Published papers

Allelopathic plants and their related phytotoxic chemicals have been explored as alter-natives for environmentally friendly weed management. Dregea volubilis (L.f.) Benth. ex Hook.f., a woody vine, is well-known for its uses in various therapeutic treatments, and several bioactive compounds have been isolated from this species. However, to date, no phytotoxic substances from D. volubilis have been reported. Hence, in this research, we aimed to investigate the allelopathic effect of D. volubilis and characterize its phytotoxic substances. Extracts of D. volubilis exhibited significant inhibitory activity against the seedling growth of Italian ryegrass (Lolium multiflorum Lam.) and cress (Lepidium sativum L.). The extracts were purified through several chromatography steps, yielding two phytotoxic substances, and using spectroscopy, their structures were determined as dehydrovomi-foliol and loliolide. The compounds inhibited the Italian ryegrass and cress seedlings with the I50 (concentrations needed for 50% inhibition) values ranging from 0.022 to 0.102 mM, for loliolide and 3.24–4.60 mM, for dehydrovomifoliol. These results suggest that both phytotoxic compounds might be responsible for the allelopathy of D. volubilis, and they may be released into the soil through the decomposition of the D. volubilis leaf and probably act as allelopathic active substances. Therefore, the extracts of D. volubilis and its related phytotoxic compounds may be applied as biological agents for controlling weeds.

Clerodendrum indicum (L.) Kuntze (Lamiaceae), an annual shrub, is renowned for being used in folk medicine in South Asian countries. Several pharmacological properties and many bioactive secondary metabolites from C. indicum have been well documented. However, the phytotoxic activities and the related phytotoxic substances with the allelopathic activity of C. indicum have not yet been reported. Thus, we explored the phytotoxic activity of C. indicum and identified its phytotoxic substance. In the experiment, the dry C. indicum leaves were extracted with aqueous methanol, and then the filtrate of the extracts was concentrated using a rotary evaporator to obtain crude extracts. The C. indicum crude extracts significantly inhibited the shoots and roots of six target species: alfalfa, cress, lettuce, barnyard grass, Italian ryegrass, and timothy. The inhibition increased when the extract concentration was increased. The crude extracts of C. indicum were separated in several chromatography steps, and a phytotoxic substance was isolated and characterized using spectroscopy as p-coumaric acid. p-Coumaric acid significantly suppressed the growth of lettuce and timothy seedlings at concentrations greater than 0.3 and 1 mM, respectively. The concentrations of p-coumaric acid required for 50% inhibition (I50) of the shoots and roots of lettuce and timothy were 0.65 and 0.17 mM, and 0.81 and 0.67 mM, respectively. This study is the first report on isolating p-coumaric acid in C. indicum. The results, therefore, suggest that p-coumaric acid may partly contribute to the phytotoxic properties of C. indicum.

Metasequoia glyptostroboides Hu et W.C. Cheng is one of the oldest living conifer species, and it has remained unchanged for millions of years compared to its fossils from the Cretaceous period. The species are cultivated in the parks, gardens, and roadsides in many countries. We investigated the allelopathy and allelopathic substances in fallen leaves of M. glyptostroboides. An aqueous methanol extract of the fallen leaves inhibited the growth of cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), Lolium multiflorum Lam., Phleum pretense L., and Vulpia myuros (L.) C.C.Gmel. The extract was then purified by several chromatographic steps, and two allelopathic substances were isolated and determined by spectral data to be (+)-rhododendrol and 9-epi-blumenol C. The compound inhibited the growth of cress and L. multiflorum. M. glyptostroboides is a deciduous perennial tree, and accumulation of its fallen leaves occur on the soil under the trees. Therefore, those allelopathic substances in the fallen leaves may be liberated into the rhizo-sphere soil during the decomposition process of fallen leaves and provide a competitive advantage for M. glyptostrob through the growth inhibition of competing plant species nearby. Therefore, M. glyptostroboides is allelopathic, and (+)-rhododendrol and 9-epi-blumenol C may be contribute to the allelopathy.

Senna garrettiana (Craib) Irwin & Barneby (Fabaceae) is a medicinal plant known to be rich in biologically active compounds that could be exploited to produce bioherbicides. The present study was conducted to explore the allelopathic potential and phytotoxic substances of S. garrettiana. Extracts of S. garrettiana leaves were found to significantly inhibit the growth of Lepidium sativum L. and Echinochloa crus-galli (L.) P. Beauv. (p ≤ 0.05). The phytotoxic substances were isolated and identified as vanillic acid and ferulic acid by bioassay-directed fractionation and spectral data analysis. The two compounds were shown to significantly inhibit the seed germination, seedling growth, and dry biomass of L. sativum. Based on the concentration required for 50% growth inhibition (defined as IC50), the roots of L. sativum were the most sensitive to the compounds, and the inhibitory effect of ferulic acid (IC50 = 0.62 mM) was >1.3 times more potent than that of vanillic acid (IC50 = 0.82 mM). In addition, a mixture of the two compounds (0.3 mM) resulted in synergistic inhibitory activity against the L. sativum roots compared with the individual compounds. These results suggest that the extracts of S. garrettiana leaves and their phytotoxic compounds have potential as candidate natural herbicides.

Odookeanynes A (1) and B (2), two acetylene-containing lipopeptides, were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa, Japan. Their structures were elucidated by spectroscopic analysis and Marfey's analysis of acid hydrolysates. Odookeanynes A (1) and B (2) dose-dependently promoted the differentiation of mouse 3T3-L1 preadipocytes in the presence of insulin.

Wedelia chinensis (Asteraceae) is a wetland herb native to India, China, and Japan. It is a valuable medicinal plant recorded to have pharmaceutical properties. However, the phytotoxic potential of Wedelia chinensis has not yet been examined. Thus, we carried out this study to establish the allelopathic effects of Wedelia chinensis and to identify its phytotoxic substances. Extracts of Wedelia chinensis exhibited high inhibitory activity against the root and shoot growth of cress, alfalfa, rapeseed, lettuce, foxtail fescue, Italian ryegrass, timothy, and barnyard grass. The inhibition was varied with species and was dependent on concentrations. The extracts were separated through several purification steps, and the two effective substances were isolated and characterized as vanillic acid and gallic acid using spectral analysis. Vanillic acid and gallic acid significantly arrested the growth of cress and Italian ryegrass seedlings. The concentrations of vanillic acid and gallic acid needed for 50% inhibition (I50 values) of the seedling growth of the cress and Italian ryegrass were 0.04–15.4 and 0.45–6.6 mM, respectively. The findings suggest that vanillic acid and gallic acid may be required for the growth inhibitory activities of Wedelia chinensis.

In forestry systems, many types of forest trees possess allelopathic properties and release a wide variety of allelochemicals that influence the growth and development of surrounding species. Dalbergia cochinchinensis, a forest tree distributed in Southeast Asia, is reputed to possess several biological properties and contain several secondary compounds. However, there have been no studies on the allelopathy of D. cochinchinensis. Therefore, D. cochinchinensis leaf extracts were examined for allelopathic potential. The present study showed that D. cochinchinensis extracts significantly inhibited the seedling growth of six test plant species: timothy, Italian ryegrass, barnyard grass, cress, alfalfa, and lettuce. Concentrations of the D. cochinchinensis extracts negatively correlated with the shoot length (r = −0.50 to −0.89) and root length (r = −0.65 to −0.89) of all the test plant species. The extracts were then purified using several chromatographic steps and the growth inhibitory substance was isolated. The chemical structure of the substance was identified through spectroscopic analysis as 3,4-dihydroxybenzoic acid (protocatechuic acid). Protocatechuic acid at a concentration higher than 3 mM significantly inhibited the growth of cress seedlings, whereas barnyard grass seedlings were inhibited at concentrations higher than 0.3 mM. As the protocatechuic acid concentration increased, the seedling growth of cress and barnyard grass was significantly reduced. I50 values showed the effectiveness of protocatechuic acid against both test plant species was apparently greater on root growth than shoot growth. Results of this study suggest that protocatechuic acid isolated from D. cochinchinensis might be responsible for its inhibitory effects.

Natural products, well known for unique chemical diversity and bioactivity, are the compounds that organisms have acquired in evolutionary history. They have attracted attentions from not only organic chemists but also scientists in related fields and have provided breakthroughs for science. Against this background, we have investigated the secondary metabolites of marine organisms, especially cyanobacteria, and have discovered various novel bioactive compounds. In this account, our recent research activities regarding the isolation, structure determination, total syntheses and biological activities of our novel natural products are described.

The allelopathic potential of plant species and their related compounds has been increas-ingly reported to be biological tools for weed control. The allelopathic potential of Garcinia xantho-chymus was assessed against several test plant species: lettuce, rapeseed, Italian ryegrass, and timo-thy. The extracts of G. xanthochymus leaves significantly inhibited all the test plants in a concentra-tion-and species-specific manner. Therefore, to identify the specific compounds involved in the allelopathic activity of the G. xanthochymus extracts, assay-guided purification was carried out and two allelopathic compounds were isolated and identified as methyl phloretate {3-(4-hydroxy-phenyl) propionic acid methyl ester} and vanillic acid (4-hydroxy-3-methoxybenzoic acid). Both of the substances significantly arrested the cress and timothy seedlings growth. I50 values (concentra-tions required for 50% inhibition) for shoots and roots growth of the cress and timothy were 113.6– 104.6 and 53.3–40.5 μM, respectively, for methyl phloretate, and 331.6–314.7 and 118.8–107.4 μM, respectively, for vanillic acid, which implied that methyl phloretate was close to 3-and 2-fold more effective than vanillic acid against cress and timothy, respectively. This report is the first on the presence of methyl phloretate in a plant and its phytotoxic property. These observations suggest that methyl phloretate and vanillic acid might participate in the phytotoxicity of G. xanthochymus extract.

We conducted an investigation to search for the possibility of phytotoxic compounds and then identify them in C. aciculatus. The aqueous methanol extracts of C. aciculatus imposed a strong suppressive effect on the shoots and roots growth of Medicago sativa and Echinochloa crus-galli. The C. aciculatus extracts were subsequently purified through chromatography and three growth-inhibitory compounds were identified by spectral data as methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde. The seedlings growth of Lepidium sativum was suppressed by methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde. The I50 values (concentration required for 50% growth inhibition) for the seedling growth of L. sativum ranged from 1.25 to 1.73 mM for methyl cis-p-coumarate, 0.74 to 1.23 mM for methyl trans-p-coumarate, and 1.05 to 1.84 mM for trans-ferulaldehyde. Our finding indicates that methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde may contribute to the phytotoxic effects of C. aciculatus.

Bromoiesol sulfates A (1) and B (2), new polyhalogenated aryl sulfates, were isolated from a Salileptolyngbya sp. marine cyanobacterium along with their hydrolyzed compounds, bromoiesols A (3) and B (4). To pick up the candidates of their structures, we used Small Molecule Accurate Recognition Technology (SMART), an artificial intelligence-based structure-prediction tool, and their structures were elucidated on the basis of single-crystal X-ray diffraction analysis of bromoiesols (3 and 4). In addition, to verify the structures, the total synthesis of bromoiesol A sulfate (1) and bromoiesol A (3) was achieved. The bromoiesol family, especially bromoiesols (3 and 4), selectively inhibited the growth of the bloodstream form of Trypanosoma brucei rhodesiense, the causative agent of human African sleeping sickness.

Kinenzoline (1), a new linear depsipeptide, was isolated from a marine Salileptolyngbya sp. cyanobacterium. Its structure was elucidated by spectroscopic analyses and degradation reactions. In addition, we achieved a total synthesis of 1 and confirmed its structure. Kinenzoline (1) showed highly selective antiproliferative activity against the causative organism of sleeping sickness, Trypanosoma brucei rhodesiense (IC50 4.5 μM), compared to normal human cells (WI-38, IC50 > 100 μM). Kinenzoline (1) is a promising lead compound for the development of new antitrypanosomal drugs.

The linear lipopeptides komesuamide (1) and odopenicillatamide (2) were isolated from Caldora penicillata a marine cyanobacterium collected in Okinawa. The structures of these compounds were established by spectroscopic analyses, and the absolute configurations were determined by HPLC analyses of the acid hydrolysates. Both compounds showed glucose uptake activity at 40 μM in cultured L6 myotubes.

Metabolomics can be used to study complex mixtures of natural products, or secondary metabolites, for many different purposes. One productive application of metabolomics that has emerged in recent years is the guiding direction for isolating molecules with structural novelty through analysis of untargeted LC-MS/MS data. The metabolomics-driven investigation and bioassay-guided fractionation of a biomass assemblage from the South China Sea dominated by a marine filamentous cyanobacteria, cf. Neolyngbya sp., has led to the discovery of a natural product in this study, wenchangamide A (1). Wenchangamide A was found to concentration-dependently cause fast-onset apoptosis in HCT116 human colon cancer cells in vitro (24 h IC50 = 38 µM). Untargeted metabolomics, by way of MS/MS molecular networking, was used further to generate a structural proposal for a new natural product analogue of 1, here coined wenchangamide B, which was present in the organic extract and bioactive sub-fractions of the biomass examined. The wenchangamides are of interest for anticancer drug discovery, and the characterization of these molecules will facilitate the future discovery of related natural products and development of synthetic analogues.

Motobamide (1), a new cyclic peptide containing a C-prenylated cyclotryptophan residue, was isolated from a marine Leptolyngbya sp. cyanobacterium. Its planar structure was established by spectroscopic and MS/MS analyses. The absolute configuration was elucidated based on a combination of chemical degradations, chiral-phase HPLC analyses, spectroscopic analyses, and computational chemistry. Motobamide (1) moderately inhibited the growth of bloodstream forms of Trypanosoma brucei rhodesiense (IC50 2.3 μM). However, it exhibited a weaker cytotoxicity against normal human cells (IC50 55 μM).

Albizia richardiana, a fast-growing, large deciduous tree belonging to the Fabaceae family, grows well in hot and humid areas but mainly grows in the tropics of the Old World. The medicinal and other uses of Albizia richardiana are well documented, but the phytotoxic effects of this tree have not yet been investigated. We conducted this study to investigate the phytotoxic activity of Albizia richardiana leaves and to identify growth inhibitory substances for controlling weeds in a sustainable way. Aqueous methanol extracts of Albizia richardiana leaves greatly suppressed the growth of cress and barnyard grass seedlings in a concentration- and species-dependent manner. Two phytotoxic substances were separated using several purification steps and characterized through spectral analysis as dehydrovomifoliol and loliolide. Dehydrovomifoliol and loliolide significantly arrested the seedling growth of cress in the concentrations of 0.1 and 0.01 mM, respectively. The extract concentrations needed for 50% growth inhibition (I50 values) of cress seedlings were 3.16-3.01 mM for dehydrovomifoliol and 0.03-0.02 mM for loliolide. The results suggest that these two allelopathic substances might play a vital role in the phytotoxicity of Albizia richardiana leaves.

To protect our environment from the toxic effect of herbicides, it is necessary to reduce the application of synthetic herbicides for controlling weeds in crop fields as well as to increase the use of natural herbicides produced from plant sources. Phytotoxic compounds obtained from plant species have gained attention as an alternate biological method for managing weeds. Albizia richardiana was previously reported to contain a number of phytotoxic substances. Therefore, we carried out this study to investigate more allelopathic substances in Albizia richardiana leaves. Aqueous methanol extracts of Albizia richardiana leaves were significantly inhabited the seedling growth of the two test species (alfalfa and timothy) in a concentration-dependent manner. The two most active substances were separated through different chromatographic steps and characterized as a novel compound 3-hydroxy-4-oxo-β-dehydroionol [(E)-6-hydroxy-3-(3-hydroxybut-1-en-1-yl)-2,4,4-trimethylcyclohexa-2,5-dien-1-one], and compound 3-oxo-α-ionone [3,5,5-trimethyl-4-(3-oxo-1-butenyl)-2-cyclohexen-1-one]. The novel compound 3-hydroxy-4-oxo-β-dehydroionol showed relatively stronger growth inhibitory potential compare to the compound 3-oxo-α-ionone and significantly restricted the seedling growth of crees and timothy. The required concentration for 50% reduction in the growth (I50 values) of the cress and timothy seedlings varied from 63.78 to 141.61 μM for 3-hydroxy-4-oxo-β-dehydroionol and 1059.60 to 1616.10 μM for 3-oxo-α-ionone. The results indicate that these two allelopathic compounds may be responsible for the phytotoxicity of Albizia richardiana leaves.

© 2020 American Chemical Society. All rights reserved. Hoshinoamide C (1), an antiparasitic lipopeptide, was isolated from the marine cyanobacterium Caldora penicillata. Its planar structure was elucidated by spectral analyses, mainly 2D NMR, and the absolute configurations of the α-amino acid moieties were determined by degradation reactions followed by chiral-phase HPLC analyses. To clarify the absolute configuration of an unusual amino acid moiety, we synthesized two possible diastereomers of hoshinoamide C and determined its absolute configuration based on a comparison of their spectroscopic data with those of the natural compound. Hoshinoamide C (1) did not exhibit any cytotoxicity against HeLa or HL60 cells at 10 μM, but inhibited the growth of the parasites responsible for malaria (IC50 0.96 μM) and African sleeping sickness (IC50 2.9 μM).

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Marine macroscopic colony-forming filamentous (MMCFF) cyanobacteria are considered as prolific producers of bioactive compounds. Thus, knowledge of the diversity of MMCFF cyanobacteria as related to bioactive compound production has become very important. However, basic taxonomic studies of MMCFF cyanobacteria are lacking. Many cyanobacterial taxa are still misidentified or undescribed. In this study, a total of 32 cyanobacterial colonies from nine coastal regions of Okinawa Prefecture were investigated for a diversity assessment. A polyphasic approach including morphological and molecular studies based on 16S rRNA gene sequences was performed to characterize Okinawan MMCFF cyanobacteria. Both morphological and molecular phylogenetic results showed that MMCFF cyanobacteria from Okinawan coasts are very diverse. We found morphotypes of Lyngbya-like, Phormidium-like, and Leptolyngbya-like groups among Okinawan cyanobacterial samples. Genetically, samples were distributed in various clades in the phylogenetic tree, including within Moorena, Okeania, Caldora, Neolyngbya, Dapis, as well as several unknown clades. In addition, cytotoxic activities of three samples from Kiyan coast were tested against HeLa cells. All three crude extracts of these samples showed strong cytotoxic activity with IC50 < 1 μg/ml.

© 2020 Taylor & Francis Group, LLC. The phytotoxic potential of the leaves and twigs of Schumannianthus dichotomus, discarded in the mat-making industry against four test plants (lettuce (Lactuca sativa L.), rapeseed (Brassica napus L.), foxtail fescue (Vulpia myuros (L.) C.C. Gmel.) and timothy (Phleum pratense L.)) was investigated and found strong phytotoxic activity. An assay-guided fractionation of S. dichotomus extarcts against cress (Lepidium sativum L.) through a series of column chromatography steps yielded two compounds, 8-(5-oxo-2,5-dihydrofuran-2-yl) octanoic acid (ODFO) and (E)-6-hydroxy-2,6-dimethylocta-2,7-dienoic acid (8-carboxylinalool). ODFO and 8-carboxylinalool showed strong phytotoxic activity against cress and timothy. The concentrations required for 50% growth inhibition (I 50 value) of the seedlings of cress and timothy were 111.94–128.01 and 36.30–91.75 µM, respectively, for ODFO, but the values were much higher at 315.98–379.13 and 107.92–148.41 µM, respectively, for 8-carboxylinalool, indicating the stronger phytotoxic activity of ODFO. This study is the first to isolate ODFO and 8-carboxylinalool from S. dichotomus and their phytotoxic potential while ODFO is firstly encountered from any natural source. The growth inhibitory activity of the identified compounds may explain their role in the phytotoxic activity of S. dichotomus, which suggests the possible use of its leaves and twigs or its active constituents as natural bioherbicides.