Chemical and Genetic Diversity of Ligularia kanaitzensis in the Hengduan Mountains Area. Chemical Relationship with L. subspicata.

Root chemicals and the sequences of the internal transcribed spacers (ITSs) were analyzed for 9 Ligularia kanaitzensis and 3 L. subspicata samples collected in northwestern Yunnan and southwestern Sichuan, China. Subspicatins A and C were isolated from two L. kanaitzensis samples. Introgression of genes responsible for these compounds from L. subspicata was suggested by their strong connection with L. subspicata/L. lamarum and the geographical proximity of the samples to L. subspicata. DNA analysis of a set of 27 L. kanaitzensis samples including those analyzed previously showed that they belong to two clades, designated A and B. Together with the presence/absence of furanoeremophilane, the 27 samples were sorted into three groups: clade A/furan, clade B/furan, and clade B/non-furan. The ancestral plant presumably belonged to clade B/non-furan, because furanoeremophilanes are biosynthesized from eremophilan-8-ones. 1ß-Angeloyloxyfukinone, a likely intermediate between fukinone and subspicatin C, was isolated for the first time. This finding allowed us to propose plausible biosynthetic pathways of subspicatins A and C.

Chemical Constituents of Ligularia Species (Asteraceae) and Their Diversity in East Asia.

More than 100 Ligularia species and those of several related genera (Cremanthodium, Cacalia, Senecio, and others) in the plant family Senecioneae grow in East Asia. For many years, researchers have studied the chemical constituents of these plants, and terpenoids, flavonoids, sterols, alkaloids, and aromatic compounds have been isolated. Among these, in particular, numerous sesquiterpenoids were reported. In this contribution, relevant chemical studies are described mainly from literature reports appearing since 2000, inclusive of investigations performed by the present authors, on the diversity in secondary metabolites of Ligularia growing in the Hengduan Mountains area of China, focusing on eremophilane sesquiterpenoids and other metabolites. Terpenoids and aromatic compounds (totaling 1049), both new and known, are listed. Genetic studies and synthesis investigations are also reviewed briefly.

Eremophilanes from Ligularia hookeri Collected in China and Structural Revision of 3ß-Acyloxyfuranoeremophilan-15,6-olide.

Chemical constituents of Ligularia hookeri (Asteraceae) collected in Yunnan and Sichuan Provinces in China were examined for the first time. Seven furanoeremophilanes, five of which were new, as well as known bisabolane- and eudesmane-type sesquiterpenoids, were isolated. Spectroscopic evidence indicates that the previously reported 3ß-(2'-methylpropenoyloxy)furanoeremophilan-15,6ß-olide should be revised to 3ß-(2'-methylpropenoyloxy)furanoeremophilan-15,6α-olide.

Synthesis of α-Methylene-γ-lactone Structure by Cyclization of ω-Formylallylsilane in Water.

Surfactant-type protonic acid-promoted intramolecular cyclization of functionalized allylsilanes was studied in water for the synthesis of α-methylene-γ-lactone compounds. ω-Formyl-ß-(acetoxymethyl)allylsilane afforded carbocyclic compounds in good yields, while the cyclization product was not obtained from the corresponding ß-ethoxycarbonyl derivative. It was found that (Z)-ß-(acetoxymethyl)allylsilane predominantly afforded the cis-product, while (E)-ß-(acetoxymethyl)allylsilane afforded both cis- and trans-products at a ratio of almost 1 : 1. The stereoselectivity of the cyclization reaction was almost the same as a protonic acid-promoted reaction in CH2Cl2 and was explained by an interaction between the C(Si)-C(alkene) bond and the carbonyl moiety. The cyclization products were converted to α-methylene-γ-lactone compounds.

Development of a TiO2/SiO2 waveguide-mode chip for an ultraviolet near-field fluorescence sensor.

Aimed at detecting fluorescent-labeled biological substances sensitively, a sensor that utilizes near-field light has attracted much attention. According to our calculations, a planar structure composed of two dielectric layers can enhance the electric field of UV near-field light effectively by inducing waveguide-mode (WM) resonance. The fluorescence intensity obtainable by a WM chip with an optimized structure is 5.5 times that obtainable by an optimized surface plasmon resonance chip. We confirmed the above by making a WM chip consisting of TiO2 and SiO2 layers on a silica glass substrate and by measuring the fluorescence intensity of a solution of quantum dots dropped on the chip.

Total Synthesis of Highly Oxygenated Bisabolane Sesquiterpene Isolated from Ligularia lankongensis: Relative and Absolute Configurations of the Natural Product.

The relative and absolute configurations of an oxygenated bisabolane natural product, isolated from Ligularia lankongensis, were determined by synthesis. All four possible stereoisomers and their tiglate analogues were synthesized from R-(-)-carvone, and their 1H and 13C NMR spectra were compared to establish the 6R,8S,10S configuration. The stereoselective synthesis of the natural product was also achieved, featuring Brown allylation, vanadium-catalyzed epoxidation, and the Mitsunobu reaction.

Terpenoids and Phenylpropanoids in Ligularia duciformis, L. kongkalingensis, L. nelumbifolia, and L. limprichtii.

The diversity in root chemicals and evolutionally neutral DNA regions in the complex of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia (the d/k/n complex) was studied using eight samples collected in central and northern Sichuan Province of China. Cacalol (14) and epicacalone (15), rearranged eremophilanes, were isolated from the complex for the first time. Two new phenylpropanoids were also obtained. Seven of the eight samples produced phenylpropanoids and the other produced lupeol alone. Two of the seven samples also produced furanoeremophilanes or their derivatives and one produced oplopanes. The geographical distribution of the sesquiterpene-producing populations suggests that the production of sesquiterpenes evolved independently in separate regions. L. limprichtii collected in northern Sichuan was also analyzed and its chemical composition and the sequence of internal transcribed spacers (ITSs) in the ribosomal RNA gene cluster were found to be similar to that in the d/k/n complex and L. yunnanensis, which are morphologically similar.

Optimization of a waveguide-mode sensing chip for an ultraviolet near-field illumination biosensor.

A waveguide-mode sensor with a planar sensing chip, consisting of two waveguiding layers and a glass substrate, is a promising candidate for a near-field illumination biosensor. Aiming at using fluorescent labeling induced by ultraviolet light, we optimize the structure of a waveguide-mode sensing chip, based on the mechanism for enhancing ultraviolet near-field light revealed by numerical calculations. Candidates of optimal materials are also presented. The chip optimized as above should be able to enhance the intensity of ultraviolet near-field light 25 times as high as an Al surface plasmon resonance sensing chip.

Chemical constituents of hybrids of Ligularia cyathiceps and L. lamarum/L. subspicata collected in China: Structures of subspicatins M, N, O1, and O2, and related compounds.

Three natural hybrids and an introgressed individual of Ligularia were evaluated based on a combination of morphology, root chemicals, and nucleotide sequences of evolutionally neutral regions to understand the chemical outcomes of hybridization and introgression. Six previously undescribed eremophilane sesquiterpenes were isolated from hybrids between L. cyathiceps and L. lamarum/L. subspicata, and benzofurans were isolated from L. subspicata for the first time. Their structures were elucidated based on spectroscopic analyses. Some compounds produced by hybrids have not been detected in either parental species, indicating that the metabolic profile was altered by hybridization and introgression.

Chemical Constituents in Hybrids of Ligularia tongolensis and L. cymbulifera: Chemical Introgression in L. tongolensis.

Two samples with morphologies intermediate between Ligularia tongolensis and L. cymbulifera were collected in Desha, Sichuan Province, and one, in Pachahai, Yunnan Province, P. R. China. The DNA sequencing confirmed that the samples were hybrids of the two species. Tetradymol (1), the major compound of L. cymbulifera not found in L. tongolensis, was isolated from the hybrid samples collected at both locations, while furanoeremophilan-15-oic acid derivative 4, a compound characteristic to L. tongolensis, was found in the Pachahai hybrid but not in the Desha hybrids. Thus, the chemical consequence of hybridization can be variable. In addition, analysis of L. tongolensis samples at Pachahai indicated that introgression has been a mechanism of generating chemical diversity in the plant. Eleven compounds including three new ones were isolated.

Chemical Lineages of Ligularia fischeri.

Six Ligularia fischeri samples, two from Sichuan (samples 1 and 2) and four from Chongqing (samples 3-6), were examined for root chemicals and the DNA sequence of the internal transcribed spacers of the ribosomal RNA gene. Samples 2 and 3 contained benzofurans. The isolation of benzofurans shows that the chemical diversity in L. fischeri is higher than previously reported. Samples 1, 4, 5, and 6 contained eremophilanes. However, the compounds were different between sample 1 and samples 4-6, indicating variation within eremophilane producers. DNA data indicated that introgression could be a mechanism of benzofuran production in sample 2 and that sample 1 and samples 4-6 were genetically separate.

Diversity in Chemical Constituents of Ligularia longihastata Collected in China.

Root constituents of Ligularia longihastata were examined for the first time. Fourteen eremophilane sesquiterpenes, including two new eremophilanes identified as 3α-isobutyroyloxy-7ßH-eremophila-9,11-dien-8-one and 3ß-angeloyloxy-1ß,10ß-epoxyfuranoeremophilan-9-one, and three eudesmanes were isolated. From samples collected in Baiyu County, Sichuan Province, furanoeremophilane derivatives were isolated, while various eremophilan-8-ones were isolated from samples collected in Kangding County, Sichuan. The results suggest chemical diversity in the species.

Chemical Constituents of Ligularia wilsoniana Collected in Chongqing, China.

The chemical constituents of Ligularia wilsoniana were studied for the first time, using four samples collected in Chongqing City, China. Two new compounds, 6ß-(2',3'-epoxy-2'-methylpropanoyloxy)-ß,10ß-epoxyfuranoeremophilane and 11αH-6ß-(2'-hydroxymethylacryloyloxy)-1ß,10ß;7ß,8ß- diepoxyeremophilanolide, as well as eight known compounds, were isolated from one of the samples, while three to four known furanoeremophilanes were isolated from the other three samples. No compound was common to the two classes of the samples, demonstrating the presence of at least two chemotypes of this species.

Two New Diterpenoids from Salvia przewarskii.

One abietane-type and one dinoricetexane-type diterpenoid, salviskin A (1) and salviskin B (2), respectively, together with fourteen known diterpenoids, were isolated from Salvia przewarskii. Structural elucidation of these compounds was performed by spectroscopic methods including 2D NMR. The compounds isolated were evaluated for their cytotoxicity against HeLa and HL-60 cells.

Chemical and Genetic Study of two Ligularia Hybrids in Shangrila County, Yunnan Province, China.

The composition of root chemicals was examined for samples of L. cyathiceps x L. duciformis, L. duciformis x L. yunnanensis, and L. yunnanensis. Various furanoeremophilanes were isolated from a sample of L. cyathiceps x L. duciformis and found to be very similar to those isolated from L. cyathiceps. Lupeol and docosyl ferulate were isolated from L. yunnanensis, L. duciformis, and L. duciformis x L. yunnanensis.

Three New Eremophilanes from a Ligularia Hybrid Collected in China.

Three new eremophilanes were isolated from a sample of Ligularia collected in Yunnan province of China and their structures were determined. The chemical - constituents including eleven eremophilanes (major constituents: 6-acyloxyfuranoeremophilan-IOß-ols) were similar to those of a hybrid of L. cyathiceps and L. subspicata, however, the base sequence of the ITS 1-5.8S-ITS2 region was typical of L. cyathiceps, indicating backcrossing.

Chemical and Genetic Diversity of Ligularia hodgsonii in China.

Ligularia hodgsonii was found to be diverse in China. Furanoeremophilanes were isolated from samples collected in Yunnan Province, while such compounds were absent from samples from Sichuan, Gansu, and Chongqing. DNA sequencing showed that the Yunnan samples were also genetically distinct. γ-Humulene and a new bisabolane sesquiterpene were isolated.

Constituents of Ligularia brassicoides Collected in China: A New Diels-Alder Adduct of Eremophilan-10ß-ol and Methacrylic Acid.

Chemical study of four samples of Ligularia brassicoides collected in Sichuan Province of China afforded 18 compounds, five of which were new. LC-MS profiles were somehow similar to each other, although the compound ratio was slightly different. One of the new compound was a Diels-Alder adduct between franoeremophilan-10ß-ol and methacrylic acid.

Four New Sesquiterpenoids from Ligularia subspicata Collected in China; Isolation of a Bakkane-type Lactone, an Eremophilane-type Lactone, and Two Ortho Esters.

A bakkane type lactone, an eremophilane type lactone, and two ortho esters were isolated from Ligularia subspicata collected in Sichuan Province, China. The ortho ester type compound has been isolated from Ligularia for the first time.

Chemotypes of Ligularia vellerea, its hybrids, and L. melanothyrsa.

Ligularia vellerea and L. melanothyrsa have been found to share chemical characteristics based on geography. In one chemotype (Shangrila type), the major components were either 15-oxygenated furanoeremophilanes or eremophilanolides; and in another (Luguhu type), either 1-angeloyloxyfuranoeremophilanes or corresponding lactones. The two L. vellerea samples were found to have experienced hybridization with either L. cyathiceps or L. kanaitzensis; however, compounds typical of L. vellerea alone were isolated from them.