Published papers

Benthic marine cyanobacteria are ubiquitous oxygenic photosynthetic bacteria and they form major components of various communities, including epiphytic, epilithic and microbial mats, in coral reef environments. Filamentous groups may form almost monospecific macroscopic colonies, and sometimes make potentially harmful algal blooms (HABs). HABs bring serious problems for marine tourism but marine filamentous cyanobacteria are good resource for drug discovery. However, cultivation and identification of such filamentous cyanobacteria are difficult and there are taxonomic confusions. In this study, we investigate 17 samples from coral reef area of 10 localities in Okinawa. Macroscopic mats or cushions of filamentous cyanobacteria were collected. The samples were divided into three parts: (1) for morphological observation, (2) for molecular work, and (3) cultivation for further study. Dominant cyanobacteira of each sample identified morphologically as Anabaena, Hydrocoleum, Lyngbya, Oscillatoria, Phormidium, Schizothrix and Symploca. Molecular results of partial 16S rDNA sequences revealed six groups that composed of heterocystous group, Leptolyngbya group, Symploca group, Moorea group, Oscillatoria group and unknown group, respectively. Some samples corroborated between morphology and molecular results but some were not. In addition, four out of six groups closely related with natural product producers. These results suggest that there are high biodiversity of marine benthic filamentous cyanobacteria in Okinawa that may become seeds of HABs as well as providing promising resources for drug discovery.

Natural products and their synthetic analogs are widely used as anticancer drugs. Many anticancer drugs originated from cytotoxic compounds, and most have been shown to induce apoptosis in cancer cells. Based on the variety of their modes of action, the identification of new cytotoxic compounds may lead to the discovery of new types of drugs. Recently, much attention has been given to the metabolites of marine organisms due to their strong cytotoxicity. For example, eribulin mesylate, a synthetic analog of halichondrin B isolated from H. okadai Kadota, has been developed as an anticancer drug. Novel cytotoxic substances, such as halichonines, bisebromoamides, and biselyngbyasides, have been isolated from marine organisms as apoptosis-inducing agents. All of these compounds may possess therapeutic potential, and thus, efforts to identify new apoptosis-inducing agents of marine origin may contribute to the discovery of new pharmaceuticals.

Ginkgo (Ginkgo biloba) is one of the oldest living tree species and has been widely used in traditional medicine. Leaf extracts of ginkgo, such as the standardized extract EGb761, have become one of the best-selling herbal products. However, no bioactive compound directed at plants has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for allelopathically active substances in ginkgo leaves. An aqueous methanol leaf extract inhibited the growth of roots and shoots of garden cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was purified by several chromatographic runs and an allelopathically active substance was isolated and identified by spectral analysis to be the novel compound 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid. The compound inhibited root and shoot growth of garden cress and timothy at concentrations greater than 3. μM. The activity of the compound was 10- to 52-fold that of nonanoic acid. These results suggest that 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid may contribute to the allelopathic effect caused by ginkgo leaf extract. The compound may also have potential as a template for the development of new plant control substances.

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.

The fern Gleichenia japonica is one of the most widely distributed fern and occurs throughout East to South Asia. The species often dominates plant communities by forming large monospecific colonies. However, the potential mechanism for this domination has not yet been described. The objective of this study was to test the hypothesis that allelochemicals are involved in the formation of G. japonica colonies. An aqueous methanol extract of G. japonica inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce (Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense). Increasing extract concentration increased the inhibition. These results suggest that G. japonica contain allelopathic substances. The extract was then purified by several chromatographies with monitoring the inhibitory activity and two growth inhibitory substances causing the allelopathic effect were isolated. The chemical structures of the two substances were determined by spectral data to be a novel compound 3- O-β-allopyranosyl-13- O-β-fucopyranosyl-3β-hydroxymanool (1) and 18- O-α- l-rhamnopyranosyl-(1→2)-β- d-glucopyranosyl-13-epitorreferol (2). These compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa (Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli) at concentrations greater than 0.1-1.0 mM. The concentrations required for 50% growth inhibition of root and shoot growth of these test plants ranged from 0.72 to 3.49 mM and 0.79 to 3.51 mM for compounds 1 and 2, respectively. Concentration of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and 5.7 mM, respectively, indicating concentrations over those required for 50% growth inhibition are potentially available under monocultural stands of these ferns. Therefore, these compounds may contribute to the allelopathic effects caused by presence of G. japonica and may thus contribute to the establishment of monocultural stands by this fern.

A highly stereoselective and stereodivergent synthesis of two possible diastereomers of (-)-gummiferol was achieved, wherein the stepwise epoxidation and Cadiot-Chodkiewicz reaction were utilized for the construction of the diepoxide moiety and triacetylene part, respectively. Detailed comparison of their 1H and 13C NMR data and specific rotation with those of the natural product unambiguously elucidated the absolute stereostructure of gummiferol. In addition, the cytotoxic activity of the synthesized gummiferol, its stereoisomers, and its truncated analogues was evaluated, which clearly indicates that (1) the absolute configuration of the diepoxide moiety has little influence on the cytotoxic activity against human cancer cells and (2) the triacetylene unit is the crucial structural element required for exerting the cytotoxic activity.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S3 cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Orthosiphon stamineus (Java tea) has been widely used as traditional herb and several bioactive compounds against animal cells have been isolated. However, no bioactive compound against plants has been reported. Therefore, we investigated possible allelopathic properties and substances in O. stamineus. Aqueous methanol extracts of O. stamineus inhibited root and hypocotyl growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) seedlings. Increasing the extract concentration increased the inhibition, which suggests that O. stamineus may have allelopathic properties. When the extract was divided into an ethyl acetate and an aqueous fraction, the ethyl acetate fraction showed the stronger inhibitory effect. Thus, the ethyl acetate phase was further purified, and the main allelopathic substance was isolated and identified as 13- epi-orthosiphol N, a novel compound, by spectral data. 13- epi-Orthosiphol N inhibited root and hypocotyl growth of cress and lettuce at concentrations greater than 10 μmol/L. The concentrations required for 50% inhibition ranged from 41 to 102 μmol/L. These results suggest that 13- epi-orthosiphol N may be an allelochemical and main contributor to the growth inhibitory effect of O. stamineus and may have potential as a template for the development of new plant control substances.

Protein kinase C (PKC) isozymes are promising targets for anticancer therapy. Bryostatin-1 (bryo-1), a unique PKC activator with little tumor-promoting activity, is currently in clinical trials for the treatment of cancer. However, its limited availability from natural sources and its synthetic complexity have hampered studies of its mode of action and structural optimization as a therapeutic agent. The development of synthetically more accessible compounds with bryo-1-like activities is thus needed. Recently, we developed a simple and less lipophilic analogue of tumor-promoting aplysiatoxin (ATX) (aplog-1) as a promising lead for bryo-1-like anticancer drugs. Structure-activity studies suggested that local hydro -phobicity around the spiroketal moiety of aplog-1 is a crucial determinant of its antiproliferative activity. The hydrophobic analogue (12,12-dimethyl-aplog-1) displayed more potent antiproliferative activity. Moreover, it showed little tumor-promoting activity and even suppressed the tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo and in vitro. Aplog-1 and bryo-1 bound selectively to novel PKC isozymes (δ, η, and θ) while tumor promoters bound to both conventional and novel PKC isozymes. These results suggest that the unique biological activities of aplog-1 and bryo-1 are ascribable in part to the ability to bind to PKCδ, but weak binding to conventional PKC isozymes might also be important.

We have focused on the identification of natural key compounds that possess biologically and medicinally intriguing functions. Some of bioactive naturally occurring compounds isolated from marine organisms were found to possess unique biological activities. Halichlorine from the marine sponge Halichondria okadai was shown to inhibit the activity of nuclear factor-κB in endothelial cells and block L-type Ca 2+ channels. Thus, it may have therapeutic potentials for diseases such as atherosclerosis and hypertension.

The fern Dicranopteris linearis Underw. (Old world forkedfern, Gleicheniaceae), the most widely distributed fern throughout tropical to temperature regions, dominates and often forms large pure colonies. Allelopathic chemical interaction of the fern was speculated to play an important role in the dominance. However, potential mechanisms, in particular, the allelopathic substance have not been reported. The objective of this study was the identification of its potential allelopathic substance and the evaluation of the ecological role of the substance. Extracts of D. linearis had an inhibitory effect on Echinochloa colonum and Avena fatua which are found near colonies of D. linearis in natural ecosystems. The extract was purified and a main inhibitory substance was isolated. The chemical structure of the substance was determined by high-resolution MS, and 1H- and 13C-NMR spectral data and optical rotation as epicatechin-(2β → O → 7,4β → 8)-epicatechin-(4β → 8)-epicatechin (cinnamtannin B-1). Cinnamtannin B-1 inhibited the shoot and root growth of A. fatua and E. colonum at concentrations greater than 0. 2-0. 5 mM, and the concentrations required for 50 % growth inhibition on shoot and root growth of these plants were 0. 34-1. 31 mM. Cinnamtannin B-1 was found in soil under the colony, at concentrations of 4. 3 and 14. 5 mM in soil at the edge of and under the colony, respectively. These concentrations were over the concentration required for 50 % growth inhibition. Therefore, cinnamtannin B-1 may work as an allelopathic agent of D. linearis and may contribute to the establishment of pure colonies of D. linearis.

An organ-specific-growth inhibitory substance was isolated from an aqueous methanol extract of red pine needles and determined by spectral data as 1-mono(16-hydroxyhexadecanoyl)glycerol. This substance inhibited root growth of cress (Lepidium sativum L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv) seedlings at concentrations greater than 0.01 and 0.03 μM, respectively. The concentrations required for 50 % growth inhibition on roots of cress and barnyard grass were 0.16 and 3.4 μM, respectively. However, the inhibitory activity of the substance on shoots of cress and barnyard grass was very weak. The endogenous concentration of 1-mono(16-hydroxyhexadecanoyl)glycerol in the pine needles was 4.6 μmol kg -1. Two related compounds, 1-monohexadecanoylglycerol and 16-hydroxyhexadecanlic acid had no activity up to 1,000 μM on cress roots and shoots. The effectiveness of 1-mono(16-hydroxyhexadecanoyl)glycerol on root growth inhibition and the occurrence of 1-mono(16-hydroxyhexadecanoyl)glycerol in pine needles suggest the substance may play an important role in the allelopathy of red pine. Root-specific-growth-inhibition by the substance may be one of the strategies for red pine to compete with neighboring plants for nutrients and space because root growth of competitive plants may be very important for their whole plant development.

The total synthesis of auripyrones was achieved by using a novel aldol-type reaction of γ-pyrone as a key step. From this synthetic work, we have established the stereostructure and absolute configuration of auripyrone B. Furthermore, the cytotoxicities of auripyrones against a panel of 39 human cancer cell lines (termed JFCR39) at the Japanese Foundation for Cancer Research were investigated. The patterns of the differential cytotoxicities of auripyrones were COMPARE negative, suggesting that they inhibit cancer cell proliferation through a novel mechanism.

A potent growth inhibitory substance was isolated from an aqueous methanol extract of cucumber (Cucumis sativus L. cv. Phung Tuong) plants and determined as (2S)-2,3-dihydro-2α-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-(1,2,3-trihydroxypropyl)benzofuran-3β-methanol (sisymbrifolin) by spectral data. Sisymbrifolin inhibited the growth of cress (Lepidium sativum) and Echinochloa crus-galli seedlings at concentrations greater than 3 μM. Concentration of sisymbrifolin in the cucumber plants was the greatest among four growth inhibitory substances, (S)-2-benzoyloxy-3-phenyl-1-propanol, 9-hydroxy-4,7-megastigmadien-9-one, (6S,7E,9S)-6,9,10-trihydroxy-4,7-megastigmadien-3-one, and sisymbrifolin found in the cucumber, whereas growth inhibitory activity of 9-hydroxy-4,7-megastigmadien-9-one against cress and E. crus-galli was the greatest. Total activities of these substances (concentration of the substance/concentration required 50 % growth inhibition) were 14. 4, 13. 2, 8. 5 and 10. 7 for (S)-2-benzoyloxy-3-phenyl-1-propanol, 9-hydroxy-4,7-megastigmadien-9-one, (6S,7E,9S)-6,9,10-trihydroxy-4,7-megastigmadien-3-one and sisymbrifolin, respectively. These total activities were about 100-fold greater than those of phenolic acids, which are often mentioned as putative allelochemicals of plants. Thus, these substances may play important roles in the allelopathy of cucumber plants through the growth inhibition of neighboring plant species.

Allelopathic activity of Piper sarmentosum Roxb. was determined and a potent growth inhibitory substance was isolated. The aqueous methanol extracts of P. sarmentosum inhibited the growth of 6-test plant species [cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), timothy (Phleum pretense L.), Italian ryegrass (Lolium multiflorum Lam.) and crabgrass (Digitaria sanguinalis L.)] to various degrees in a dose-dependent manner. The active substance 3-phenylpropionic acid was purified from the aqueous methanol extract of P. sarmentosum and identified by 1H-, 13C-NMR and MS. The threshold of 3-phenylpropionic acid for growth inhibition was 1 μM. The concentrations required for 50% growth inhibition of cress and lettuce were 1.2-9.3 μM and the concentrations required for 50% growth inhibition of timothy and Italian ryegrass were 4.7-51.8 μM. The endogenous concentration of 3-phenylpropionic acid in P. sarmentosum was 121 μmol/kg. Considering the endogenous level and the inhibitory activity, 3-phenylpropionic acid may work as allelopathic substance in P. sarmentosum through growth inhibition of neighbouring plant species.