JP2006176480A - L-histidine decarboxylase inhibitor - Google Patents
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本発明は、Ｌ−ヒスチジン脱炭酸酵素活性の上昇に起因する種々の疾患の予防又は治療薬に関する。 The present invention relates to a preventive or therapeutic agent for various diseases caused by an increase in L-histidine decarboxylase activity.
ヒスタミンは炎症、アレルギー、胃酸分泌、神経伝達ならびに腫瘍細胞の転移・増殖といった反応を調節する生体アミンである。ヒスタミン産生細胞としては肥満細胞や、好塩基球、ＥＣＬ細胞（ｅｎｔｅｒｏｃｈｒｏｍａｆｆｉｎ−ｌｉｋｅ ｃｅｌｌ）などがあり、これら細胞内にはヒスタミン生合成酵素であるヒスチジン脱炭酸酵素（Ｌ−ｈｉｓｔｉｄｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ；ＨＤＣ）が存在している。ＨＤＣは誘導性の酵素であり、刺激に応じて数倍から百倍以上にも酵素活性が上昇し、種々のアレルギー疾患や炎症、消化性潰瘍などを引き起こす。 Histamine is a biogenic amine that regulates reactions such as inflammation, allergy, gastric acid secretion, neurotransmission, and metastasis / proliferation of tumor cells. Histamine-producing cells include mast cells, basophils, ECL cells (enterochromaffin-like cells), and histidine decarboxylase (HDC), which is a histamine biosynthetic enzyme, is present in these cells. is doing. HDC is an inducible enzyme, and its enzyme activity increases several to one hundred times or more in response to a stimulus, causing various allergic diseases, inflammation, peptic ulcer and the like.
本発明の目的は、ＨＤＣ阻害活性を有する生薬を探索し、副作用の少ないＨＤＣ阻害剤、引いては、アレルギー疾患、炎症性疾患、消化性潰瘍あるいはヒスタミンの産生により引き起こされる病的状態などを低減させうる薬剤を提供することである。 The object of the present invention is to search for herbal medicines having HDC inhibitory activity and reduce HDC inhibitors with few side effects, and thus reduce allergic diseases, inflammatory diseases, peptic ulcers or pathological conditions caused by production of histamine It is to provide a drug that can be allowed.
本発明者らは前記課題を解決するために多くの生薬のＨＤＣ阻害活性を探索した結果、粉防已をはじめとする数種の生薬がＨＤＣ阻害活性を有することを見出した。
かかる知見に基づき完成した本発明の一つの態様は、粉防已、茶葉、麻黄、丁子、蘇木、牡丹皮、大黄、黄ゴン及び甘草からなる群から選ばれる少なくとも１種を有効成分とするＬ−ヒスチジン脱炭酸酵素阻害剤である。As a result of searching for the HDC inhibitory activity of many herbal medicines in order to solve the above problems, the present inventors have found that several kinds of herbal medicines including powder-preventive have HDC inhibitory activity.
One embodiment of the present invention completed on the basis of such knowledge has, as an active ingredient, at least one selected from the group consisting of powder-prevented tea, tea leaves, mahuang, clove, sugi, peony skin, Daihuang, yellow gon and licorice. L-histidine decarboxylase inhibitor.
本発明者らはさらに粉防已からのＨＤＣ阻害物質の単離を目的として、分画、精製、構造解析を行い、粉防已より単離したｂｉｓｂｅｎｚｙｌｉｓｏｑｕｉｎｏｌｉｎｅ型アルカロイドがＨＤＣ阻害活性を有するとの知見を得た。
かかる知見に基づき完成した本発明の一つの態様は、下記式で表されるｂｉｓｂｅｎｚｙｌｉｓｏｑｕｉｎｏｌｉｎｅ化合物を有効成分とするＬ−ヒスチジン脱炭酸酵素阻害剤である。The present inventors further conducted fractionation, purification, and structural analysis for the purpose of isolating an HDC inhibitory substance from powdered powder, and found that the bisbenziliquinoquinoline-type alkaloid isolated from powdered powder has HDC inhibitory activity. Got.
One embodiment of the present invention completed based on such findings is an L-histidine decarboxylase inhibitor containing a bisbenziliquinoquinine compound represented by the following formula as an active ingredient.
本発明における「粉防已」とは、主に抗リュウマチ作用を期待し処方に配合される生薬である。「粉防已」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。粉防已の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり２．０〜３．０ｇであり、好ましくは２．５〜３．０ｇである。 The “powder-prevented” in the present invention is a herbal medicine that is blended in a prescription mainly for anti-rheumatic activity. The “powder already” may include not only the raw powder but also an extract such as an extract, and the form thereof is not particularly limited. The effective dose (powder equivalent amount) of the powdered powder is approximately 2.0 to 3.0 g per day for an adult, and preferably 2.5 to 3.0 g.
本発明における「茶葉」とは、利尿、脂肪分解抑制、清頭目を期待し処方に配合される生薬である。「茶葉」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。茶葉の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり１．５〜２．０ｇであり、好ましくは１．５ｇである。 “Tea leaf” in the present invention is a herbal medicine blended into a prescription in anticipation of diuresis, suppression of lipolysis, and clean eyes. “Tea leaves” may include not only the raw powder but also extracts such as extracts, and the form thereof is not particularly limited. The effective dose (raw drug equivalent amount) of tea leaves is approximately 1.5 to 2.0 g per day, preferably 1.5 g, per adult.
本発明における「麻黄」とは、主に解熱、鎮咳、鎮痛を期待し処方に配合される生薬である。「麻黄」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。麻黄の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり１．２〜６．０ｇであり、好ましくは３．０〜４．０ｇである。 The “mao” in the present invention is a herbal medicine blended in a prescription mainly for antipyretic, antitussive and analgesic. The “mao” may include not only the raw powder but also an extract such as an extract, and the form is not particularly limited. The effective dose (raw substance equivalent amount) of Mao is about 1.2 to 6.0 g per day, preferably 3.0 to 4.0 g per adult.
本発明における「丁子」とは、主に抗菌、抗ウイルス作用、子宮収縮作用を期待し処方に配合される生薬である。「丁子」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。丁子の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり１．０〜１．５ｇであり、好ましくは１．５ｇである。 The “clove” in the present invention is a herbal medicine blended in a prescription mainly with antibacterial, antiviral and uterine contraction effects. “Chome” may include not only the raw powder but also an extract such as an extract, and the form thereof is not particularly limited. The effective dose of clove (the amount equivalent to the drug substance) is approximately 1.0 to 1.5 g, preferably 1.5 g, per day for one adult.
本発明における「蘇木」とは、主に高脂血症改善、止血を期待し処方に配合される生薬である。「蘇木」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。蘇木の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり２．０〜２．５ｇであり、好ましくは２．０ｇである。 “Soki” in the present invention is a herbal medicine that is blended in a prescription mainly in anticipation of improvement of hyperlipidemia and hemostasis. “Soki” is not limited to the bulk powder, and may be an extract such as an extract, and the form is not particularly limited. The effective dose of Soki (the amount equivalent to the drug substance) is approximately 2.0 to 2.5 g per day per adult, and preferably 2.0 g.
本発明における「牡丹皮」とは、婦人用薬の主薬として用いられ、末梢血液循環の改善、更年期障害の改善や瀉下効果を期待し処方に配合される。「牡丹皮」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。牡丹皮の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり２．０〜５．０ｇであり、好ましくは４．０〜５．０ｇである。 The “peony skin” in the present invention is used as a main drug for women's medicine and is formulated in the prescription with the expectation of improvement of peripheral blood circulation, improvement of climacteric disorder, and axillary effect. The “peony skin” may include not only the raw powder but also an extract such as an extract, and the form thereof is not particularly limited. The effective dose of peony skin (raw drug equivalent amount) is approximately 2.0 to 5.0 g, preferably 4.0 to 5.0 g per day per adult.
本発明における「大黄」とは、主に抗菌、抗炎症作用や瀉下作用を期待し処方に配合される生薬である。「大黄」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。大黄の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり０．５〜４．０ｇであり、好ましくは２．０〜３．０ｇである。 In the present invention, “Daigo” is a herbal medicine blended in a prescription mainly in anticipation of antibacterial, anti-inflammatory action and axillary action. “Dai yellow” is not limited to the bulk powder but may be an extract such as an extract, and the form thereof is not particularly limited. The effective dose of Dai-Yo (the amount equivalent to the drug substance) is approximately 0.5 to 4.0 g, preferably 2.0 to 3.0 g per day per adult.
本発明における「黄ゴン」とは、主に利胆作用、抗炎症作用や肝障害予防作用を期待し処方に配合される生薬である。「黄ゴン」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。黄ゴンの有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり１．５〜３．０ｇであり、好ましくは２．０〜３．０ｇである。 The “yellow gon” in the present invention is a herbal medicine blended in a prescription mainly in anticipation of a bile action, an anti-inflammatory action and a liver damage prevention action. “Yellow Gon” may include not only the bulk powder but also an extract such as an extract, and the form thereof is not particularly limited. The effective dose of Huang Gon (the amount equivalent to the drug substance) is approximately 1.5 to 3.0 g per day for adults, preferably 2.0 to 3.0 g.
本発明における「甘草」とは、主に清熱解毒、潤肺止咳、鎮痙を期待し処方に配合される生薬である。「甘草」は、原末のみならず、エキス等の抽出物あってもよく、その形態には特に限定はない。甘草の有効投与量（原生薬換算量）は、おおよそ成人１人に対して１日当たり１．０〜６．０ｇであり、好ましくは１．０〜３．０ｇである。 The “licorice” in the present invention is a herbal medicine blended in a prescription mainly in anticipation of clean fever detoxification, pulmonary antitussive and antispasmodic. The “licorice” may include not only the raw powder but also an extract such as an extract, and the form thereof is not particularly limited. The effective dose of licorice (the equivalent amount of crude drug) is approximately 1.0 to 6.0 g, preferably 1.0 to 3.0 g per day per adult.
本発明において前記各生薬は単独で配合するのみならず、数種を組み合わせて配合してもよい。
本発明における「Ｌ−ヒスチジン脱炭酸酵素阻害剤」は、例えば、粉防已などのエキスを含有する内服用固形製剤（錠剤、散剤など）を調製し、１日１乃至数回患者に経口投与することができる。In the present invention, each herbal medicine may be blended not only alone but also in combination of several kinds.
The “L-histidine decarboxylase inhibitor” in the present invention is prepared, for example, as a solid preparation for internal use (tablets, powders, etc.) containing an extract such as powdered powder and orally administered to a patient once or several times a day. can do.
本発明により、Ｌ−ヒスチジン脱炭酸酵素活性の上昇に起因する種々の疾患（アレルギー疾患、炎症性疾患、睡眠、消化性潰瘍、腫瘍（転移抑制）、脳代謝改善あるいはヒスタミンの産生により引き起こされる病的状態等）の予防又は治療薬を提供することが可能となった。 According to the present invention, various diseases caused by increased L-histidine decarboxylase activity (allergic diseases, inflammatory diseases, sleep, peptic ulcer, tumor (metastasis suppression), improved brain metabolism or histamine production) It has become possible to provide preventive or therapeutic drugs.
以下、実験例を挙げて本発明を詳細に説明する。
１．酵素調製
雄Ｗｉｓｔａｒ系ラット５匹よりＨＤＣを調製した。操作は氷冷下、または、４℃で行った。富山医科薬科大学実験動物委員会の基準に基づいた動物に苦痛を与えない条件下（ｄｉｅｔｈｙｌｅｔｈｅｒで麻酔）で、開腹し胃を摘出した。２０倍容の０．０１ｍＭ ｐｙｒｉｄｏｘａｌ−５’−ｐｈｏｓｐｈａｔｅ（ＰＬＰ）、１００μｇ／ｍＬ ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌ ｆｌｕｏｒｉｄｅ（ＰＭＳＦ）、０．１ｍＭ ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅｔｅｔｒａａｃｅｔａｔｅ（ＥＤＴＡ）、６ｍＭ ２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、０．２５Ｍ ｓｕｃｒｏｓｅ含有０．１Ｍ ｐｏｔａｓｓｉｕｍ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．３）でＷａｒｉｎｇ Ｂｌｅｎｄｅｒを用いてホモジナイズした。このホモジネイトを４８，０００×ｇで１０分間遠心分離し、得られた上清に硫酸アンモニウムを２５％飽和になるように徐々に加え、完全に溶解させた。３０分撹拌後、１０，０００×ｇで３０分間遠心分離を行った。さらに、得られた上清に硫酸アンモニウムを５５％飽和になるように徐々に加え塩析を行った。６０分撹拌後、再度１０，０００×ｇで３０分間遠心分離した。得られた沈殿を０．０１ｍＭ ＰＬＰ、１００μｇ／ｍＬ ＰＭＳＦ、０．１ｍＭ ＥＤＴＡ、６ｍＭ ２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ含有０．１Ｍ ｐｏｔａｓｓｉｕｍ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ （ｐＨＨ６．８）で懸濁させ同様のｂｕｆｆｅｒで一晩透析した。得られた溶液を粗酵素として、４℃で保存し、適宜実験に使用した。Hereinafter, the present invention will be described in detail with reference to experimental examples.
1. Enzyme preparation HDC was prepared from 5 male Wistar rats. The operation was performed under ice cooling or at 4 ° C. Under conditions that did not cause pain to the animal based on the criteria of the Toyama Medical and Pharmaceutical University Experimental Animal Committee (anesthesia with dietylether), the abdomen was opened and the stomach was removed. 20 volumes of 0.01 mM pyridoxal-5′-phosphate (PLP), 100 μg / mL phenylmethylsulfuryl fluoride (PMSF), 0.1 mM ethylenediaminetetraacetate (EDTA), 6 mM 2-mercaptoethanol 0.2 (PH 7.3) was homogenized using a Waring Blender. This homogenate was centrifuged at 48,000 × g for 10 minutes, and ammonium sulfate was gradually added to the resulting supernatant to 25% saturation to completely dissolve it. After stirring for 30 minutes, centrifugation was performed at 10,000 × g for 30 minutes. Furthermore, ammonium sulfate was gradually added to the obtained supernatant to 55% saturation, and salting out was performed. After stirring for 60 minutes, it was centrifuged again at 10,000 × g for 30 minutes. The resulting precipitate was suspended in 0.01 mM PLP, 100 μg / mL PMSF, 0.1 mM EDTA, 0.1 mM potassium phosphate buffer (pH H 6.8) containing 6 mM 2-mercaptoethanol, and dialyzed overnight with the same buffer. The obtained solution was stored as a crude enzyme at 4 ° C. and used appropriately in experiments.
２．Ｌ Ｈｉｓｔｉｄｉｎｅ脱炭酸酵素阻害活性測定
０．１Ｍ ｐｏｔａｓｓｉｕｍ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ６．８）２４０μＬ、０．０１ｍＭ ＰＬＰ １２０μＬ、ｂｏｖｉｎｅ ｓｅｒｕｍａｌｂｕｍｉｎ（ＢＳＡ；１０ｍｇ／ｍＬ）１２０μＬ、ａｌｋａｌｏｉｄ（１０ｍｇ／ｍＬ）１２０μＬ、に粗酵素３００μＬ加え撹拌した。３７℃、１５分プレインキュベートし、その後、基質である１０ｍＭ ｈｉｓｔｉｄｉｎｅ ３００μＬを加え、３７℃、１２０分インキュベートした。ＨＤＣ活性は、生成したｈｉｓｔａｍｉｎｅをＰ−ｃｅｌｌｕｌｏｓｅカラムにて分離し、ο−ｐｈｔｈａｌａｌｄｅｈｙｄｅ法に基づきｈｉｓｔａｍｉｎｅを定量し、阻害活性を測定した。2. L Histidine decarboxylase inhibitory activity measurement 0.1 M potassium phosphate buffer (pH 6.8) 240 μL, 0.01 mM PLP 120 μL, bovine serumbumin (BSA; 10 mg / mL) 120 μL, alkaline (10 mg / mL) 120 μL, crude enzyme Added and stirred. After incubating at 37 ° C. for 15 minutes, 300 μL of 10 mM histidine as a substrate was added and incubated at 37 ° C. for 120 minutes. For the HDC activity, the produced histamine was separated by a P-cellulose column, histamine was quantified based on the o-phtaldehyde method, and the inhibitory activity was measured.
（スクリーニング生薬）
スクリーニングに用いた生薬は次のとおりである。
蘇葉、枇杷葉、杏仁、サンザシ、紫根、荊芥、連翹、山梔子、防風、桔梗、細辛、竜胆、クコシ、牛膝、括楼根、附子、ヨクイニン、大棗、茯苓、沢瀉、ビャクシ、柴胡、当帰、麦芽、苦参、酸棗仁、忍冬、天麻、粳米、天門冬、薄荷、前胡、括楼仁、唐独活、木通、桑白皮、天南星、シツリシ、竜眼肉、五味子、茶葉、升麻、半夏、麦門冬、菊花、蝉退、蒼朮、呉茱萸、山椒、枳穀、陳皮、白朮、麻子仁、延胡索、枳実、粉防已、貝母、何首烏、蓮肉、丁子、辛夷、蘇木、牛蒡子、良姜、縮砂、紅花、麻黄、竹葉、カッコウ、黄柏、胡麻、ウコン、橘皮、杜仲、生姜、小麦、桃仁、冬瓜子、猪苓、竹茹、山茱萸、羌活、芍薬、人参、青皮、香附子、地骨皮、乾姜、牡丹皮、山薬、遠志、甘草、木香、センキュウ、インチンコウ、艾葉、地黄、厚朴、大黄、車前子、釣藤鈎、桂皮、オウゴン、黄耆、威霊仙、知母、呉茱萸、レイシ、黄連(Screening crude drug)
The crude drugs used for screening are as follows.
Soha, Aoba, Apricot, Hawthorn, Murasakine, Aoi, Rei, Yamashiko, Windbreak, Kikyo, Spicy, Dragon, Kokushi, Beef Knee, Giraffe, Neko, Yokuinin, Daegu, Aoi, Sawaso, Beakshi, Shiba Hu, Toki, Malt, Ginseng, Acid 棗仁, Shinobu Winter, Tenma, Satsume Rice, Tenmon Winter, Lightweight, Mae Hu, Kanrojin, Tang Dynasty, Wood Street, Mulberry White Bark, Tennan Star, Shitsuri, Longan Meat, Goriko, Tea Leaves , Hemp, half-summer, barley winter, chrysanthemum flower, retirement, persimmon, wu han, yam, cereals, chenseed, white birch, maikojin, yankogong, coconut, powdered already, shellfish mother, garlic, lotus meat, clove, Hot pepper, Sugi, Gyudon, Ryokan, Shredded sand, Safflower, Mao, Bamboo leaves, Cuckoo, Yellow cocoon, Sesame, Turmeric, Tachibana skin, Tochu, Ginger, Wheat, Peach seed, Winter eggplant, Kaki, Bamboo candy, Yam , Salmon, glaze, carrots, green skin, garlic, ground bones, psoriasis, peony skin, mountain medicine, ambition, licorice, wood incense, nematode, ginseng, green leaves, ground yellow Magnolia bark, rhubarb, psyllium, Tsurifujikagi, cinnamon, Scutellaria root, Astragalus, Wei Ryozen, intellectual mother, Wu Fructus, litchi, Coptidis
３．生薬熱水抽出画分およびアルカロイド画分の調製
各生薬１００ｇを８００ｍＬの水で４０分間煎じ、シルクの布で濾過した。その生薬に再び４００ｍＬの水を加え４０分間煎じ、濾過した後凍結乾燥させた。アルカロイド画分は、生薬熱水エキスを得た後、上清を陽イオン交換カラムＡｍｂｅｒｌｉｔｅ ＩＲ−１２０Ｂ（Ｈ＋ｆｏｒｍ）に付し、樹脂の５倍量の０．５Ｍアンモニア溶液で溶出させた後、濃縮乾固することにより得た。3. Preparation of herbal medicine hot water extract fraction and alkaloid fraction 100 g of each herbal medicine was decocted with 800 mL of water for 40 minutes and filtered through a silk cloth. 400 mL of water was added to the herbal medicine again, decocted for 40 minutes, filtered and lyophilized. The alkaloid fraction was obtained after obtaining a crude drug hot water extract, and then applying the supernatant to a cation exchange column Amberlite IR-120B (H + form) and eluting it with a 0.5 M ammonia solution 5 times the amount of the resin. Obtained by concentration to dryness.
上記各生薬（１１２種）の熱水可溶性画分を終濃度１ｍｇ／ｍＬになるように調製してＨＤＣ阻害活性を測定した。その中で粉防已、茶葉、ボクソク、麻黄、丁子、檳榔子、蘇木、牡丹皮、大黄、黄ゴン、甘草に明らかなＨＤＣ阻害活性が認められた。さらにこれら生薬のアルカロイド画分を調製し、同様にＨＤＣ阻害活性を測定したところ、粉防已、丁子では熱水抽出画分より活性の上昇が認められた（図１）。 The hot water soluble fraction of each of the above herbal medicines (112 types) was prepared to a final concentration of 1 mg / mL, and the HDC inhibitory activity was measured. Among them, clear HDC inhibitory activity was observed in powder proof, tea leaves, boxok, mao, clove, eggplant, sugi, peony skin, Daio, yellow Gon, and licorice. Furthermore, when alkaloid fractions of these herbal medicines were prepared and the HDC inhibitory activity was measured in the same manner, an increase in activity was observed in the powdered powder and clove than in the hot water extraction fraction (FIG. 1).
４．粉防已からの活性物質の単離精製
粉防已２ｋｇ（乾燥重量）を熱水で１時間抽出を行なった。抽出液を冷却後、沈殿物をセライトで濾過した。濾液をクロロホルムで分配しクロロホルム層を得た。得られたクロロホルム層を濃縮後、Ｓｉｌｉｃａ ｇｅｌ ６０カラムにアプライし、クロロホルム：メタノール：水（１７：３：０．２→４０：１０：１→６：４：１）で順次溶出した。顕著な阻害効果が認められたクロロホルム：メタノール：水（４０：１０：１）で溶出される区分を減圧下で濃縮し、１．２０ｇの茶色のアメ状固体を得た。これをＳｅｐｈａｄｅｘ ＬＨ−２０（１．９×９２ｃｍ）にアプライし、１分画が１０ｍｌとなるように１００％メタノールで溶出し分画した。同カラムおよびＳｉｌｉｃａ ｇｅｌ ６０カラムにてクロマトを繰り返し、活性画分を減圧濃縮した。得られた活性化合物（１０９．０ｍｇ）は、アルカロイドの呈色試薬であるドラーゲンドルフに陽性であり、ＴＬＣ上においてシングルスポットであった。4). Isolation and purification of active substance from powdered powder 2 kg of powdered powder (dry weight) was extracted with hot water for 1 hour. After cooling the extract, the precipitate was filtered through celite. The filtrate was partitioned with chloroform to obtain a chloroform layer. The obtained chloroform layer was concentrated, applied to a Silica gel 60 column, and eluted sequentially with chloroform: methanol: water (17: 3: 0.2 → 40: 10: 1 → 6: 4: 1). The fraction eluted with chloroform: methanol: water (40: 10: 1) in which a remarkable inhibitory effect was observed was concentrated under reduced pressure to obtain 1.20 g of a brown candy-like solid. This was applied to Sephadex LH-20 (1.9 × 92 cm), and fractionated by eluting with 100% methanol so that one fraction was 10 ml. Chromatography was repeated using the same column and Silica gel 60 column, and the active fraction was concentrated under reduced pressure. The obtained active compound (109.0 mg) was positive for Dragendorf, a color reagent for alkaloids, and was a single spot on TLC.
粉防已中の活性本体はｂｉｓｂｅｎｚｙｌｉｓｏｑｕｉｎｏｌｉｎｅ系アルカロイドであるＦａｎｇｃｈｉｎｏｌｉｎｅであった（下記式）。ＦａｎｇｃｈｉｎｏｌｉｎｅのＨＤＣに対する阻害活性を測定した結果、ＩＣ５０値は９４．３μｇ／ｍＬであった。またＨＤＣに対する阻害様式と阻害定数をＬｉｎｅｗｅａｖｅｒ−Ｂｕｒｋｐｌｏｔにより検討した結果、阻害様式は拮抗阻害でありＫｉ値は１６７μＭであった。The active body in the powder control was Fangchinoline, a bisbenziliquinoquine alkaloid (the following formula). As a result of measuring the inhibitory activity of Fanchinolin on HDC, the IC 50 value was 94.3 μg / mL. Moreover, as a result of examining the inhibition mode and inhibition constant with respect to HDC by Lineweaver-Burkplot, the inhibition mode was competitive inhibition, and Ki value was 167 micromol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011152100A1 (en) * | 2010-06-01 | 2011-12-08 | ピアス株式会社 | Mesenchymal stem cell inducer, and method for induction of mesenchymal stem cell |
| CN112138011A (en) * | 2020-10-10 | 2020-12-29 | 上海中医药大学 | Application of fangchinoline in preparation of medicine for preventing and treating ulcerative colitis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011152100A1 (en) * | 2010-06-01 | 2011-12-08 | ピアス株式会社 | Mesenchymal stem cell inducer, and method for induction of mesenchymal stem cell |
| US9458429B2 (en) | 2010-06-01 | 2016-10-04 | Pias Corporation | Mesenchymal stem cell attractant and method for attracting mesenchymal stem cell |
| CN112138011A (en) * | 2020-10-10 | 2020-12-29 | 上海中医药大学 | Application of fangchinoline in preparation of medicine for preventing and treating ulcerative colitis |
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