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| 1.
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論文
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Shi, Wen ; Kitaoka, Fumiyo ; Doui, Misato ; Miyake, Katsunori ; Sasaki, Yohei ; Kakiuchi, Nobuko ; Mikage, Masayuki ; 堂井, 美里 ; 垣内, 信子 ; 御影, 雅幸
概要:
金沢大学医薬保健研究域薬学系<br />日本薬局方ではアカネ科 Uncaria sinensis Havil., U. rhynchophylla Miq. 及び U. macrophylla Wall. の鉤を釣藤鉤として規定している。
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釣藤鉤には異なる薬効を示すインドール及びオキシインドールアルカロイドが含まれているが， これらの組成は原植物の違いにより特徴的に異なるとされる。 しかし， Uncaria 属植物のアルカロイド組成に影響を与える要因については報告されていない。 本研究では， 広域で採集した Uncaria 属植物の種を DNA 塩基配列により正確に同定した後に， アルカロイド （rhynchophylline, geissoschizine methyl ether 及び hirsutine） 含量を HPLC 法により測定した。 その結果， 降水量が低い地域に生育する U. rhynchophyllaの鉤及び茎にアルカロイド含量が高い傾向が認められた。 また， 同一種であっても， 栽培品と野生品のアルカロイド組成は異なり， 市場品 「釣藤鉤」 には rhynchophylline 優位のタイプと geissoschizine methyl ether と hirsutine 優位のタイプの 2 種が確認された。 加えて， 釣藤鉤の原植物を正確に同定するために， DNA 塩基配列解析及び PCR-RFLP 法を用いた分子系統学的手法を確立した。<br />The Japanese Pharmacopoeia states that the crude drug Chotoko is composed of the hooks of Uncaria rhynchophylla Miq., U. sinensis Havil., and U. macrophylla Wall., all of which belong to the Rubiaceae family. It has been reported that the indole and oxindole type alkaloids contained in Chotoko have different pharmacological effects, and Chotoko products derived from different species are composed of different alkaloids. However, there are no reports about the factors affecting the chemical compositions of Uncaria plants. In this study, we analyzed the alkaloid contents (the rhynchophylline, geissoschizine methyl ether, and hirsutine contents) of Uncaria samples collected from a broad range of sites by HPLC after identifying their species by DNA sequence analysis. As a result, we found that the hooks and small stems of U. rhynchophylla grown in habitats with lower annual precipitation levels tended to display higher alkaloid contents. We also found that the alkaloid compositions of cultivated Uncaria plants were different from those of wild plants, even those belonging to the same species, and crude Chotoko products displayed two types of alkaloid profile, the rhynchophylline-rich type and the geissoschizine methyl ether and hirsutine-rich type. Moreover, in order to accurately identify the botanical origin of Chotoko, we established a method involving molecular genetics techniques; i.e., DNA sequence analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). © 2012, Medical and Pharmaceutical Society for WAKAN-YAKU. All rights reserved.
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| 2.
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Atsumi, Toshiyuki ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
金沢大学大学院自然科学研究科生理活性物質科学<br />金沢大学薬学部<br />We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS
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1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype. © 2007 Pharmaceutical Society of Japan.
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| 3.
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Kobira, Sayuri ; Atsumi, Toshiyuki ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
発行後1年より全文公開<br />Poria, a dried sclerotium of Wolfiporiacocos Ryvarden et Gilbertson (Polyporaceae) has been used as a c
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rude drug in both Chinese and Japanese (Kampo) traditional medicines. Recently, cultivated products of Chinese Poria strains have accounted for most of the market, while the cultivation of Japanese Poria strains has not been successful. Aiming to determine the relationship between the differences in cultivation characteristics and genetic polymorphism, we conducted a field cultivation experiment, a rot test, and rapid amplification of polymorphic DNA (RAPD) analysis of Poria strains collected from China and Japan: 3 Chinese and 7 Japanese strains. In field cultivation, although there was no marked inferiority of Japanese strains to Chinese ones in either mycelium propagation or the rate of sclerotium formation, Chinese strains formed whiter sclerotia with a mean size more than twice that of Japanese ones. Representatives of Chinese and Japanese strains, Yunnan and Kaimondake, respectively, were tested for wood-rotting ability. More wood was utilized and the wood color was darker in trials of the Yunnan strain. Amplifications of total DNA of these 10 fungal strains with 2 primers, PC-6 and PC-11, in RAPD analysis showed a difference in the amplicon profile between Japanese and Chinese strains, suggesting differences in their genetic background. © 2011 The Japanese Society of Pharmacognosy and Springer.
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| 4.
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Kakiuchi, Nobuko ; Iwaki, Naoko ; Mikage, Masayuki ; Xiao, Huai ; Wang, Zhigang ; Hattori, Masao
概要:
Aiming to examine whether the genetic background of the crude drugs derived from four Yunnanese Swertia plants and their
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chemical constituent profiles correlate, we analyzed the nucleotide sequences of their nuclear ribosomal DNA regions including ITS1, 5.8S ribosomal RNA gene, and ITS2, together with those of Japanese S. japonica and S. pseudochinensis from Hebei Province. The result that two of the Yunnanese Swertia plants, S. binchuanensis and S. punicea, were genetically similar may explain their similarity in chemical constituent profiles. On the other hand, in spite of differences in chemical profile, S. decora and S. pseudochinensis were genetically close. The other Yunnanese Swertia plants, S. delavayi, and S. japonica, stood at intermediate positions between these two genetically similar pairs. The result suggests that although genetic background would have an influence, environmental factors, e.g., soil and weather conditions, might be critical for their production of secondary metabolites. © The Japanese Society of Pharmacognosy and Springer Japan 2013.
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| 5.
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Doui, Misato ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
中国では， 漢方生薬 「大黄」 に， 特定の薬効の向上や副作用の低減を目的に種々の加工を施してきた。 その内の一つに熟大黄 （JD） が挙げられる。 古代， JD の調製法は 「蒸す （蒸）」 と 「酒をかけた後蒸す （酒蒸）」 の 2 種
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あったが， 現在は酒蒸のみとなり， それに伴って JD の薬効が変化した可能性がある。 本研究では， 蒸 （St） 品と酒蒸品の薬効を解明するために， 中国の本草書並びに医方書の記載内容を調査し， また， 修治品の主成分含量を調査した。 酒蒸大黄として， 16 % エタノール溶液 （黄酒の代用） を吹きかけた後， 蒸したもの （LSp+ St） 及び16 % エタノール溶液に十分浸した後， 蒸したもの （LSo + St） の 2 種を調製した。 本草考証の結果， JD は古来， 解毒， 駆血， 潤腸作用を期待してきたことが明らかになった。 また， 成分研究の結果， St と LSp + Stはsennoside のみを減少させる修治法であり， LSo + St はanthraquinone 含量を増加し， sennoside 含量を減少させる修治法であることが明らかになった。 すなわち， 抗菌， 抗炎症など解毒作用に繋がる anthraquinone 含量が増加する修治法は LSo + St のみであるが， 調製した全修治品で sennoside 含量が低下することから， 相対的に駆血など他の作用が向上する可能性がある。 一方， 潤腸作用の向上については本研究の測定成分からは明らかにならなかった。
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| 6.
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論文
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Doui, Misato ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
金沢大学医薬保健研究域薬学系<br />漢方生薬 「大黄」 は中国において古来， 酒を用いた修治 （酒製） が施されてきた。 酒製に使用する酒は主にアルコール度が低い黄酒とアルコール度が高い白酒の 2 種があり， 大黄の酒製には黄酒が用いら
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れてきたが， その理由は明確でない。 本研究では， 蒸留水， 16 % エタノール溶液， 50 % エタノール溶液または黄酒の一種である紹興酒を用いて酒浸大黄を調製し， 主成分含量 （sennoside A, sennoside B, aloe-emodin, rhein, emodin, chrysophanol, physcion, lindleyin, isolindleyin, tannin 類） の変化を調査した結果， これらの変化はほとんどアルコール度に依存していることが明らかになった。 すなわち， 16 % エタノール溶液または紹興酒で修治した時， sennoside 含量が減少し， anthraquinone 含量が増加した。 それ故， 瀉下作用を抑制し， 抗菌， 抗炎症作用が向上する可能性がある酒浸大黄を調製するために黄酒のようなアルコール度が低い酒を使用してきたと考えられる。
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| 7.
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Doui, Misato ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
漢方生薬「大黄」は中国において古来，酒洗（LDi），酒浸（LSo）及び酒炒（LH）と呼ばれる酒を用いる修治が行われてきた。我々はこれまでに，古文献の記載内容から，酒炒品は瀉下作用を示す LDi 品または駆瘀血作用を示す LSo 品と同様の薬
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効を期待して用いられていたと考察した。また，現在は駆瘀血作用を期待し，「酒大黄」と称する酒噴霧した LH 品（LSpH）が用いられているが，「酒炒大黄」の本質は科学的に解明されていない。そこで本研究では，16 % エタノール溶液（黄酒の代用）を用いて LDi, LSo, 酒噴霧（LSp）, LDi＋加熱（LDiH), LSo＋加熱（LSoH）, 及び LSpH 品を調製し主成分含量の変化を調査した。LDiH, LSoH 及び LSpH は瀉下作用を示す sennoside 含量が有意に低下した。一方，抗炎症作用を示す anthraquinone, lindleyin について，LSoH 及び LSpH のanthraquinone含量は LSo 同様に増加し，LSoH 及び LSpH の lindleyin 含量は LSo に比して保持されていた。以上，瀉下作用が向上する酒炒品は存在しないことが明らかになった。また，LSoH 及び LSpH は，LSo に比して抗炎症作用が期待できる可能性がある。
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| 8.
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Inoko, Ai ; Kakiuchi, Nobuko ; Yoshimitsu, Michiyo ; Cai, Shaoqing ; Mikage, Masayuki
概要:
金沢大学医薬保健研究域薬学系<br />Wild Ephedra plants growing near the Tibetan border of Yunnan and Sichuan Provinces and north-centra
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l Sichuan were surveyed and their DNA and ephedrine alkaloids content were analyzed. By analysis of internal transcribed spacer 1 (ITS) 1 DNA, E. likiangensis was found to be the dominant species in these regions, which clustered into 2 major groups in the phylogenic tree. Most Ephedra plants in these regions of ordinal size contained ephedrine and pseudoephedrine of more than 0.7%, the requirement for Japanese Pharmacopoeia 15th edition, suggesting that they have potential for crude drug production of Ephedra herbs. © 2009 Pharmaceutical Society of Japan.
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| 9.
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論文
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Okutsu, Kayu ; Yoshimitsu, Michiyo ; Kakiuchi, Nobuko ; Mikage, Masayuki
概要:
金沢大学医薬保健研究域薬学系<br />Ayurvedaで古くから薬用に供される「Asava」は,生薬を発酵させて製する薬酒である。我が国における一般的な薬酒は生薬を酒に浸漬させてつくられ,これら両者の大きな違いは発酵という過程を経ているか
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否かという点である。発酵が薬酒の性質にどのように影響を与えるかを明らかにするため,本研究では生姜と大棗を用い,それぞれからAsava製法とチンキ剤製法により薬酒を作製し,揮発性成分比をGC-MSにより測定した。Asava製法については,スリランカにて調査した結果に従った。その結果,Asava製法で作製した薬酒の揮発性成分比は,phenylethanolが30%を占めたのに対し,チンキ剤製法では2%以下と大きく異なっていた。またAsava製法で作製した生姜の薬酒はアルデヒド類(geranial,neral)が1%以下であり,作製中にgeraniol,nerolに還元されたことが示唆された。さらにAsava製法で作製した大棗の薬酒には,zizybeosideのアグリコンであるbenzyl alcoholの含有が認められたことから,アルコール発酵による配糖体の加水分解が示唆された。In Ayurvedic Medicine, some crude drugs and sugar are mixed and fermented, and developed into a herbal liquor called "Asava". During the alcoholic fermentation, the constituents of the crude drug would change chemically. There would thus be a chemical difference between Asava and tincture prepared by soaking the crude drug in alcohol. To investigate the differences between Asava and tincture, we attempted to prepare Asava in our laboratory with ginger rhizomes or jujube fruits and compared the volatile compounds of Asava with those of tincture by GC-MS. The alcohol contents of Ginger Asava and Jujube Asava prepared in the laboratory were 11% and 9.0%, respectively, and were almost the same as Asavas obtained in Sri Lanka. A characteristic difference between the Asava and the tincture was the relative content of phenylethanol (Asava: >30%, tincture: <2%), which is known to be a metabolite of phenylalanine during alcoholic fermentation. In the Ginger Asava, only trace aldehydes such as geranial and neral were found, whereas they were abundant in ginger tincture. Geranial and neral seemed to be reduced to geraniol and nerol. In Jujube Asava, we detected benzyl alcohol that was not detected in jujube tincture. Benzyl alcohol is an aglycon of zizybeoside I or II that was a component of jujube. Therefore, it seemed that glycosides are hydrolyzed by alcoholic fermentation. Hydrolysis of glycosides might help absorption of effective aglycones for the aged.
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| 10.
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Kitaoka, Fumiyo ; Kakiuchi, Nobuko ; Long, Changfeng ; Itoga, Mai ; Yoshimatsu, Hideki ; Mitsue, Akihiro ; Atsumi, Toshiyuki ; Mouri, Chika ; Mikage, Masayuki
概要:
金沢大学医薬保健研究域薬学系<br />The stem of the Akebia plant, "Mokutsu", is a crude diuretic and antiphlogistic drug. Japanese produ
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cts prepared from wild Akebia plants cover most of the Mokutsu market. Two Akebia plants, Akebia quinata Decaisne (Aq) and A. trifoliata Koidzumi (At) of Lardizabalaceae, are standardized as Mokutsu in Japanese pharmacopoeia. These two Akebia plants along with A. × pentaphylla Makino (Ap), which is considered a hybrid with the morphology of Aq and At, can be distinguished by DNA sequence analysis of internal transcribed spacers 1 and 2 (ITS) of nuclear ribosome DNA. Here, we report the results of molecular genetic analysis of Akebia plants grown in various wild habitats in Japan. We found that each of three Akebia plants could be distinguished in terms of their locality according to their nucleotide sequence in ITS, specifically at positions 91, 128, 133, 134, and 221. Plants with a comparable habitat had similar nucleotide sequences at these five points. We also found Aq with ITS and nucleotide deletion at position 86 that was distributed only around Awajishima in Shikoku (A), Harimanada (B), and Kinki (C), including the chief production center of Akebia Caulis. The results of these ITS sequences enabled discrimination of plants originating from Akebia Caulis. © 2009 The Japanese Society of Pharmacognosy and Springer.
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