Published papers

Kagimminols A (1) and B (2), new cembrene-type diterpenoids, were isolated from an Okeania sp. marine cyanobacterium. By combining DP4 analysis with an efficient NMR chemical shift calculation protocol, we clarified the relative configurations of 1 and 2 without consuming precious natural products. We determined the absolute configurations by a comparison of theoretical electronic circular dichroism (ECD) spectra with experimental spectra, and the absolute configuration of 1 was verified experimentally. Finally, we found that 1 and 2 showed selective growth-inhibitory activity against the causative agent of human African trypanosomiasis. This study exemplifies that computational chemistry is an efficient tool for clarifying the configurations of natural products possessing tautomers in equilibrium.

The total synthesis of acetylene-containing lipopeptides odookeanynes A (1) and B (2), isolated from the marine cyanobacterium Okeania sp., has been achieved. A biological evaluation using synthetic odookeanynes revealed their potent antitrypanosomal activity. This report not only describes a synthetic pathway for structurally related compounds for SAR studies but also offers new insights into their potential as antiparasitic agents.

Ikoamide (1) is a highly N-methylated antimalarial lipopeptide that was isolated from a marine cyanobacterium, an Okeania sp. in 2018, which shows strong antimalarial activity without cytotoxicity against human cancer cell lines. To establish a synthetic method for obtaining enough ikoamide for its biological evaluations, we have established a total synthesis of ikoamide. The synthetic method presented here lays the foundation for the development of novel ikoamide analogues, which may lead to a discovery of pharmaceutically unique antimalarial drug leads.

The linear lipopeptides okeaniamide A (1) and okeaniamide B (2) were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa. The structures of these compounds were established by spectroscopic analyses, and the absolute configurations were elucidated based on a combination of chemical degradations, Marfey’s analysis, and derivatization reactions. Okeaniamide A (1) and okeaniamide B (2) dose-dependently promoted the differentiation of mouse 3T3-L1 preadipocytes in the presence of insulin.

Plant parts and extracts that are rich in bioactive substances with allelopathic potential can be explored as a possible alternative to herbicides for natural weed control in sustainable agriculture. In the present study, we investigated the allelopathic potential of Marsdenia tenacissima leaves and its active substances. Aqueous methanol extracts of M. tenacissima showed significant inhibitory activities against the growth of lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), timothy (Phleum pratense L.), and barnyard grass (Echinochloa crusgalli (L.) Beauv.). The extracts were purified through various chromatography steps, and one active substance was isolated and determined by spectral data to be a novel compound, assigned as steroidal glycoside 3 (8-dehydroxy-11β-O-acetyl-12β-O-tigloyl-17β-marsdenin). Steroidal glycoside 3 significantly inhibited the seedling growth of cress at a concentration of 0.03 mM. The concentrations needed for 50% growth inhibition of the cress shoots and roots were 0.25 and 0.03 mM, respectively. These results suggest that steroidal glycoside 3 may be responsible for the allelopathy of M. tenacissima leaves.

Osmanthus fragrans Lour. has been cultivated for more than 2500 years because of the fragrance and color of the flowers. The flowers and roots have been used in tea, liquors, foods, and traditional Chinese medicine. The species contains more than 180 compounds including terpenoids, phenylpropanoids, polyphenols, flavonoids, and sterols. However, there has been limited information available on the allelopathic properties and allelopathic substances of O. fragrans. We investigated the allelopathy and allelopathic substances of O. fragrans and Osmanthus heterophyllus (G.Don) P.S. Green, as well as Osmanthus × fortunei Carrière, which is the hybrid species between O. fragrans and O. heterophyllus. The leaf extracts of O. fragrans, O. heterophyllus, and O. × fortunei suppressed the growth of cress (Lepidium sativum L.), alfalfa (Medicago sativa L.), Lolium multiflorum Lam., and Vulpia myuros (L.) C.C.Gmel with the extract concentration dependently. The extract of the hybrid species O. × fortune was the most active among the extracts. The main allelopathic substances of O. × fortunei and O. fragrans were isolated and identified as (+)-pinoresinol and 10-acetoxyligustroside, respectively. (+)-Pinoresinol was also found in the fallen leaves of O. × fortunei. Both compounds showed an allelopathic activity on the growth of cress and L. multiflorum. On the other hand, several allelopathic substances including (+)-pinoresinol may be involved in the allelopathy of O. heterophyllus. O. fragrans, O. heterophyllus, and O. × fortunei are evergreen trees. but their senescent leaves fall and cover the soil under the trees. It is possible that those allelopathic substances are liberated through the decomposition process of the leaves into their rhizosphere soil, and that they accumulate in the soil and provide a competitive advantage to the species through the inhibition of the growth of the neighboring competing plants. Therefore, the leaves of these Osmanthus species are allelopathic and potentially useful for weed management options in some agriculture settings to reduce commercial herbicide dependency for the developing sustainable agriculture systems.

The phytotoxic potential of plants and the effect of their active components on another plant species is being explored as a potential alternative to synthetic herbicides for weed control. In the current study, we investigated the phytotoxic potential of the leaves of Dregea volubilis (L.f.) Benth. ex Hook.f. against four test plants [timothy (Phleum pratense L.), barnyard grass (Echinochloa crus-galli (L.) P. Beauv), lettuce (Lactuca sativa L.), and alfalfa (Medicago sativa L.)] and observed significant growth inhibition on those plants at concentrations >3 mg D.W. equivalent extract mL−1. A bioassay-governed purification of the D. volubilis extracts using different chromatography phases produced two growth inhibitory compounds, 3-hydroxy-α-ionone (compound 1) and 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one (compound 2). The compounds retarded the growth of barnyard grass and cress (Lepidium sativum L.) with I50 (concentration required for 50% growth suppression) values ranging from 0.098 to 0.450 mM for 3-hydroxy-α-ionone and 0.029 to 0.420 mM for 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one. Thus, the extracts and identified compounds may have the possibility to be utilized as bioagents for weed control.

Polygonum chinense Linn. is a medicinal and invasive plant that belongs to the family Polygonaceae. The pharmacological activities and phytochemical constituents of Polygonum chinense are well reported, but the allelopathic effects and potent allelopathic substances of P. chinense remain to be investigated. Hence, this experiment was conducted to separate and characterize potentially allelopathic substances from an extract of the Polygonum chinense plant. The Polygonum chinense plant extracts highly suppressed the growth of cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), barnyard grass (Echinochloa crusgalli (L.) P. Beauv.), and timothy grass (Phleum pratense L.) seedlings in a species- and concentration-dependent way. Two active substances were separated using a series of purification procedures and determined through spectral analysis as (−)-3-hydroxy-β-ionone and (−)-3-hydroxy-7,8-dihydro-β-ionone. These two compounds significantly suppressed the seedling growth of Lepidium sativum (cress) at concentrations of 0.01 and 1 mM, respectively. The extract concentrations necessary for 50% growth inhibition (I50 values) of the cress hypocotyls and roots were 0.05 and 0.07 mM for (−)-3-hydroxy-β-ionone, respectively, and 0.42 and 1.29 mM for (−)-3-hydroxy-7,8-β-ionone, respectively. These findings suggest that these two compounds are in charge of the inhibitory effects of the Polygonum chinense extract and may serve as weed control agents.

Two new natural products were isolated from the marine cyanobacterium Rivularia sp. collected in Japan. Hennaminal possesses a very rare functional group, β,β-diamino unsaturated ketone, which has only been found in bohemamine-type natural products so far. Hennamide possesses a reactive N-acyl pyrrolinone moiety, which induces self-dimerization. The isolation and structure determination supported by computational chemistry and total synthesis, as well as the antitrypanosomal activities of hennaminal and hennamide are described.

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

Caldorazole (1) is a novel polyketide that was isolated from a marine cyanobacterium in 2022. It is a unique natural product that exhibits potent inhibitory activity against mitochondrial respiratory chain complex I despite having no chiral centers. To establish a method for obtaining caldorazole without relying on biological resources and for constructing a useful synthetic route for studies of its structure-activity relationship, we achieved the first total synthesis of caldorazole using a convergent synthetic route.

Phytotoxic substances in plants that may serve as alternative natural herbicides for controlling weeds are required for sustainable agriculture. We explored the phytotoxic activities of aqueous methanol extracts of Senna garrettiana (Craib) Irwin & Barneby leaves and the active substances they contain. The results revealed that the S. garrettiana leaf extracts had significant phytotoxic effects on three dicotyledons (Lepidium sativum L., Lactuca sativa L., and Medicago sativa L.) and two monocotyledons (Phleum pratense L. and Lolium multiflorum Lam.). An bioassay-guided isolation process yielded three active substances; caffeic acid, methyl caffeate, and (S)-6-hydroxymellein inhibited the seed germination, seedling growth, and biomass accumulation of L. sativum in a concentration-dependent manner. Based on the concentration required for 50% growth inhibition (IC50), (S)-6-hydroxymellein had the highest inhibitory effects on L. sativum in all test parameters, followed by methyl caffeate and caffeic acid. The L. sativum roots were the most susceptible to (S)-6-hydroxymellein (IC50 = 383 µM) and caffeic acid (IC50 = 2627 µM), whereas methyl caffeate (IC50 = 1361 µM) had the greatest effect on the L. sativum shoots. Thus, three isolated compounds may explain the phytotoxic effects of the S. garrettiana extracts. Consequently, caffeic acid, methyl caffeate, and (S)-6-hydroxymellein could be potential candidates for the future production of bioherbicides.

The plant Plumbago rosea Linn., belonging to the Plumbaginaceae family, is an important medicinal herb distributed in part of Southeast Asia, and there are many reports of its pharmacological properties. However, the allelopathic activities of P. rosea have not been examined. Thus, the present study was conducted to assess the allelopathic activity of P. rosea and to identify its allelopathic substances. The aqueous methanol stem extract of P. rosea significantly suppressed the seedling growth of barnyard grass (Echinochloa crus-galli L. P. Beauv.), Italian ryegrass (Lolium multiflorum Lam.), timothy (Phleum pretense L.), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), and alfalfa (Medicago sativa L.). The extract of P. rosea was then purified through chromatographic steps, and two active substances were isolated and determined as 7,4′,5′-tri-O-methyl dihydroquercetin and 7,4′,5′-tri-O-methylampelopsin. The two compounds significantly inhibited the seedling growth of cress, with 7,4′,5′-tri-O-methylampelopsin showing a greater inhibitory effect than 7,4′,5′-tri-O-methyl dihydroquercetin. This result may be due to the 3′-OH group in 7,4′,5′-tri-O-methylampelopsin. The effective concentrations of both compounds required for 50% growth inhibition (EC50 values) of cress seedlings were 0.24 mM and 0.59 mM for root and shoot, and 0.07 mM and 0.21 mM, respectively. These findings suggest that the two compounds may contribute to the allelopathic effect of P. rosea and could be used as a natural source of allelopathic substances.

© International Society for Horticultural Science. All rights reserved. The yield of kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson] often decreases after 20 years of cultivation. Replanted young kiwifruit plants cannot grow well in fields where aged trees were previously cultivated. Phytotoxicity is considered one possible reason for this problem. However, allelopathic or phytotoxic substances from kiwifruit have not been reported. This study aimed to isolate and identify potentially allelopathic active substances in kiwifruit leaves. Extracts from kiwifruit leaves inhibited the growth of cress, suggesting that kiwifruit leaf extracts may contain growth-inhibitory substances. The extracts were then purified using a silica-gel column, Sephadex LH-20 column, ODS cartridge, and HPLC. The inhibitory activity of all fractions obtained in each purification step was determined by the cress bioassay. Quercitrin was finally isolated and identified as a growth-inhibitory substance. This substance inhibited cress root growth by more than 50% of control at 3 mM. The aglycone of quercitrin, quercetin, did not significantly inhibit cress growth. Quercitrin may work as an allelopathic substance and cause the kiwifruit replant problem. Further study is in progress to investigate the influence of kiwifruit leaf extracts and these flavonoids on kiwifruit seedlings.

Medicinal plants are rich sources of bioactive substances that can be used to develop environmentally friendly weed control alternatives. Marsdenia tenacissima (Roxb.) Moon is a traditional medicinal plant well known for its pharmacological activities and several bioactive compounds. However, its allelopathy and related substances have not been reported. Hence, the present study was conducted to explore the allelopathic potential and substances from M. tenacissima leaves. Aqueous methanol extracts of M. tenacissima showed significant inhibitory activities against the growth of cress (Lepidium sativum L.) and Italian ryegrass (Lolium multiflorum Lam.). The extracts were purified through various chromatography steps, and two allelopathic substances were isolated and determined by spectral data to be steroidal glycoside 1 (5,6-dihydrogen-11α-O-acetyl-12β-O-tigloyl-17β-marsdenin), a novel compound, and steroidal glycoside 2 (5,6-dihydrogen-11α,12β-di-O-tigloyl-17β-marsdenin). Both compounds significantly inhibited the growth of cress seedlings. Steroidal glycoside 1 showed 1.6-and 4-times greater growth inhibitory potential against the cress shoots and roots than steroidal glycoside 2. The concentrations needed for 50% growth inhibition of the cress seedling shoots and roots were 0.46 and 0.03 mM for steroidal glycoside 1, respectively, and 0.74 and 0.12 mM for steroidal glycoside 2, respectively. Therefore, these results suggest that steroidal glycosides 1 and 2 may be responsible for the allelopathy of the M. tenacissima leaves.

A 68 μg amount of an acyclic polyketide, named beru'amide, was isolated from a marine cyanobacterium Okeania sp. Beru'amide contains six unique moieties in its relatively small skeleton. By applying several cutting-edge techniques, including DFT-based chemical shift calculations, we achieved the structure determination and the total synthesis of this highly functionalized scarce natural product. Furthermore, beru'amide was shown to have strong antitrypanosomal activity. 2022 American Chemical Society.

Caldorazole (1) was isolated from the marine cyanobacterium Caldora sp. collected on Ishigaki Island, Okinawa, Japan. Its structure was determined to be a new polyketide that contained two thiazole rings and an O-methylenolpyruvamide moiety. Caldorazole (1) showed strong cytotoxicity toward tumor cells that had been seeded at a high density. Cell death induced by 1 in HeLa and A431 cells was also observed only in the presence of the glycolysis blocker 2-deoxy-d-glucose (2DG). Co-treatment with 1 and 2DG remarkably decreased ATP levels in these cells. Furthermore, 1 selectively inhibited complex I in the mitochondrial respiratory chain. Thus, 1 was demonstrated to exert cytotoxicity toward human tumor cells by blocking mitochondrial respiration. 2022 American Chemical Society.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As its function is associated with various biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium Leptochromothrix valpauliae and named iezoside (1). The structure of iezoside (1) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (Ki 7.1 nM). In this article, we report our comprehensive analysis of iezoside (1), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC50 6.7 ± 0.4 nM against HeLa cells).

Lingaoamide (1), a new cyclic heptapeptide natural product, was isolated from a freshwater Oscillatoria cyanobacterium. The producing organism was collected in Chinese water spinach paddy farm fields in Lingao County, Hainan, China. The structure of 1 was determined by conventional mass spectrometry, NMR spectroscopy, and chemical degradation studies. Lingaoamide concentration-dependently up-regulates the biogenesis of pro-pathogenic melanin pigments (melanogenesis) in the rice blast fungus, Magnaporthe grisea, when tested in vitro. This molecule also exhibits weak in vitro inhibition (MIC 32 μg/mL) of Gram-negative Pseudomonas aeruginosa and Gram-positive Bacillus pumilus, and does not appear to impact plant growth or immune response in vivo using the model organism, Arabidopsis thaliana.

Iheyamide A ( 1 ) is an antitrypanosomal linear peptide isolated from aDapissp. marine cyanobacterium by our group in 2020, and based on structure-activity relationships of its natural analogues, the C-terminal pyrrolinone moiety has been identified as the phamacophore for its antiparasitic activity. Further, we isolated this pyrrolinone moiety by itself as a new natural product from the marine cyanobacterium and named it iheyanone ( 2 ). As expected, iheyanone ( 2 ) showed antitrypanosomal activity, but its potency was weaker than iheyamide A ( 1 ). To clarify more detailed structure-activity relationships, we completed a total synthesis of iheyamide A ( 1 ) along with iheyanone ( 2 ) and evaluated the antitrypanosomal activities of several synthetic intermediates. As a result, we found that the longer the peptide chain, the stronger the antitrypanosomal activity. As iheyamide A ( 1 ) showed selective toxicity againstTrypanosoma brucei rhodesiense, these findings can provide design guidelines for antitrypanosomal drugs.