Masayuki Mikage

Kanazawa University, Kanazawa, Ishikawa, Japan

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Publications (53)42.11 Total impact

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    ABSTRACT: The stems of Akebia plants, Akebiae Caulis, have long been used in traditional Chinese and Japanese medicines, and are mainly produced in western Japan. Three Akebia plants, Akebia quinata (AQ), A. trifoliata (AT), and A. pentaphylla (AP) grow wild in Japan. With the aim of carrying out molecular biological identification of Akebia plant species and discriminating Akebiae Caulis from other related crude drugs originating from non-Akebia plants, sequencing analysis of Akebia plants collected from various parts of Japan and the southern Korean Peninsula was performed. Specimens identified morphologically as AQ and AT had their respective common internal transcribed spacer one (ITS1) sequences, which could be distinguished. Cloning experiments of AP specimens showed that their ITS1 contained both common sequences of AQ and AT as well as their chimera. These chimeric sequences were not identical between AP specimens, suggesting that AP is not a species with uniform DNA sequences but a group of individuals with hybrid genomes of AQ and AT. Based on the sequences of Akebia species found here, we propose polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) methods to discriminate Akebiae Caulis from the related crude drugs and to distinguish three Akebia plants. Comparison of triterpene-rich fractions of extracts from Akebia plants by TLC showed that AP had an intermediate profile of AQ and AT.
    Biological & Pharmaceutical Bulletin 05/2009; 32(4):665-70. DOI:10.1248/bpb.32.665 · 1.83 Impact Factor
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    ABSTRACT: The stem of the Akebia plant, "Mokutsu", is a crude diuretic and antiphlogistic drug. Japanese products 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. x 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.
    Journal of Natural Medicines 04/2009; 63(3):368-74. DOI:10.1007/s11418-009-0318-8 · 1.59 Impact Factor
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    ABSTRACT: Antioxidants in herbs or crude herbal formulations are well known. The antioxidative effect of a Kampo formulation, Bofu-tsusho-san (BTS), was studied in healthy human subjects by means of an ex vivo study of the lag time of low-density lipoprotein (LDL) oxidation. Eighteen (18) healthy young males (22 +/- 3 years old), who were screened out from 38 males by strict screening tests of serum lipids, were randomly assigned into 3 groups (active BTS 7.5 g/day, placebo of BTS 7.5 g/day, and vitamin E 500 mg and vitamin C 1000 mg/day). The BTS groups were assigned in a double-blind manner. These 3 groups took each test drug, and the antioxidant effects of their plasma were estimated by assessing the susceptibility of LDL to oxidation induced by 2-2'-azobis (4-methoxy-2,4-dimethyl-valeronitrile) via an ex vivo study. The antioxidative activity was evaluated by measuring the lag time of the production of conjugated dienes. Along with lag time, plasma ephedrine, plasma baicalin, serum lipid peroxide, serum free fatty acids (nonesterified fatty acids), urinary 8(OH)dG/creatinine, blood pressure, and heart rate were monitored for 24 hours. Although lag time of the placebo group revealed no significant changes, the active BTS group showed some tendency of prolonged plasma lag time (p = 0.08). However, urinary 8(OH)dG/creatinine and serum lipid peroxide did not show no significant changes. The physiologic changes were compatible with the pharmacokinetic results of ephedrine and its sympathomimetic effect in the active BTS group. Although the systemic antioxidative effects of BTS have not been ascertained, the antioxidative potentiality on LDL oxidation was suggested by the ex vivo study.
    Journal of alternative and complementary medicine (New York, N.Y.) 04/2009; 15(3):267-74. DOI:10.1089/acm.2007.0713 · 1.59 Impact Factor
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    ABSTRACT: TLC and HPLC were used to identify possible chemical markers for evaluating the quality of the crude drug "Pogostemoni herba" (aerial part of Pogostemon cablin), which is a component of Kampo medicines. In addition to the reported patchouli alcohol and 2-hydroxy-6-methyl-3-(4-methylpentanoyl)-4-pyrone, three phenylethanoids were isolated from this plant material for the first time: acteoside, isoacteoside, and crenatoside. The usefulness of these compounds as indicators of the crude commercial drug under various TLC conditions was examined, and patchouli alcohol was found to give a definite spot with a reproducible Rf value. Therefore, we propose TLC of the methanol (MeOH) extract using patchouli alcohol as a marker as a convenient method for identifying the crude drug Pogostemoni herba.
    Yakugaku zasshi journal of the Pharmaceutical Society of Japan 01/2009; 128(12):1833-7. DOI:10.1248/yakushi.128.1833 · 0.26 Impact Factor
  • Taketo Okada · Masayuki Mikage · Setsuko Sekita ·
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    ABSTRACT: The cDNAs (Espals) encoding phenylalanine ammonia-lyase (PAL) were cloned from Ephedra sinica by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and by 5' and 3'-rapid amplification of cDNA ends (RACE). 2166 bp of the open reading frame (ORF) encoded 722 amino acids; sequence analyses of Espal clones suggested that at least four isoforms of EsPAL (EsPAL1, 2, 3, 4) existed, with nine amino acids substitution in their sequences. Phylogenetic analysis of EsPAL and PALs from other plant species revealed that EsPAL and Pinus PAL formed a gymnosperm-type PAL subfamily. The recombinant EsPAL1 to 4 functionally catalyzed a PAL reaction and their K(m), V(max), K(cat) and K(cat)/K(m) values did not show significant differences. Semi-quantitative RT-PCR analysis indicated that the expression of Espal genes in the roots was higher than in the plant's aerial parts. In addition, the activity of PAL in the roots was also higher than in the aerial parts. These results suggest that Espal genes are expressed in the whole plant but are dominant in the roots rather than in the aerial parts.
    Biological & Pharmaceutical Bulletin 01/2009; 31(12):2194-9. · 1.83 Impact Factor
  • Misato DOUI · Masayuki MIKAGE ·

    Kampo Medicine 01/2009; 60(4):429-434. DOI:10.3937/kampomed.60.429
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    Naoko Fushimi · Lili Wang · Shunsuke Ebisui · Shaoqing Cai · Masayuki Mikage ·
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    ABSTRACT: マオウ科のEphedra sinica Stapf, E. intermedia Schrenk et C.A.Meyerおよび中国産のE. distachya L.の種間差を明らかにする目的で,中国の主として内蒙古自治区と青海省で採集したマオウ属植物を外部および内部形態学的に検討した。その結果,E. sinicaと中国産のE. distachyaは同一種であること,E. sinicaとE. intermediaは内部形態的に茎の皮層中の繊維束数と髄中の繊維数の2要素を考慮することにより明確に区別できること,この形質によってこれら2種に由来する市場品や毬果のない押葉標本の種同定か可能であること,E. sinicaが分布するとされる青海省東部で採集したマオウ属植物はすべてE. intermediaでありE. sinicaは確認できなかったこと,青海省産E. intermediaの毬果のない小型株がE. sinicaと同定されたと考えられること,よって青海省東部の竜羊峡地区などで採集される麻黄の原植物はE. intermediaと考えられること,などが明らかになった。 Ephedra plants collected mainly in Inner Mongolia and Qinghai Province, China, were studied morphologically to clarify the specific differences between E. sinica Stapf, E. intermedia Schrenk et C.A.Meyer, and E. distachya L. recorded in China. The results obtained were as follows: E. sinica and E. distachya recorded in China were the same species; E. sinica and E. intermedia could be stem-anatomically distinguished from each other by examining both the number of fiber bundles in the cortex and the number of fibers in the pith into account, and the species of commercial samples of Ma-huang as well as sterile herbarium specimens that include no cones were identified by this method; All Ephedra plants collected in the eastern region of Qinghai Province, where E. sinica was reported to be distributed, were identified as E. intermedia; The limited plant of E. intermedia without cones collected in the east of Qinghai Province might possibly have been identified as E. sinica in the past; The plant origin of the Ma-huang produced in Qinghai Province, which is collected in the eastern regions such as Longyangxia district, was thought to be E. intermedia.
    07/2008; 25(3).
  • Masayuki Mikage · Yukari Hutagi ·
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    ABSTRACT: The historical change in the botanical origin of the Chinese herbal drug Shan-zhu-yu, San-syu-yu in Japanese) was studied herbologically. The results obtained were as follows: The original plant, described in Shen-nong-ben-cao-jing written in the Hou-Han Dynasty (A.D. 25-220) and Wu-pu-ben-cao written in the Wei dynasty (A.D. 220-265), was Prunus pseudocerasus or an allied species such as P. tomentosa. However, the original plant described in Ming-yi-bie-lu, written in the same era, was thought to be Cornus officinalis; the fruit of which is used commonly today as Shan-zhu-yu. In addition to Prunus and Cornus, the plants of the genus Berberis, of the family Berberidaceae, and Zanthoxylum of Rutaceae were also used in China until the Ming Dynasty. The plants of Elaeagnus, of the family Elaeagnaceae, were possibly used in ancient Japan. Cornus officinalis was determined to be the origin of Shan-zhu-yu during the Qing Dynasty, China.
    Yakushigaku zasshi. The Journal of Japanese history of pharmacy 02/2008; 43(1):33-9.
  • Taketo Okada · Masayuki Mikage · Setsuko Sekita ·

    Biological & Pharmaceutical Bulletin 01/2008; 31(12):2194-2199. DOI:10.1248/bpb.31.2194 · 1.83 Impact Factor
  • Masayuki MIKAGE · Hiroko ENDO ·

    Kampo Medicine 01/2008; 59(1):25-34. DOI:10.3937/kampomed.59.25
  • Masayuki MIKAGE · Hiroko ENDO · Shigeki KATSUKI · Nobuko KAKIUCHI ·

    Kampo Medicine 01/2008; 59(2):279-285. DOI:10.3937/kampomed.59.279
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    Hiroyuki Motomura · Shuichi Noshiro · Masayuki Mikage ·
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    ABSTRACT: Plants of Ephedra normally have vessels, but are known to become nearly vessel-less in some alpine localities. Previous studies implied that wood formation in Ephedra differs fundamentally from that in dicotyledons in which vessel-bearing and vessel-less taxa are systematically distinct. Using E. pachyclada in the Mustang district of Nepal, growing in an altitudinal range of over 2000 m, variation in wood formation and adaptation to alpine environment was studied in this normally vessel-bearing species. Variation in wood anatomy and wood formation was observed with conventional optical microscopy. The lengths of three kinds of tracheary elements were measured and statistically analysed against habitat altitude and plant size of the individuals studied. In E. pachyclada three kinds of tracheary elements, vessel elements, tracheids and fibre-tracheids, were nearly equal in length within individuals showing no elongation after differentiation from cambial initials. Tracheary element lengths among individuals had a negative correlation with altitude and a positive correlation with plant size. Multivariate analyses showed that altitude has a stronger correlation with tracheary element lengths than plant height or stem diameter. Moreover, several individuals from high elevations completely lacked vessels, and vessel formation fluctuated even in individuals from lower elevations. Wood anatomical trends in E. pachyclada are considered as an adaptation to extremely dry conditions in high mountains. Fluctuation in vessel formation in individuals from low elevations indicated that vessels differentiate only when their lateral expansion is allowed. These results showed that E. pachyclada has a different system of wood formation from dicotyledons and supported the opinion that the wood structure of Gnetales is fundamentally different from that of angiosperms.
    Annals of Botany 09/2007; 100(2):315-24. DOI:10.1093/aob/mcm111 · 3.65 Impact Factor
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    Toshiyuki Atsumi · Nobuko Kakiuchi · Masayuki Mikage ·
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    ABSTRACT: We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 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.
    Biological & Pharmaceutical Bulletin 09/2007; 30(8):1472-6. DOI:10.1248/bpb.30.1472 · 1.83 Impact Factor
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    ABSTRACT: More than 400 species of medicinal plants grow in the Northern Areas of Pakistan, including Ephedra plants. To investigate the wild Ephedra plant resources in the area, we surveyed the medicinal plants and collected 71 specimens from 18 collecting sites to analyze their genetic variation. The DNA sequences of the internal transcribed spacers 1 and 2 (ITS 1 and 2) of nuclear ribosomal DNA and a noncoding sequence of chloroplast DNA (trn L/F) were analyzed. This DNA data analysis and external morphological features were used to confirm the species of the specimens, and it was found that E. intermedia was the major species in the area and that E. gerardiana and E. przewalskii were present sporadically. Although it inhabits a relatively small area in comparison with the northwestern Chinese provinces, the DNA sequence of E. intermedia in the Northern Areas of Pakistan was significantly more heterogeneous than the same species grown in those neighboring regions. Most of the E. intermedia specimens contained more than 0.7% ephedrine alkaloids, fulfilling the requirement of the Japanese Pharmacopoeia; thus, the Ephedra plants in the area are a genetic and medicinal resource of great importance.
    Journal of Natural Medicines 06/2007; 61(3):357-365. DOI:10.1007/s11418-007-0134-y · 1.59 Impact Factor
  • Kazuhiro Matsuyama · Sanae Tatsukawa · Nobuko Kakiuchi · Masayuki Mikage ·
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    ABSTRACT: The tuberous root of aconite, Aconiti tuber, contains tonic but poisonous aconite alkaloids, such as aconitine, hipaconitine and mesaconitine. The standardization of the cultivation protocols must be established, since the quality of the crude drug, especially the content of aconite alkaloids, is influenced by cultivation conditions. Here, we report the effects of planting depth, disbudding and root pruning on the content of the aconite alkaloids as well as on the weight of the root tuber of aconite. The daughter root tubers of shallow-planted plants showed a significantly higher content of aconitine, hipaconitine and sum of aconite alkaloids per unit weight than those of deeply planted root tubers (control group), while the average weight per root tuber was lower than that of the control group. The total weight of the daughter root tubers per plant of the disbudding group was significantly higher than that of other groups, including the root-pruning group whose average weight per tuber was the highest. In conclusion, disbudding is effective in achieving both higher content of aconite alkaloids and heavier total weight of the root tuber.
    Journal of Natural Medicines 03/2007; 61(2):127-130. DOI:10.1007/s11418-006-0028-4 · 1.59 Impact Factor
  • Masayuki Mikage · Akane Ochimori ·
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    ABSTRACT: The Chinese crude drug Qiang-hua was listed as an alias of Du-hua in Shen-nong-ben-cao-jing, an herbal journal written during the Han Dynasty, China. Du-hua and Qiang-hua are recognized as different herbs in China these days; the main botanical origin of Du-hua is Angelica spp. and that of Qiang-hua is Notopterygium spp., of the family Umbelliferae. To make clear the botanical origins of Du-hua and Qiang-hua in ancient China, the authors made a herbological study. The findings were as follows: the name of Qiang-hua was given to the genuine Du-hua, which is produced in Qiang Province, an ancient province located in northwest China; the botanical origin of Qiang-hua is presumed to be Notopterygium incisum Ting ex H.T. Chang and N. forbesii Boissieu based on both the morphology and habitat written in ancient herbal journals. Both species are prescribed as having the plant origin of Qiang-hua in the present Chinese Pharmacopoeia. Therefore, we concluded that Du-hua and Qiang-hua were essentially the same drug, and were originally derived from the Notopterygium species in ancient China.
    Yakushigaku zasshi. The Journal of Japanese history of pharmacy 02/2007; 42(1):17-21.
  • Kayu Okutsu · Michiyo Yoshimitsu · Nobuko Kakiuchi · Masayuki Mikage ·
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    ABSTRACT: 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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    ABSTRACT: Progression of the desertification in northern China has been causing damage to wild Ephedra plants on which we depend for most of supply of the traditional herbal medicine, "Ma huang." The Chinese government encourages the cultivation of Ephedra plants, and Ephedra fields have been reclaimed in the original Ephedra habitats in recent years. We surveyed 7 Ephedra fields that have been recently developed in the Inner Mongolia Autonomous Region and Ningxia Hui Autonomous Region to collect information on Ephedra plant cultivation, especially pertaining to crop species. Specimens taken from those Ephedra fields were genetically and morphologically analyzed, and their ephedrine alkaloid content was examined. DNA analyses of Ephedra specimens, including DNA sequencing of ITS (internal transcribing sequence of nuclear ribosomal DNA) and trn L/F (intron of trnL and intergenic spacer between the trnL and trnF of chloroplast DNA) region and species-specific amplification of trn L/F were conducted to identify Ephedra species. Based on the results of DNA sequencing and morphological determination, the crops grown in 6 fields ware identified as Ephedra sinica, while co-planting of E. sinica and E. intermedia was found in one field where a higher appearance rate of plants with varied morphology from wild Ephedra plants was observed. Furthermore, direct sequencing of the PCR product of the trn L/F region of some specimens from the field and their species-specific PCR showed ambivalent result. Cloning and sequencing of the PCR product of the trn L/F region of those specimens DNA suggested their heteroplasmy, containing both E. sinica- and E. intermedia-type chloroplasts. On the other hand, the profile of the ephedrine alkaloid content was clearly correlated with the result of direct sequencing of the trn L/F region; the specimens showing the E. sinica-type sequence contained more ephedrine than pseudoephedrine, and the specimens of the E. intermedia-type more pseudoephedrine.
    Biological & Pharmaceutical Bulletin 05/2006; 29(4):746-9. DOI:10.1248/bpb.29.746 · 1.83 Impact Factor
  • Chieko Yoshizawa · Makiko Kitade · Masayuki Mikage ·
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    ABSTRACT: As we previously reported, ma-huang ([Chinese characters: see text], Ephedrae Herba) has been sometimes used together with mu-zei ([Chinese characters: see text], Equiseti Herba) in medieval China and Japan. We herbologically studied this confusion and found that, in China, the confusion was found in literature in the Song dynasty, and Li Shi-Zhen recorded in Ben-cao-gang-mu that both drugs were morphologically and medicinally the same in the Ming dynasty. Though the main reason why the plant of the genus Equisetum, especially E. ramosissimum Desf., had been substituted for Ephedra plants is thought to be their morphological similarity, the doctors who lived in the area where no Ephedra plants grew might have used Equisetum plants as ma-huang based on Li's description. Owing to this confusion in China, the plants of E. ramosissimum were sometimes imported to Japan as ma-huang, and it caused the tentative use of E. ramosissimum as ma-huang in the middle of the Edo era in Japan.
    Yakushigaku zasshi. The Journal of Japanese history of pharmacy 02/2006; 41(1):9-17.
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    Changfeng Long · Nobuko Kakiuchi · Guoyue Zhong · Masayuki Mikage ·
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    ABSTRACT: The resources of wild Ephedra plants in the Xinjiang Uygur Autonomous Region were surveyed. Ephedra plants mainly grow on the fringes of the Taklimakan Desert and Gureban-tonggute Desert. We found six genotypes of Ephedra przewalskii growing widely in Xinjiang. Three genotypes of Ephedra intermedia were limited to the northern and eastern parts, and Ephedra regeliana scattered in the northern part of Xinjiang. These Ephedra specimens were analyzed for DNA sequences of nuclear ribosomal DNA, internal transcribed spacers 1 and 2, chloroplastic DNA, trnL intron and trnL-trnF intergenic spacer. Intraspecific variation of the nucleotide sequence in E. przewalskii was found in different habitats. Norephedrine, ephedrine, pseudoephedrine, and methylephedrine contents of the specimens were determined. Although Ephedra intermedia of all three genotypes contained ephedrine alkaloids, ephedrine alkaloids were not detected in E. regeliana and E. przewalskii.
    Biological & Pharmaceutical Bulletin 03/2005; 28(2):285-8. DOI:10.1248/bpb.28.285 · 1.83 Impact Factor

Publication Stats

157 Citations
42.11 Total Impact Points


  • 2002-2014
    • Kanazawa University
      • Graduate School of Natural Science and Technology
      Kanazawa, Ishikawa, Japan
  • 2011
    • Peking University
      • School of Pharmaceutical Sciences
      Beijing, Beijing Shi, China