 2.0 Hz, 19), 1.64 (q, 2H, CH2, J = 4.5 Hz, 7), 1.75 (q, 2H, CH2, J = 2.0 Hz, 5), 2.00 (d, 2H, NCH2, 13), 2.26 (d, 2H, CH2, 6), 2.47 (t, 2H, NCH2, J = 5.5 Hz, 17), 3.27 (t, 2H, CH2, J = 8.0 Hz, 22), 6.02 (q, 1H, CH, 3) and 6.36 (d, 1H, CH, 2). 13C-NMR (500 MHz, MeOD-d4): 14.00 (C- 21), 15.48 (C-20), 20.16 (C-4), 20.39 (C-19), 22.37 (C-18), 24.53 (C-5), 31.77 (C-7), 34.02 (C-15), 34.50 (C- 1), 37.79 (C-6), 39.90 (C-16), 40.36 (C-8), 42.03 (C-11), 43.39 (C-14), 55.96 (C-9), 63.34 (C-12), 65.83 (C-17), 68.81 (C-13), 108.85 (C-22), 122.20 (C-2), 135.00 (C- 3), 150.77 (C-10)
The physicochemical properties of TMOQ are shown below.
HREIMS m / z 317.55 [M + ] (calcd for C 22 H 39 N, 317.2) .IR v (KBr) cm -1 : 1024, 1121, 1456, 1507, 1541, 1558, 1646, 1698, 1748, 2360, 2927, 3448. 1 H-NMR (500 MHz, MeOD-d 4 ): 0.75 (q, 3H, CH 3 , J = 10.5 Hz, 20), 0.85 (t, 3H, CH 3 , J = 1.5 Hz, 18 ), 0.89 (q, 3H, CH 3 , J = 4.0 Hz, 21), 0.94 (t, 3H, CH 3 , J = 2.5 Hz, 1), 1.09 (t, 2H, CH 2 , J = 2.5, 8 ), 1.17 (q, 2H, CH 2, 11), 1.22 (q, 2H, CH 2 , J = 1.5 Hz, 14), 1.28 (s, 2H, CH 2 , 14), 1.39 (q, 1H, NCH , J = 2.5 Hz, 12), 1.43 (q, 2H, CH 2 , J = 2.2 Hz, 4), 1.58 (t, 3H, CH 3 , J = 2.0 Hz, 19), 1.64 (q, 2H, CH 2 , J = 4.5 Hz, 7), 1.75 (q, 2H, CH 2 , J = 2.0 Hz, 5), 2.00 (d, 2H, NCH 2 , 13), 2.26 (d, 2H, CH 2 , 6) , 2.47 (t, 2H, NCH 2 , J = 5.5 Hz, 17), 3.27 (t, 2H, CH 2 , J = 8.0 Hz, 22), 6.02 (q, 1H, CH, 3) and 6.36 (d, 13 C-NMR (500 MHz, MeOD-d 4 ): 14.00 (C-21), 15.48 (C-20), 20.16 (C-4), 20.39 (C-19), 22.37 (C-18), 24.53 (C-5), 31.77 (C-7), 34.02 (C-15), 34.50 (C- 1), 37.79 (C-6), 39.90 (C-16), 40.36 ( C-8), 42.03 (C-1 1), 43.39 (C-14), 55.96 (C-9), 63.34 (C-12), 65.83 (C-17), 68.81 (C-13), 108.85 (C-22), 122.20 (C-2 ), 135.00 (C-3), 150.77 (C-10)
実 施 例 ３
α−グルコシダーゼ活性阻害試験：
α−グルコシダーゼ活性阻害試験は、Ａｈｍａｄ ｅｔ ａｌ. （２０１１）が報告した方法を多少改変した手法により行った。まず、９６穴プレートに異なる濃度サンプル１５μＬと酵素溶液１４０μＬ（α−グルコシダーゼ０．００７３Ｕ/ｍＬ；１００ｍＭ ＮａＣｌ含有０．０５Ｍ リン酸ナトリウム緩衝液）を加え、１５分間、３７℃でインキュベートした。その後、０．０５Ｍ リン酸ナトリウム緩衝液（ｐＨ６．８）に０．７ｍＭ ＰＮＰ−Ｇを含む溶液２５μＬをそれぞれのウェルに加えた。α-グルコシダーゼ活性は、酵素加水分解によりＰＮＧ−Ｐ（マルトース）から形成されるグルコース量を分光光度計（ＵＶ ｍｉｎｉ １２４０, Ｓｈｉｍａｄｚｕ, Ｋｙｏｔｏ, Ｊａｐａｎ）を用いて、４０５ｎｍで測定することにより決定した。陽性コントロールとして、ケルセチン及びカテキンを使用した。
Example 3
α-Glucosidase activity inhibition test:
The α-glucosidase activity inhibition test was carried out by a method slightly modified from the method reported by Ahmad et al. (2011). First, 15 μL of a sample having a different concentration and 140 μL of an enzyme solution (α-glucosidase 0.0073 U / mL; 0.05 mM sodium phosphate buffer containing 100 mM NaCl) were added to a 96-well plate and incubated at 37 ° C. for 15 minutes. Thereafter, 25 μL of a solution containing 0.7 mM PNP-G in 0.05 M sodium phosphate buffer (pH 6.8) was added to each well. The α-glucosidase activity was determined by measuring the amount of glucose formed from PNG-P (maltose) by enzymatic hydrolysis at 405 nm using a spectrophotometer (UV mini 1240, Shimadzu, Kyoto, Japan). Quercetin and catechin were used as positive controls.
結果：
ＴＭＯＱとＭＴＤのＩＣ５０は、それぞれ１．６２および１．６４μｇ／ｍＬであった。これら化合物は、陽性コントロールのケルセチン（１．６２μｇ／ｍＬ）と同程度の阻害効果を有することが分かった。結果を図３に示す。
result:
The IC 50 for TMOQ and MTD were 1.62 and 1.64 μg / mL, respectively. These compounds were found to have an inhibitory effect comparable to that of the positive control quercetin (1.62 μg / mL). The results are shown in FIG.
実 施 例 ４
トリプシン活性阻害試験：
トリプシン活性阻害試験は、基質としてＢＡＰＮＡを用い、ＷＡＴＩら（２００９）の方法で測定した。まず、サンプル１００μＬ、２０μｇ／ｍＬのトリプシン２００μＬおよび蒸留水１００μＬを含んだ溶液を３７℃で、１０分間インキュベートした。これに、あらかじめ３７℃でプレウォームした０．４ｍｇ／ｍＬのＢＡＰＮＡ５００μＬを加え、反応を開始させた。１０分間、３７℃でインキュベーションした後、１００μＬの３０％酢酸（ｖ／ｖ）を加えて反応を終了させ、遠心分離（１０分、４℃、２０００ｇ）にかけた。トリプシン活性は、吸光度４１０ｎｍでｐ−ニトロアニリン量を測定することにより決定した。陽性コントロールとして、ケルセチン及びカテキンを使用した。
Example 4
Trypsin activity inhibition test:
The trypsin activity inhibition test was measured by the method of WATI et al. (2009) using BAPNA as a substrate. First, a solution containing 100 μL of a sample, 200 μL of 20 μg / mL trypsin and 100 μL of distilled water was incubated at 37 ° C. for 10 minutes. To this, 500 μL of 0.4 mg / mL BAPNA pre-warmed at 37 ° C. was added to start the reaction. After incubation at 37 ° C. for 10 minutes, 100 μL of 30% acetic acid (v / v) was added to terminate the reaction, followed by centrifugation (10 minutes, 4 ° C., 2000 g). Trypsin activity was determined by measuring the amount of p-nitroaniline at an absorbance of 410 nm. Quercetin and catechin were used as positive controls.
結果：
ＴＭＯＱとＭＴＤのＩＣ５０は、それぞれ３１．７５および４１．４８μｇ／ｍＬであった。ＴＭＯＱは、陽性コントロールであるケルセチン（３４．６８μｇ／ｍＬ）よりも阻害効果が高いことが分かった。結果を図４に示す。
result:
The IC 50 for TMOQ and MTD were 31.75 and 41.48 μg / mL, respectively. TMOQ was found to have a higher inhibitory effect than quercetin (34.68 μg / mL), which is a positive control. The results are shown in FIG.
実 施 例 ５
一酸化窒素生成阻害実験：
一酸化窒素阻害実験はＧｒｉｅｓｓ Ｉｌｌｏｓｖｏｙ ｒｅａｃｔｉｏｎ（Ｇｏｖｉｎｄａｒａｊａｎら、２００３）を用いて行った。まず、１０ｍＭ ＳＮＰ２００μＬ、リン酸緩衝生理食塩水５０μＬ及びサンプル５０μＬを含んだ反応混合溶液３００μＬを１５０分間、２５℃でインキュベートした。次いで、反応混合溶液５０μＬを０．３３％スルファニル酸（２０％氷酢酸中）１００μＬと混合し、５分間室温に置いて完全にジアゾ化させ、ＮＥＤＨ１００μＬを添加し、混合して２５℃で、３０分放置した。放置後の吸光度を５４０ｎｍで測定した。陽性コントロールとして、カテキンとケルセチンを使用した。
Example 5
Nitric oxide production inhibition experiment:
Nitric oxide inhibition experiments were performed using Griess Illosvoy reaction (Govindarajan et al., 2003). First, 300 μL of a reaction mixed solution containing 200 μL of 10 mM SNP, 50 μL of phosphate buffered saline and 50 μL of sample was incubated at 25 ° C. for 150 minutes. Next, 50 μL of the reaction mixture was mixed with 100 μL of 0.33% sulfanilic acid (in 20% glacial acetic acid), allowed to completely diazotize for 5 minutes at room temperature, 100 μL of NEDH was added, mixed and mixed at 25 ° C., 30 Left for a minute. The absorbance after standing was measured at 540 nm. Catechin and quercetin were used as positive controls.
結果：
ＴＭＯＱのＩＣ５０は１５．７０μｇ／ｍＬであった。また、ＭＴＤのＩＣ５０は、３０μｇ／ｍＬであった。ＴＭＯＱは、陽性コントロールのケルセチン（１８．９０μｇ／ｍＬ）より高い効果を有することが分かった。結果を図５に示す。
result:
IC 50 of TMOQ was 15.70μg / mL. Further, IC 50 of MTD was 30 [mu] g / mL. TMOQ was found to have a higher effect than the positive control quercetin (18.90 μg / mL). The results are shown in FIG.
実 施 例 ６
キサンチンオキシダーゼ活性阻害試験：
０．１ｍＭ ＥＤＴＡ含有５０ｍＭリン酸ナトリウムバッファー（ｐＨ７．４）に、キサンチン１００μＭ、馬心臓シトクロムｃ２５μＭ及び異なる濃度のサンプルを添加し、反応混合液を作成した。この反応混合液に、キサンチンオキシダーゼ(０．０７Ｕ／ｍＬ)を添加し、反応を開始させた。２分後、分光光度計を用いて５５０ｎｍで還元されたシトクロムｃを測定した。生成したスーパーオキシドの量は、ε＝２１，１００ Ｍ−１ｃｍ−１を使用して計算した。陽性コントロールとしてＬ−ＮＡＭＥ（ＮＧ-ニトロアルギニンメチルエステル）を使用した。
Example 6
Xanthine oxidase activity inhibition test:
To 50 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM EDTA,
結果：
ＴＭＯＱのＩＣ５０は１４．５１μＭであった。また、ＭＴＤのＩＣ５０は、１４０μＭであった。ＴＭＯＱは、陽性コントコロールのＬ−ＮＡＭＥ(ＩＣ５０＝１２．８８μＭ)とほぼ同等の効果を有することが分かった。結果を図６に示す。
result:
IC 50 of TMOQ was 14.51μM. In addition, IC 50 of MTD was 140μM. TMOQ was found to have almost the same effect as positive control col L-NAME (IC 50 = 12.88 μM). The results are shown in FIG.
実 施 例 ７
ＬＤＬ酸化活性阻害試験：
ＬＤＬ酸化活性阻害試験は、ＲａｔｔａｎとＡｒａｄ（１９９８）の方法により行った。まず、１０ｍＭ ＰＢＳ（ｐＨ７．４）によりＬＤＬを２２０μｇ／ｍＬに調整し、一定量の異なる濃度のサンプルを添加した。ＬＤＬの酸化反応は、５５μＭ ＣｕＳＯ４の添加で開始し、３７℃で２４時間インキュベートした。その後、1Ｍ ＥＤＴＡ５０μＬを添加して反応を停止させ、Ｓｔｅｉｎｂｒｅｃｈｅｒら（１９８４）の方法によりＴＢＡＲＳ活性のため２０℃でサンプルを静置した。酸化後、ＬＤＬに０．６７％ＴＢＡ１．５ｍＬと２０％ＴＣＡ１．５ｍＬを混合し、１００℃、３０分でサンプルを静置した。反応物は３０分、２５℃で保管され、１５分、４℃、２００ｇで遠心分離した。上澄を５３２ｎｍで吸光度を測定した。
Example 7
LDL oxidation activity inhibition test:
The LDL oxidation activity inhibition test was performed by the method of Rattan and Arad (1998). First, LDL was adjusted to 220 μg / mL with 10 mM PBS (pH 7.4), and a certain amount of samples with different concentrations were added. The oxidation reaction of LDL was started by the addition of 55 μM CuSO 4 and incubated at 37 ° C. for 24 hours. Thereafter, 50 μL of 1M EDTA was added to stop the reaction, and the sample was allowed to stand at 20 ° C. for TBARS activity according to the method of Steinbrecher et al. (1984). After oxidation, 1.5 mL of 0.67% TBA and 1.5 mL of 20% TCA were mixed in LDL, and the sample was allowed to stand at 100 ° C. for 30 minutes. The reaction was stored for 30 minutes at 25 ° C. and centrifuged for 15 minutes at 4 ° C. and 200 g. The absorbance of the supernatant was measured at 532 nm.
結果：
ＭＴＤのＩＣ５０は、２０μＭであった。ＴＭＯＱのＩＣ５０は、２．１０μＭであり、陽性コントロールのケルセチンよりも高い効果を示した。結果を図７に示す。
result:
The MTD IC 50 was 20 μM. IC 50 of TMOQ is 2.10MyuM, it showed higher effect than quercetin positive control. The results are shown in FIG.
実 施 例 ８
ラジカル生成阻害試験：
（１）ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）の準備：
ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）を以下の方法で準備した。
ＤＳファーマバイオメディカル株式会社（大阪府、日本）から、ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）(Lot:121210/ 255902-1R, No.12002053)とＣＳＣ完全組み換え培地を購入した。細胞は付属因子を含んだＣＳＣ培地で維持され、３７℃、５％ＣＯ２の加湿雰囲気で増殖させた。
Example 8
Radical formation inhibition test:
(1) Preparation of human umbilical vein endothelial cells (HUVEC):
Human umbilical vein endothelial cells (HUVEC) were prepared by the following method.
Human umbilical vein endothelial cells (HUVEC) (Lot: 121210 / 255902-1R, No. 12002053) and CSC complete recombinant medium were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Cells were maintained in CSC medium containing accessory factors and grown in a humidified atmosphere at 37 ° C., 5% CO 2 .
ゼラチンでコーティングしたフラスコに、２０％ＦＢＳ、５Ｕ／ｍＬヘパリン、１ｍＬ Ｌ−グルタミン＋ストレプトマイシン＋ペニシリン、１μｇ／ｍＬヒドロコルチソン、５０μｇ／ｍＬ内皮細胞成長サプリメント及び１０μｇ／ｍＬヒト表皮成長因子を添加した培地１９９を注ぎ、同培地に２〜４継代したＨＵＶＥＣ細胞（１０６細胞）及び各サンプル５０μＭを加え、２４時間インキュベートした。インキュベート後、培地を除去し、細胞を５ｍＬＰＢＳで洗浄した。５ｍＬ０．２５％トリプシン-ＥＤＴＡで細胞を収集し、遠心分離し（５分、１０００ｇ、４℃）、細胞を回収した。 20% FBS, 5 U / mL heparin, 1 mL L-glutamine + streptomycin + penicillin, 1 μg / mL hydrocortisone, 50 μg / mL endothelial cell growth supplement and 10 μg / mL human epidermal growth factor were added to a gelatin coated flask. Medium 199 was poured, HUVEC cells (10 6 cells) passaged 2 to 4 and 50 μM of each sample were added to the same medium, and incubated for 24 hours. After incubation, the medium was removed and the cells were washed with 5 mL PBS. Cells were harvested with 5 mL 0.25% trypsin-EDTA, centrifuged (5 min, 1000 g, 4 ° C.) and cells were harvested.
（２）Ｏ２ ・−生産の阻害試験：
上記回収した細胞（ＨＵＶＥＣ）を、５０ｍＭリン酸バッファー（ｐＨ７．４）によって洗浄し、４０μＭフェリシトクロムｃ含有ＨＥＰＥＳ緩衝等張塩培地（１３３ｍＭ ＮａＣｌ、６．５ｍＭ ＫＣｌ、１ｍＭ ＣａＣｌ２、１ｍＭ ＭｇＣｌ２、５．５ｍＭグルコース、５０μＭ Ｌ−アルギニン、２０ｍＭ ＨＥＰＥＳ、ｐＨ ７．４）を用いて、６時間、３７℃で１μＭアンジオテンシンＩＩを作用させた。フェリシトクロムｃの減少は、上澄みの５５０ｎｍにおける吸光度によって測定された。Ｏ２ ・−放出はＳＯＤ（スーパーオキシドジスムターゼ；２００Ｕ／ｍＬ）添加とＳＯＤ無添加を用意しその差から計算された（Ｓｔｅｆｆｅｎら、２００８）。
(2) O 2 . - Production inhibition test:
The collected cells (HUVEC) were washed with 50 mM phosphate buffer (pH 7.4), and 40 μM ferricytochrome c-containing HEPES buffered isotonic salt medium (133 mM NaCl, 6.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 μM angiotensin II was allowed to act at 37 ° C. for 6 hours using 5.5 mM glucose, 50 μM L-arginine, 20 mM HEPES, pH 7.4). The decrease in ferricytochrome c was measured by the absorbance at 550 nm of the supernatant. The release of O 2 · − was calculated from the difference between SOD (superoxide dismutase; 200 U / mL) added and no SOD added (Steffen et al., 2008).
結果：
ＭＴＤ及びＴＭＯＱのＯ２ ・−生産は、サンプル未処理のコントロールに対して、９５．９４±０．３９及び８４．２３±０．５２％であった。結果を図８に示す。
result:
The MT 2 and TMOQ O 2 − production was 95.94 ± 0.39 and 84.23 ± 0.52% relative to the untreated sample control. The results are shown in FIG.
実 施 例 ９
活性酸素種（ＲＯＳ）及び一酸化窒素（ＮＯ）生成阻害試験：
（１）細胞として、３Ｔ３−Ｌ１細胞（アメリカン・タイプ・カルチュア・コレクション（ＡＴＣＣ）から入手）を用い、これを、２％のグルタミンと、１０（ｖ／ｖ）％のウシ胎児血清（ＣＳ）を含むダルベッコ改変イーグル培地（ＤＭＥＭ）中、コンフルエントになるまで培養した。コンフルエンシーに達した２日後に、細胞は、さらに２日間、１０％ＦＢＳ、０.５ｍＭ３−イソブチル−１−メトキシキサンチン（ＩＢＭＸ）、１μＭデキサメサゾンおよび１０μＭインシュリンを含むＤＭＥＭ培地中で培養し脂肪細胞に分化するように刺激された。細胞は、それから更に、２日間、１０％ＦＢＳと１０μｇ／ｍＬインシュリンを含むＤＭＥＭ中で維持され、更に４日間、１０％ＦＢＳのみを含むＤＭＥＭで培養された。
この結果、細胞の９０％以上は、脂質滴が蓄積された３Ｔ３−Ｌ１脂肪細胞に分化していた。分化した３Ｔ３−Ｌ１細胞は、異なった濃度の試験化合物で処理され、試験中を通して５％のＣＯ２を含む加湿されたインキュベーター中で、３７℃に維持した。
Example 9
Reactive oxygen species (ROS) and nitric oxide (NO) production inhibition test:
(1) 3T3-L1 cells (obtained from American Type Culture Collection (ATCC)) were used as cells, and these were used as 2% glutamine and 10 (v / v)% fetal calf serum (CS). In Dulbecco's modified Eagle medium (DMEM) containing Two days after reaching confluency, the cells were cultured in DMEM medium containing 10% FBS, 0.5 mM 3-isobutyl-1-methoxyxanthine (IBMX), 1 μM dexamethasone and 10 μM insulin for 2 days. Stimulated to differentiate. The cells were then maintained in DMEM containing 10% FBS and 10 μg / mL insulin for a further 2 days and cultured in DMEM containing only 10% FBS for a further 4 days.
As a result, 90% or more of the cells were differentiated into 3T3-L1 adipocytes in which lipid droplets were accumulated. Differentiated 3T3-L1 cells were treated with different concentrations of test compound and maintained at 37 ° C. in a humidified incubator containing 5% CO 2 throughout the test.
（２）３Ｔ３−Ｌ１細胞生存性
細胞生存率を、ＭＴＴ（３−（４，５−ジメチルチアゾール−２−イル）−２，５−ジフェニルテトラゾリウムブロミド）アッセイにより測定した。３Ｔ３−Ｌ１前脂肪細胞を９６ウェルプレートに１×１０４細胞／ウェルの密度で播種し、培養培地中で培養した。次いで、細胞を１００又は２５０μｇ／ｍＬの濃度の試験化合物により処理した。７２時間後、暗所にて細胞を３７℃で４時間ＭＴＴ溶液でインキュベートした。上清を吸引し、ジメチルスルホキシド（ＤＭＳＯ）を各ウェルに添加し、プレートを撹拌してホルマザン結晶生成物を溶解させた。マイクロプレート分光光度計（Bio-Rad Laboratories, Inc.）を用いて５７０ｎｍにおける吸光度を測定した。未処理の場合を１００％として、細胞生存率を求めた。結果を図９に示す。
(2) 3T3-L1 cell viability Cell viability was measured by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay. 3T3-L1 preadipocytes were seeded in 96 well plates at a density of 1 × 10 4 cells / well and cultured in culture medium. Cells were then treated with test compounds at a concentration of 100 or 250 μg / mL. After 72 hours, the cells were incubated with MTT solution for 4 hours at 37 ° C. in the dark. The supernatant was aspirated, dimethyl sulfoxide (DMSO) was added to each well, and the plate was agitated to dissolve the formazan crystal product. Absorbance at 570 nm was measured using a microplate spectrophotometer (Bio-Rad Laboratories, Inc.). The cell viability was determined with the untreated case as 100%. The results are shown in FIG.
（３）細胞内活性酸素種（ＲＯＳ）測定
細胞を９６ウェルプレートに２×１０６細胞／ｍＬの密度で播種し、上記と同様にしてコンフルエントまで培養し、分化させた。ＲＯＳ生成は、ニトロブルーテトラゾリウム（ＮＢＴ）アッセイによって検出した（Oliveira, H.R.; Verlengia, R.; Carvalho, C.R.; Britto L.R.; Curi, R.; Carpinelli, A.R. Pancreatic β-cells express phagocyte-like NAD(P)H oxidase. J. Diabetes. 2003, 52, 1457-1463.）。ＮＢＴは、ＲＯＳにより還元され、ホルマザンと呼ばれる暗青色で不溶性形態になる。分化後、細胞を１０又は２０μｇ／ｍＬの濃度の試験化合物とともに２４時間インキュベートした。次いで、細胞を０．２％ＮＢＴ含有ＰＢＳ１００μＬ中で９０分間インキュベートした。暗青色のホルマザンを５０％酢酸に溶解し、５７０ｎｍにおける吸光度を測定した。結果を図１０に示す。
(3) Measurement of intracellular reactive oxygen species (ROS) Cells were seeded in a 96-well plate at a density of 2 × 10 6 cells / mL, cultured to confluence and differentiated as described above. ROS production was detected by a nitroblue tetrazolium (NBT) assay (Oliveira, HR; Verlengia, R .; Carvalho, CR; Britto LR; Curi, R .; Carpinelli, AR Pancreatic β-cells express phagocyte-like NAD (P ) H oxidase. J. Diabetes. 2003, 52, 1457-1463.). NBT is reduced by ROS to a dark blue insoluble form called formazan. After differentiation, cells were incubated for 24 hours with test compounds at a concentration of 10 or 20 μg / mL. Cells were then incubated for 90 minutes in 100 μL PBS containing 0.2% NBT. Dark blue formazan was dissolved in 50% acetic acid, and the absorbance at 570 nm was measured. The results are shown in FIG.
（４）細胞内一酸化窒素（ＮＯ）生成測定
上記と同様にして、細胞を９６ウェルプレートに播種し、分化させた。亜硝酸塩生成（ＮＯ２）アッセイを用いて測定した( Fang, X.K.; Gao, J.; Zhu, D.N. Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity. J. Life Sci. 2008, 82, 615-622.)。細胞を１０又は２０μｇ／ｍＬの濃度の試験化合物とともに２４時間インキュベートした。上清（１００μＬ）及びグリース試薬（１００μＬ、１％スルファニルアミドと０．１％ナフチルエチレンジアミン二塩酸塩含有５％リン酸の１：１混合物（ｖ／ｖ））を、９６ウェルプレート中で混合し、室温で１０分間インキュベートした。マイクロプレート分光光度計を用いて５４０ｎｍにおける吸光度を測定し、亜硝酸ナトリウムで作成した標準曲線により亜硝酸塩濃度を推定した。結果を図１１に示す。
(4) Measurement of intracellular nitric oxide (NO) production In the same manner as described above, cells were seeded in a 96-well plate and differentiated. Measured using a nitrite production (NO 2 ) assay (Fang, XK; Gao, J .; Zhu, DN Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity. J. Life Sci. 2008, 82, 615-622.). Cells were incubated with test compounds at a concentration of 10 or 20 μg / mL for 24 hours. Supernatant (100 μL) and Grease reagent (100 μL, 1: 1 mixture of 1% sulfanilamide and 5% phosphoric acid containing 0.1% naphthylethylenediamine dihydrochloride (v / v)) were mixed in a 96 well plate. Incubated for 10 minutes at room temperature. Absorbance at 540 nm was measured using a microplate spectrophotometer, and the nitrite concentration was estimated from a standard curve prepared with sodium nitrite. The results are shown in FIG.
結果：
ＭＴＤ及びＴＭＯＱは、脂肪細胞におけるＲＯＳ生成を強く阻害した。濃度２０μｇ／ｍＬにおける阻害率は、ＭＴＤが５９．５±１．９０％，ＴＭＯＱは５２．５±１．１０％であった。またＮＯ生成も有意に抑制し、ＮＯ生成量は、ＭＴＤにより７１．１±０．８１％、ＴＭＯＱにより５７．７±０．５８％に減少した。一方、ＭＴＤ及びＴＭＯＱは、１００μｇ／ｍＬの濃度で３Ｔ３−Ｌ１細胞生存性に対してほとんど影響を示さず、２５０μｇ／ｍＬでもわずかな生存率の低下しか認められなかった（ＭＴＤ：６．９±２．４９％、ＴＭＯＱ：６．９±１．２８％）。
result:
MTD and TMOQ strongly inhibited ROS production in adipocytes. The inhibition rate at a concentration of 20 μg / mL was 59.5 ± 1.90% for MTD and 52.5 ± 1.10% for TMOQ. NO production was also significantly suppressed, and the NO production amount was reduced to 71.1 ± 0.81% by MTD and 57.7 ± 0.58% by TMOQ. On the other hand, MTD and TMOQ showed little effect on 3T3-L1 cell viability at a concentration of 100 μg / mL, and only a slight decrease in viability was observed at 250 μg / mL (MTD: 6.9 ±). 2.49%, TMOQ: 6.9 ± 1.28%).
実 施 例 １０
メラニン生成抑制作用：
（１）マウスＢ１６Ｆ１０メラノーマ細胞（ＡＴＣＣより入手）を、３７℃にて、１０％熱不活化ウシ胎児血清（ＦＢＳ）および１％ペニシリン／ストレプトマイシン（１０．０００Ｕ／１００μｇ／ｍＬ）を加えたＤＭＥＭ中で５％ＣＯ２の加湿雰囲気下で培養した。
Example 10
Inhibition of melanin production:
(1) Mouse B16F10 melanoma cells (obtained from ATCC) in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin (10.000 U / 100 μg / mL) at 37 ° C. And in a humidified atmosphere of 5% CO 2 .
（２）Ｂ１６Ｆ１０細胞生存性
細胞生存率はＭＴＴアッセイを用いて測定した（Campos, P.M.; da Silva Horinouchi, C.D.; da Silveira Prudente, A.; Cechinel-Filho, V.; de Almeida Cabrini, D.; Otuki, M.F. Effect of a Garcinia gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells. J. Ethnopharmacol. 2013, 148, 199-204.）。Ｂ１６Ｆ１０細胞を９６ウェルプレートに７×１０３細胞／ウェルの密度で播種した。４８時間培養後、細胞を１００又は２００μｇ／ｍＬの濃度の試験化合物、又は５００μＭコウジ酸溶液で処理し、３７℃でさらに４８時間インキュベートした。インキュベーション後、培地を除去し、細胞をリン酸緩衝液で２回洗浄し、３７℃、３時間ＭＴＴ溶液（０．５ｍｇ／ｍＬ）でインキュベートした。培地を捨てて、エタノール２００μＬを添加した。マイクロプレート分光光度計（Bio-Rad Laboratories, Inc.）を用いて５７０ｎｍにおける吸光度を測定した。結果を図１２に示す。
(2) B16F10 cell viability Cell viability was measured using MTT assay (Campos, PM; da Silva Horinouchi, CD; da Silveira Prudente, A .; Cechinel-Filho, V .; de Almeida Cabrini, D .; Otuki, MF Effect of a Garcinia gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells. J. Ethnopharmacol. 2013, 148, 199-204.). B16F10 cells were seeded in 96 well plates at a density of 7 × 10 3 cells / well. After culturing for 48 hours, the cells were treated with a test compound at a concentration of 100 or 200 μg / mL, or a 500 μM kojic acid solution, and incubated at 37 ° C. for an additional 48 hours. After incubation, the medium was removed and the cells were washed twice with phosphate buffer and incubated with MTT solution (0.5 mg / mL) at 37 ° C. for 3 hours. The medium was discarded and 200 μL of ethanol was added. Absorbance at 570 nm was measured using a microplate spectrophotometer (Bio-Rad Laboratories, Inc.). The results are shown in FIG.
（３）メラニン含有量測定
メラニン含有量はYoonらの方法に従って測定した（Yoon, N.Y.; Eom, T-K.; Kim, M-M.; Kim, S-K. Inhibitory effect of Phlorotannins isolated from Ecklonia cava on mushroom tyrosianse activity and melanin formation in mouse B16F10 melanoma cells. J. Agri. Food. Chem. 2009, 57, 4124-4129.）。すなわち、Ｂ１６Ｆ１０細胞を７×１０３細胞／ウェルの密度で９６ウェルプレートに播種した。４８時間培養後、細胞を２０又は５０μｇ／ｍＬの試験化合物、又は５００μＭコウジ酸で処理した。１時間後、１００μＭイソブチル−１−メチルキサンチン（ＩＢＭＸ）を加え、さらに３７℃で４８時間インキュベートした。細胞をリン酸緩衝液で２回洗浄し、次いで１０％ＤＭＳＯを含むＮａＯＨ（１Ｎ）１００μＬに溶解させた。サンプルを８０℃で１時間インキュベートし、メラニンを可溶化するために混合した。混合ホモジネートの４９０ｎｍにおける光学濃度を測定した。対照群において実験期間中に生成するメラニンの総量を１００％とし、処置群における阻害率を計算した。結果を図１３に示す。
(3) Melanin content measurement Melanin content was measured according to the method of Yoon et al. (Yoon, NY; Eom, TK .; Kim, MM .; Kim, SK. Inhibitory effect of Phlorotannins isolated from Ecklonia cava on mushroom tyrosianse activity and melanin formation in mouse B16F10 melanoma cells. J. Agri. Food. Chem. 2009, 57, 4124-4129.). That is, B16F10 cells were seeded in a 96-well plate at a density of 7 × 10 3 cells / well. After culturing for 48 hours, the cells were treated with 20 or 50 μg / mL test compound or 500 μM kojic acid. After 1 hour, 100 μM isobutyl-1-methylxanthine (IBMX) was added and further incubated at 37 ° C. for 48 hours. Cells were washed twice with phosphate buffer and then lysed in 100 μL of NaOH (1N) containing 10% DMSO. Samples were incubated at 80 ° C. for 1 hour and mixed to solubilize melanin. The optical density at 490 nm of the mixed homogenate was measured. The total amount of melanin produced during the experimental period in the control group was taken as 100%, and the inhibition rate in the treatment group was calculated. The results are shown in FIG.
（４）細胞内チロシナーゼ活性
Liらの方法を若干修正してチロシナーゼ活性を測定した（Li, X.; Guo, L.; Sun, Y.; Zhou, J.; Gu, Y.; Li Y. Baicalein inhibits melanogenesis through activation of the ERK signaling pathway. Inter. J. Mol. Med. 2010, 25, 923-927.）。Ｂ１６Ｆ１０細胞を９６ウェルプレートに７×１０３細胞／ウェルの密度で播種した。４８時間培養後、細胞を２０又は５０μｇ／ｍＬの試験化合物、又は５００μＭコウジ酸で処理した。１時間後、１００μＭ イソブチル−１−メチルキサンチン（ＩＢＭＸ）を加え、さらに３７℃で４８時間インキュベートした。次いで、細胞を氷冷リン酸緩衝液で洗浄し、１％トリトン−Ｘ（９０μＬ／ウェル）含有リン酸緩衝液（ｐＨ６．８）で溶解した。プレートを−８０℃で３０分間凍結した。解凍、混合した後、１％Ｌ−ＤＯＰＡ １０μＬを各ウェルに加えた。３７℃、２時間インキュベーションした後、４９０ｎｍにおける吸光度を測定した。結果を図１４に示す。
(4) Intracellular tyrosinase activity
Tyrosinase activity was measured with a slight modification of Li et al. (Li, X .; Guo, L .; Sun, Y .; Zhou, J .; Gu, Y .; Li Y. Baicalein inhibits melanogenesis through activation of the ERK signaling pathway. Inter. J. Mol. Med. 2010, 25, 923-927.). B16F10 cells were seeded in 96 well plates at a density of 7 × 10 3 cells / well. After culturing for 48 hours, cells were treated with 20 or 50 μg / mL test compound or 500 μM kojic acid. After 1 hour, 100 μM isobutyl-1-methylxanthine (IBMX) was added and further incubated at 37 ° C. for 48 hours. The cells were then washed with ice-cold phosphate buffer and lysed with phosphate buffer (pH 6.8) containing 1% Triton-X (90 μL / well). Plates were frozen at -80 ° C for 30 minutes. After thawing and mixing, 10 μL of 1% L-DOPA was added to each well. After incubation at 37 ° C. for 2 hours, the absorbance at 490 nm was measured. The results are shown in FIG.
結果：
３−イソブチル−１−メチルキサンチン（ＩＢＭＸ）は、チロシナーゼを活性化する強力なメラニン生成刺激因子である。チロシナーゼはメラニン生成における重要かつ律速段階に関与する酵素であり、チロシンの水酸化により３，４−ジヒドロキシフェニルアラニン（Ｌ−ＤＯＰＡ）を生成し、次にＬ−ＤＯＰＡの酸化によりドーパキノンとなる。メラニン産生刺激因子であるＩＢＭＸの存在下、ＭＴＤ及びＴＭＯＱのメラニン生成抑制作用を評価した。
Ｂ１６Ｆ１０メラノーマ細胞を、４８時間ＩＢＭＸの存在下ＭＴＤ及びＴＭＯＱ（２０又は５０μｇ／ｍＬ）で処理したところ、図１３に示されるように、いずれも有意にメラニン含有量を減少させた。５０μｇ／ｍＬでは、ＭＴＤ、ＴＭＯＱのメラニン生成抑制率はそれぞれ７８．９±０．８２％、５８．７±８．８９％であり、陽性対照であるコウジ酸（５００μＭ、５０．５±４．９４％）よりも優れたメラニン生成抑制作用を示した。
result:
3-isobutyl-1-methylxanthine (IBMX) is a potent melanogenesis stimulator that activates tyrosinase. Tyrosinase is an enzyme involved in an important and rate-determining step in the production of melanin. It produces 3,4-dihydroxyphenylalanine (L-DOPA) by hydroxylation of tyrosine and then becomes dopaquinone by oxidation of L-DOPA. In the presence of IBMX, which is a melanin production stimulating factor, the melanin production inhibitory action of MTD and TMOQ was evaluated.
Treatment of B16F10 melanoma cells with MTD and TMOQ (20 or 50 μg / mL) in the presence of IBMX for 48 hours, both significantly reduced melanin content, as shown in FIG. At 50 μg / mL, the inhibition rate of melanin production by MTD and TMOQ was 78.9 ± 0.82% and 58.7 ± 8.89%, respectively, and kojic acid (500 μM, 50.5 ± 4. 94%) was superior to melanin production.
ＭＴＤ及びＴＭＯＱが細胞内チロシナーゼ活性を阻害するか否かを評価するために、Ｂ１６Ｆ１０メラノーマ細胞をＭＴＤ及びＴＭＯＱ(２０又は５０μｇ／ｍＬ)で４８時間処理し、Ｌ−ＤＯＰＡを加えてインキュベーションした。２０μｇ／ｍＬにおけるＭＴＤ、ＴＭＯＱのチロシナーゼ阻害率は、それぞれ７５．６±６．５６％、４６．２±２．５９％であった。５０μｇ／ｍＬにおける阻害率は、ＭＴＤが８２．５±５．８１％、ＴＭＯＱが６１．２±０．３４％であり、陽性対照であるコウジ酸（５３．４±１．３８％）よりも強い阻害活性を示した。一方、ＭＴＤ及びＴＭＯＱは、Ｂ１６Ｆ１０メラノーマ細胞の生存性にほとんど影響を与えなかった。 In order to assess whether MTD and TMOQ inhibit intracellular tyrosinase activity, B16F10 melanoma cells were treated with MTD and TMOQ (20 or 50 μg / mL) for 48 hours and incubated with L-DOPA. The tyrosinase inhibition rates of MTD and TMOQ at 20 μg / mL were 75.6 ± 6.56% and 46.2 ± 2.59%, respectively. The inhibition rate at 50 μg / mL is 82.5 ± 5.81% for MTD and 61.2 ± 0.34% for TMOQ, which is higher than that of kojic acid (53.4 ± 1.38%) as a positive control. Strong inhibitory activity was shown. On the other hand, MTD and TMOQ had little effect on the viability of B16F10 melanoma cells.
本発明の新規化合物であるＭＴＤやＴＭＯＱは、α‐グルコシダーゼ及びトリプシンの活性阻害剤や一酸化窒素生成阻害剤として利用可能である。また、ラジカル阻害剤及びＬＤＬ酸化阻害剤としても利用可能である。特に、同新規化合物を糖尿病、動脈硬化、肥満及びメタボリックシンドロームの治療又は予防を目的とした飲食品、医薬品及び医薬部外品等や美白化粧料等の有効成分として利用することも可能である。 MTD and TMOQ, which are novel compounds of the present invention, can be used as α-glucosidase and trypsin activity inhibitors and nitric oxide production inhibitors. It can also be used as a radical inhibitor and an LDL oxidation inhibitor. In particular, the novel compound can also be used as an active ingredient in foods, beverages, pharmaceuticals, quasi drugs, and whitening cosmetics for the purpose of treating or preventing diabetes, arteriosclerosis, obesity and metabolic syndrome.
従って、新たな医薬品等の開発に極めて有効なものである。
以 上
Therefore, it is extremely effective for the development of new medicines.
that's all
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| JP2008013481A (en) * | 2006-07-05 | 2008-01-24 | Univ Of Tokushima | Tyrosinase activity inhibitor and lipoxygenase activity inhibitor derived from alpinia speciosa |
| JP2011506485A (en) * | 2007-12-19 | 2011-03-03 | ５５ ファルマ ドラッグ ディスカバリー アンド ディベロップメント アーゲー | Octahydroquinolidine for the treatment of diabetes |
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| JP2008013481A (en) * | 2006-07-05 | 2008-01-24 | Univ Of Tokushima | Tyrosinase activity inhibitor and lipoxygenase activity inhibitor derived from alpinia speciosa |
| JP2011506485A (en) * | 2007-12-19 | 2011-03-03 | ５５ ファルマ ドラッグ ディスカバリー アンド ディベロップメント アーゲー | Octahydroquinolidine for the treatment of diabetes |
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| KUBO,H. ET AL: "The hypoglycemic effect of (7R*,9aS*)-7-phenyl-octahydroquinolizin-2-one in mice", BIOLOGICAL & PHARMACEUTICAL BULLETIN, vol. 23, no. 9, JPN6019006842, 2000, pages 1114 - 1117 * |
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