Isolation of phenylisoserine methyl ester from the leaves of Taxus cuspidata var. nana

From methanolic extracts of leaves of kyaraboku, Taxus cuspidata var. nana, phenylisoserine methyl ester (3) was isolated along with taxinine (1), taxol (2), sciadopitysin (4), ginkgetin (5), isorhamnetin (6), and quercetin (7). This is the first time that phenylisoserine methyl ester has been isolated from T. cuspidata var. nana. Compound 3 was also isolated from the ethanolic extracts of leaves of T. cuspidata var. nana. Furthermore, compound 3 was identified in methanolic extracts from the bark of this tree.     

Constituents from the roots of <Emphasis Type="Italic">Taxus cuspidata</Emphasis>

The known propelargonidin, afzelechin-(48)-afzelechin (1), the known lignans 7-hydroxynortrachelogenin (2), epinortrachelogenin (3), nortrachelogenin (4), hydroxymatairesinol (5), allohydroxymatairesinol (6), matairesinol (7), oxomatairesinol (8), and isotaxiresinol (9), and the known taxoids taxinine M (10), taxayuntin (11), and 10-deacetyltaxol (12), and 10-deacetylbaccatin III (13) were isolated from the roots of Taxus cuspidata (Japanese yew, Taxaceae). The propelargonidin was isolated from Taxus spp. for the first time, and was detected in the roots, bark, and twigs.     

Suppression of phytopathogenic fungi by hexane extract of Nepenthes ventricosa x maxima leaf

The hexane extract of Nepenthes ventricosa x maxima leaf exhibited antifungal activity against Alternaria alternata, Aspergillus niger, Bipolaris oryzae, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani, Rhizopus stolonifer var. stolonifer and Sclerotinia sclerotiorum.     

Structure identification for compound I separated and purified fromtaxoids-produced endophytic fungi （Alternaria. alternata var. taxi 1011）

Endophytic fungi are widely found in almost all kinds of plants. Many endophytic fungi can produce some physio-logical active compounds, which are same to or analog to those isolated from their hosts. Producing physiological active compounds through microbial fermentation can give a new way to resolve resource limitation and to find out alternative source.Through the methods of organic solvent extraction, thin layer chromatography (TLC) and column chromatography, compound I was isolated, purified from the liquid fermentation metabolites of the taxoids-produced endophytic fungi (Altemaria. altemata var.taxi 1011 Y. Xiang et LU An-guo) that was screened from the bark of Taxus. cuspidata Sieb.et Zucc.. Compound I was identified as one kind of taxoids type Ⅲ, based on the analyzing results by using the methods of ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). This study provides a completed method for separation and purification of the endophytic fungi as well as structure identification of its fermentation metabolite     

The polar neutral and basic taxoids isolated from needles and twigs of Taxus cuspidata and their biological activity

Twelve basic taxoids and 22 neutral taxoids were isolated from basic and polar neutral fractions of the extracts of needles and twigs of Taxus cuspidata. Among them, taxine NA-13, 3,11-cyclotaxinine NN-1, taxinine NN-6, 11(15→1)abeo-taxinine NN-1, taxine NA-8, and taxine NA-4 were isolated first from natural sources by us. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). 7-Epitaxol, 7-epicephalomannine, taxinine NN-6, taxine NA-2, taxuspine H, and taxagifine were active toward VA-13 cells and 7-epitaxol, 7-epicephalomannine, taxinine NN-1, 9,10-deacetyltaxinine, and taxagifine were active toward HepG2 cells. The multidrug-resistant (MDR) cancer reversal activity of isolated compounds was evaluated on the basis of the amount of vincristine (VCR) accumulated in MDR human ovarian cancer 2780 AD. Taxine NA-8, taxine NA-2, 5-cinnamoyl-10-acetyltaxicin II, and taxinine NN-1 indicated stronger MDR cancer reversal activity than verapamil. The result of primary screening based on 39 human cancer cell lines suggests that taxinine NN-1 belongs to a new mechanistic class and is a new anticancer agents. 7-Epicephalomannine was found to be an effective anticancer agent with tubulin as its molecular target, which is the same as paclitaxel. Part of this work was presented at the 74th Annual Meeting of the Chemical Society of Japan, Tanabe, March 1998, and the 41st Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics, Gifu, December 1998. The results of this work were also presented as patent documents at PCT Int: Appl WO 2001007040 A1, February 2001; Appl WO 2000-Jp 5036, 27 July 2000. Priority: JP 1999-214273, 28 July 1999; JP 1999-224652, 6 August 1999; JP 2000-76404, 14 March 2000     

Comparison of terpenes in extracts from the resin and the bark of the resinous stem canker of Chamaecyparis obtusa and Thujopsis dolabrata var. hondae

A monoterpene and 15 diterpenes were isolated from the ethyl acetate extracts of the bark-glued resin from the resinous stem canker ofThujopsis dolabrata var.hondae Makino. A monoterpene (nezukone20) and 4 diterpenes (acetyl torulosol5, acetyl isocupressic acid8, acetyl abietinol11, and 7-methoxytotarol18) were characteristic constituents of the ethyl acetate extracts but were absent in then-hexane extracts from the resinous stem canker ofT. dolabrata var.hondae. These terpenes were first isolated fromT. dolabrata var.hondae andT. dolabrata. The available literature suggests that diterpene18 is a new compound. The resinous stem canker ofChamaecyparis obtusa Endlicher contained larger amounts of manool1,trans-communic acid6, and ferruginol12 and smaller amounts of isocupressic acid7 and abietinol10 than the resinous stem canker ofT. dolabrata var.hondae. The concentration of18 was less than 2% in the extracts, and the resinous stem canker ofC. obtusa lacked this compound. The resinous stem cankers ofC. obtusa andT. dolabrata var.hondae provided extracts 15.6 and 4.96 times, respectively, heavier than the healthy ones. Large differences in the ratios and compositions of terpenes were also observed between the resinous stem canker and the healthy trees. Terpenes isolated from the extracts contained many kinds of diterpene, especially the labdane-type diterpenes, in these diseased trees. These results suggest that the presence of labdane-type diterpenes is closely associated with the resinous stem canker or the causal fungi of this disease.Part of this report was presented at the 50th annual meeting of the Japan Wood Research Society, Kyoto, April 2000     

Nursery soil cover influences germination of Thujopsis dolabrata var. hondai seeds

We examined the influence of nursery soil cover on germination of Thujopsis dolabrata var. hondai. Seeds were sown under seeding bed conditions: (1) nursery soil (andosol) without soil cover, (2) Kanuma pumice without soil cover, (3) nursery soil with nursery soil cover, and (4) nursery soil with sterilized sand cover. Germination percentages were compared between these conditions. The germination percentage (8.8%) at 10 weeks after sowing under condition 3 was significantly lower than under conditions 1, 2, and 4 (56.8%, 52.0%, and 47.2%, respectively). Of the nongerminated seeds under condition 3, 75.0% were decayed and several fungi were isolated from decayed seeds. An inoculation test of isolated fungi Cylindrocarpon tenue, Cylindrocarpon sp., Fusarium oxysporum, and Botrytis sp. to seeds showed that these fungi caused seed decay. Our results indicated that nursery soil cover may not be suitable for T. dolabrata var. hondai seeds sown in nursery soil because of seed decay by pathogens.     

林地土壤及其真菌对毛红椿种子发芽及幼苗保存的影响

[目的]通过不同群落毛红椿天然林林地土壤及其真菌对种子萌发和幼苗存活影响的研究,探讨影响毛红椿天然更新的障碍因素。[方法]在江西九连山国家级自然保护区内的3个毛红椿天然优势群落中,分别取距离毛红椿母树3个不同距离(2.5、5.0、7.5 m)处的根区土壤和远离毛红椿母树(25 m以外)的非根区土壤,以非林地土为对照开展室内模拟播种和以根区土壤真菌的水或根系分泌物悬液对毛红椿幼苗进行灌根接种,观察种子萌发、幼苗存活和幼苗感病情况。[结果]各群落土壤中种子发芽均呈先上升后下降规律,且基本在第8 10天达到高峰;林地土与非林地土种子发芽率差异不显著,但林地根区土幼苗的存活率极显著低于非林地土和林地非根区土;距母树3个不同距离根区土的幼苗存活率差异不显著。灌根接种不同处理间幼苗存活率差异极显著,具体表现为接种RS2、RS5根区真菌的幼苗感病率显著高于根系分泌物及无菌水空白对照,且RS2真菌的根系分泌物悬浮液幼苗感病率显著高于其无菌水悬浮液。[结论]毛红椿种子萌发不受土壤环境的影响,但幼苗建成受根区范围内土壤的影响,根系分泌物和致病菌的互作显著降低幼苗保存率。由此推断,毛红椿根区土壤内存在幼苗的潜在致病菌及根系分泌物可增强其致病性。     

Dihydroroseoside, a new cyclohexanone glucoside, from the leaves of shirakamba (Betula platyphylla Sukatchev var.japonica Hara)

A new cyclohexanone glucoside (II) and a known cyclohexenone glucoside roseoside [I, (6S, 9S)-6-hydroxy-6-(9--D-glucopyranosyloxy-trans-7-butenyl)-1,5,5-trimethyl-1-cyclohexenone] were isolated from an ethanol extract of shirakamba (Betula platyphylla Sukatchev var.japonica Hara) leaves. The structure ofII was determined to be (6S, 9R)-6-hydroxy-6-(9-O--D-glucopyranosyloxy-trans-7-butenyl)-1, 5,5-trimethyl-1-cyclohexanone by¹H-NMR and¹³C-NMR spectroscopic analyses. It was named dihydroroseoside.     

红花尔基沙地樟子松天然林枯立木特征分析

[目的]了解沙地樟子松天然纯林中枯立木的数量及空间结构特征,探究枯立木形成的原因,为樟子松林的保护和经营提供依据。[方法]在沙地樟子松天然纯林中设置2块1 hm~2的大样地,用全站仪对样地中所有胸径大于5 cm的立木进行定位并进行全面调查;对调查样地的基本特征,枯立木的数量特征及径级分布进行了分析,提出了用于表达林分中枯立木微环境的活立木比的概念,并采用林分空间结构参数一元分布和二元分布分析方法,对枯立木与其最近4株相邻木的关系进行分析。[结果]2块不同密度的樟子松天然纯林下更新幼苗和枯立木数量相差较大,密度较小(样地1)的样地更新幼苗和枯立木较少,而密度较大的样地(样地2)中枯立木达到200棵,林下更新幼苗数量达到15 280株·hm~(-2);樟子松天然纯林样地内枯立木主要以小径级木为主,胸径集中在11 cm以下;样地1枯立木径级连续分布,幅度较窄;样地2中的枯立木径级幅度较宽,但在20 22 cm缺刻,有2株大于23 cm的枯立木;2块样地中枯立木的分布格局均为随机分布,样地1中枯立木周围的4株相邻立木大多为活立木,且胸径较枯立木大;样地2中,只有一半的枯立木周围的最近4株立木为活立木,且有三分之一以上的枯立木胸径不是最小的,枯立木有连续分布的现象。2块样地中枯立木的角尺度-大小比数二元分布特征的差异不明显,而角尺度-活立木比二元分布特征和大小比数-活立木比二元分布特征差异明显,样地1中枯立木的最近4株随机分布于其周围的相邻木为活立木且胸径大于枯立木的比例明显高于样地2,而枯立木最近4株随机分布于其周围的相邻木有枯立木的比例明显小于样地2。[结论]樟子松天然纯林枯立木以小径级林木为主,枯立木的数量与林分密度相关,林木竞争是林木死亡的主要原因,密度过大也会产生病虫害,因此,对天然樟子松纯林要进行适度经营,保持合理密度。     

Susceptibility of adult trees of the endangered species Pinus armandii var. amamiana to pine wilt disease in the field

Adult trees of Pinus armandii var. amamiana (PAAm) and P. thunbergii grown in the field were inoculated with 100000 or 1000 of the nematode Bursaphelenchus xylophilus to evaluate their susceptibility to pine wilt disease. PAAm trees inoculated with 100000 nematodes started to show disease symptoms 2 weeks after inoculation, and all died within 29 weeks. Although the PAAm trees inoculated with 1000 nematodes tended to show delayed disease symptoms compared with those inoculated with 100000 nematodes, all of them died within 33 weeks after inoculation. All P. thunbergii trees inoculated with 1000 nematodes had died 6 weeks after inoculation. In the nematode-inoculated PAAm trees, death of branches distal to the nematode inoculation site was the first visible symptom, followed by the systemic discoloration of needles, whereas the whole tree wilted simultaneously in P. thunbergii trees. In nematode-inoculated PAAm trees, the period from inoculation to death was longer than that in P. thunbergii. These results suggest that adult PAAm trees are susceptible to pine wilt disease, but are less vulnerable than P. thunbergii.     

Distinction of resin compounds between the healthy bark and the resinous stem canker of <Emphasis Type="Italic">Thujopsis dolabrata</Emphasis> var. <Emphasis Type="Italic">hondae</Emphasis>

Methyl esters of higher fatty acids were detected from the healthy bark of Thujopsis dolabrata var. hondae but not from the bark of the resinous stem canker of T. dolabrata var. hondae. This difference enabled us to distinguish healthy trees from diseased ones. Fourteen diterpenes were also isolated from the n-hexane extracts of the bark-glued resin taken from the resinous stem canker of T. dolabrata var. hondae. Of these diterpenes, abietane diterpenes [abieta-7,13-diene (1), abietinol (2), dehydroabietinol (4)], pimarane diterpene [sandaracopimaric acid (8)], and labdane diterpenes [manool (10), torulosol (11), torulosal (12), cupressic acid (13)] were first isolated from T. dolabrata var. hondae.Part of this paper was presented at the 47th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Photodiscoloration of western hemlock (Tsuga heterophylla) sapwood III Early stage of photodiscoloration reaction with lignans

The reaction during the early stage of photodiscoloration of constituents in western hemlock [Tsuga heterophylla (Raf. Sarg., Pinaceae] sapwood was investigated with chemical methods. The main photodiscoloring constituents, hydroxymatairesinol, allohydroxymatairesinol, -conidendrin, and oxomatairesinol, were used as substrates for light-irradiation experiments in vitro. The structures of photodiscoloration reaction products were elucidated by isolation and instrumental analyses and/or co-high-performance liquid chromatography analyses with authentic specimens. The experiment was undertaken to distinguish each series of liquid phases using chloroform, water (both including a trace of methanol), and methanol, and the solid phase. The reaction products allohydroxymatairesi (2), oxomatairesinol (3), -conidendrin (4), allo-7-methoxymatairesinol (5), 7-methoxymatairesinol (6), and vanillin (7) were isolated or detected in the reaction mixture of a hydroxymatairesinol system. The reaction products hydroxymatairesinol (1), 3, 4, 5, 6, and 7 were confirmed in the reaction system of allohydroxymatairesinol, which was an epimer of hydroxymatairesinol. Product 3 was confirmed from the -conidendrin system, and reaction product 7 was confirmed from oxomatairesinol. The photodiscoloration reaction of western hemlock sapwood could be initiated by the formation of phenoxy radicals from the respective constituents. The reaction was then presumed to progress via formation of a quinonemethide intermediate in many of them. It was suggested that the reactive species, such as phenoxy radical or quinonemethide intermediate, formed by lightirradiation might be converted to quinone derivatives and colored oligomers. Products 1, 2, 3, 4, and 7, formed from substrates such as hydroxymatairesinol, allohydroxymatairesinol, -conidendrin, and oxomatairesinol, were the same as the original metabolic constituents of western hemlock. Therefore it was concluded that the photodiscoloration of western hemlock depends not on the quantitative level of a few respective metabolites but, rather, on the coexistence of many metabolites.Part of this paper was presented at the 46th Annual Meeting of Japan Wood Research Society at Kumamoto, April 1996     

沙地樟子松天然纯林的结构特征

[目的]为了解红花尔基地区沙地樟子松天然纯林的结构特征,指导沙地樟子松的保护与经营。[方法]在红花尔基地区设置2块100 m×100 m的樟子松天然纯林固定样地,利用样地内每木定位调查数据和分析统计软件进行一元分布及二元分布特征分析。[结果](1)樟子松天然林纯林直径分布为单峰或多峰山状分布,垂直结构简单,只有乔木层和草本层。(2)樟子松天然纯林的林木分布格局为均匀分布,接近随机分布,林木分布格局类型与林分密度无关;林分中樟子松个体竞争激烈,多数单元中林木呈较密集状态。(3)2块样地中随机分布状态下的林木多数为中等密集或比较密集,不同分布状态下的林木优劣性差异较小;低密度樟子松天然纯林中多数密集状态的林木为绝对优势木或优势木,而高密度林分中林木密集度分布与林木大小无关。[结论]红花尔基沙地樟子松天然林结构不合理,应选择病腐木及聚集分布的个体作为潜在调整对象,进行密度调整和结构优化。     

Virulence of ophiostomatoid fungi associated with the spruce bark beetleIps typographus f.japonicus in Yezo spruce

Yezo spruce trees (Picea jezoensis), approximately 40-year-old were inoculated with eight ophiostomatoid fungi associated withIps typographus f.japonicus to compare relative virulence of the fungi. Among them,Ophistoma penicillatum formed the longest necrotic lesion on inner bark around inoculation points, followed byO. aenigmaticum, Ceratocystis polonica, andO. bicolor, whileC. polonica formed a larger dry zone in sapwood than the other fungi. Yezo spruce trees were also mass inoculated withC. polonica, O. penicillatum, O. piceae singly or mixed to demonstrate the ability of the fungi to kill Yezo spruce trees. The trees inoculated withC. polonica, O. penicillatum or the mixed inoculum showed discoloration of needles in the early summer of the next year and died by autumn. However, the trees inoculated withO. piceae or the control inocula did not die, except for one tree. These results indicated thatC. polonica andO. penicillatum were more virulent thanO. piceae and suggested that they might be at least partially responsible for the mortality of the beetle-infested Yezo spruce trees. Part of this study was supported by the Sumitomo Foundation, Japan to Y. Yamaoka and I. Takahashi. Part of this study was presented at the 107th meeting of the Japanese Forestry Society, April 1–4, 1996, Tsukuba, Ibaraki, at the 42nd annual meeting of the Mycological Society of Japan, May 16–17, 1998, Kyoto, and at the 110th meeting of the Japanese Forestry Society, April 2–5, 1999, Matsuyama, Ehime. Contribution No. 143, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Phenolic constituents ofTaxus cuspidata I: lignans from the roots

The phenolic constituents of the roots ofTaxus cuspidata (Japanese yew) were investigated. Four lignans, [(+)-taxiresinol (1), (+)-lariciresinol (2), (–)-secoisolariciresinol (3), and (+)-pinoresinol (4)] were isolated and identified. The assignment of proton and carbon atoms for the lignans were finally solved by one- and twodimensional-nuclear magnetic resonance spectra. The enantiomeric excess of these lignans were determined by chiral high-performance liquid Chromatographic analyses. (+)-Lariciresinol and (–)-secoisolariciresinol were optically pure; (+)-taxiresinol was also suggested to be optically pure, although (+)-pinoresinol was not (77% enantiomeric excess).     

Iridoids from the roots of Valeriana jatamansi and their neuroprotective effects

Three new iridoids, valeriandoids A–C (1–3), together with three known analogues (4–6), were isolated from the roots of Valeriana jatamansi. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HRESIMS, 1D and 2D NMR) and by comparison of their NMR data with those of related compounds. All the isolated compounds were evaluated for their neuroprotective effects and compounds 1, 3, 4 and 6 showed moderate neuroprotective effects.     

Antifungal properties of methanol extract and its active compounds from Brickellia rosmarinifolia Vent

A new isocoumarin, 7-hydroxyl-4-methyl isocoumarine (1), together with three known monoterpenes, (3R, 4R, 6S)-3, 6-dihydroxy-1-menthene (2), (+)-(1R, 3S, 4R, 6S)-6-hydroxymenthol (3) and 4-isopropyl-1-methylcyclohex-2-ene-1, 6-diol (4), was isolated from the methanol extract of Brickellia rosmarinifolia. The structures were determined by spectroscopic means. Compounds 1, 2, 3 and 4 showed antifungal activities against Colletotrichum musae and Peronophythora litchii in vitro.     

Chemical composition of the green alga Codium Divaricatum Holmes

A new sterol, 24-R-stigmasta-4,25-diene-3β,6β-diol (1), along with three known compounds (2–3), was isolated from the green alga Codium divaricatum Holmes, a traditional Chinese medicine, which is efficacious against cancer. All structures were determined by spectroscopic methods and comparison with related known compounds. Single-crystal X-ray crystallography allowed us to confirm the structure of 1. To our knowledge, the compound 1 is reported as the first from natural source, and compounds 2, 4 have not been isolated from green algae before.     

neo-Clerodane diterpenes from Ajuga ciliata Bunge and their neuroprotective activities

Three new (1–3) and three known (4–6) neo-clerodane diterpenes have been isolated from the whole plants of Ajuga ciliata Bunge. The structures of the new compounds were elucidated as (12S)-1β,6α,19-triacetoxy-18-chloro-4α,12-dihydroxy-neo-clerod-13-en-15,16-olide (1), (12S,2′S)-12,19-diacetoxy-18-chloro-4α,6α-dihydroxy-1β-(2-methylbutanoyloxy)-neo-clerod-13-en-15,16-olide (2), and (12S)-6α,18,19-triacetoxy-4α,12-dihydroxy-1β-tigloyloxy-neo-clerod-13-en-15,16-olide (3), on the basis of spectroscopic data analysis. All the diterpenes were evaluated for the neuroprotective effects against MPP⁺-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells and compounds 2–5 exhibited moderate neuroprotective effects.