Progress 10/01/99 to 09/30/04
Outputs
By bioassay-guided fractionation using Aedes aegypti larvae, four novel mosquito larvicidal saponins, pachyelaside A - D, were effectively isolated from the root bark of Pachyelasma tessmannii (Leguminosae) using recycling high pressure liquid chromatography. The structure of cinnamoyl saponins were established on the basis of extensive NMR spectroscopic and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric studies. These new saponins possess the most complex sugar chains among the saponins characterized from Pachyelasma species, and were found to exhibit potent mosquito larvicidal property against A. aegypti larvae. In addition, the same saponins were found to have potent mosquito larvicidal activity against Culex tarsalis, the principal vector of the Western equine and St. Louis encephalitis viruses common throughout the Western US. Despite their excellent mosquito larvicidal activity, these triterpene saponins are difficult to obtain in
quantities and complex molecules to synthesize. To cross these hurdles, a series of aliphatic primary alkanols was tested as a model for their mosquito larvicidal activity, since saponins are usually considered as surface active agents (surfactants). The alkanols tested show activity against C. tarsalis larvae. Alkanols are among the most versatile of all organic compounds; free and esterified alkanols occur widely in nature, and more importantly, they are stable, colorless, inexpensive, biodegradable, and essentially nontoxic to humans. The fact that they are already approved for use in food products at concentrations comparable to the doses found may facilitate their approval as insecticides. Continuing to search for alternative insect control agents, novel limonoids were characterized from the methanol extract of Croton jatrophoides (Euphorbiaceae) as potent antifeedants against the second-instar larvae of Pectinophora gossypiella and Spodoptera frugiperda. The structures were
determined mainly on the basis of extensive nuclear magnetic resonance (NMR) spectroscopic analyses. There is no doubt that many plant secondary metabolites affect insect behavior, development and reproduction. Identifying these substances is an important first step in understanding the effect of plants on insect life at the molecular level. Information about growing environment of living plants, including defenses against insects often provides hints for the choosing bioassays. However, there is usually no such information available when the plants are sold at market places. The methanol extract of the dried flower of Heterotheca inuloides (Compositae) was found to exhibit insect growth inhibitory activity against the second-instar larvae of P. gossypiella using an artificial diet feeding assay. The bioassay guided fractionation led to isolation of the active principle, a sesquiterpenoid, inuloidin.
Impacts
The results obtained may provide more scientifically sound and environmentally acceptable species specific insect control agents.
Publications
- NIHEI, K., YING, B. P., MURAKAMI, H., MATSUDA, N., HASHIMOTO, M. and KUBO, I. 2005. Pachyelasides A, B, C, and D; Novel molluscicidal triterpene saponins from Pachyelasma tessmannii. J. Agric. Food Chem. 53:608-613.
- NIHEI, K., ASAKA, Y., MINE, Y. and KUBO, I. 2005. Insect antifeedants from tropical plants: Structures of zumsen, zumsenin and zumsenol. J. Nat. Prod. 68:244-247.
- KUBO, I. 2004. New concept to search for alternate insect control agents. In Naturally Occurring Bioactive Compounds: A New and Safe Alternative for Management of Pests and Diseases (M. C. Carpinella and M. Rai, eds.) Elsevier, in press.
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Progress 01/01/03 to 12/31/03
Outputs
A series of (2E)-alkenals were applied for control of larval stage mosquitoes. By applying the (2E)-alkenals as soluble solutions rather than as insoluble monolayers, and by trapping larvae under glass in assays that isolated them from the surface phenomena believed to be responsible for death by suffocation, the data obtained have shown that the action of (2E)-alkenals against mosquito larvae is biochemical in nature, similar to those described for alkanols. Eight novel A-seco limonoids were isolated from methanolic extract of Croton jatrophoides (Euphorbiaceae) and all showed potent antifeedant activity against two lepidopteran larvae; pink bollworm, Pectinophora gossypiella, and fall armyworm, Spodoptera frugiperda. The structures of seven of them were determined using a variety of spectroscopic methods including NMR, MS, and circular dichroism. They are classified into two types; zumsin-type having A'-B trans-fused ring and dumsin-type possessing A'-B cis-fused
ring. The result suggests two unique biosynthetic process after the A-ring oxidative expansion in this plant. In our continuing search for insect antifeedants from plants, three new diterpenes, ent-isopimaran-8B, 15R, 16-triol, ent-isopimaran-16-acetoxy-8B, 15R-diol, and ent-isopimaran-8B-hydroxy-15R, 16-acetonide, were isolated from the active fraction of the leaves of Rabdosia forrestii (Labiatae). They were designated as forrestins H-J and the structures were established as ent-isopimarane diterpenoids on the basis of NMR spectroscopic analysis, as well as consideration of biogenesis. In addition, the modified Mosher's method has established the absolute configuration of the secondary hydroxyl group at C-15 as the R-configuration.
Impacts
The results obtained may provide more scientifically sound and environmentally acceptable species specific insect control agents.
Publications
- Kubo, I., Kinst-Hori, I., Nihei, K., Puente, F. S. Takasaki, M., Cespedes, C. L. And Calderon, J. S. 2003. Tyrosinase inhibitors from galls of Rhus javanica leaves and their effects on insects. Z. Naturforsch. C: J. Biosci. 58c:719-725.
- Kubo, I., Fujita, K., Kubo, A., Nihei, K. And Lunde, C. S. 2003. Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae. J. Agric. Food Chem. 51:3951-3957.
- Kubo, I., Xu, Y-L. And Shimizu, K. 2003. ent-Isopimarane diterpenoids of the leaves from Rabdosia forrestii. Fitoterapia 74:643-649.
- Nihei, K., Asaka, Y., Mine, Y., Ito, C., Furukawa, H., Ju-Ichi, M. And Kubo, I. 2004. Insect antifeedants from tropical plants III: Structures of dumnin and dumsenin. J. Agric. Food Chem. In Press.
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Progress 01/01/02 to 12/31/02
Outputs
A novel A-seco limonoid was isolated from methanolic extract of Croton jatrophoides and designated as zumsin. This compound showed potent antifeedant activity against two lepidopteran larvae, pink bollworm, Pectinophora gossypiella, and fall armyworm, Spodoptera frugiperda. The structure of zumsin was determined using variety of spectroscopic methods including NMR, MS, and circular dichroism. The structure consists of an A'-B trans-fused ring while dumsin, a constituent of the same source, maintains A'-B cis-fused suggests two unique biosynthetic processes after A ring oxidative expansion. Quinolizidine alkaloids, found in the leaves of Cytisus monspessulanus L. (Leguminosae), were characterized in the cuticle of larvae of the pyralid moth Uresiphita reversalis (Lepidoptea: Pyralidae) when the latter were fed on this weed. By GC-MS analysis of the methanolic extracts of the cuticle, four quinolizidine alkaloids, N-methylcytisine, cytisine, aphylline and anagyrine,
were identified as possible defense substances. In addition, the quinolizidine alkaloid, (+)-2,3-dehydro-10-oxo-alpha-isosparteine was characterized in both the insect and host plant.
Impacts
The results obtained may provide more scientifically sound and environmentally acceptable species specific insect control agents.
Publications
- NIHEI, K., HANKE, F. J., ASAKA, Y., MATSUMOTO, T. and KUBO, I. 2002. Insect antifeedants from tropical plants II: Structure of zumsin. J. Agric. Food Chem. 50:5048-5052.
- NIHEI, K., SHIBATA, K. and KUBO, I. 2002. (+)-2,3-Dehydro-10-oxo-*-isosparteine in Uresiphita reversalis larvae fed on Cytisus monspessulanus leaves. Phytochemistry 61:987-990.
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Progress 01/01/01 to 12/31/01
Outputs
As a defense mechanism of the leaves of Rhus javanica (Anacardiaceae) against the aphid Melaphis chinensis (Aphididae) attack, tannic acid is rapidly accumulated and forms galls along the midrib of the leaves resulting in a unique natural medicine Gallae Rhois. Tannic acid was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase (EC 1.14.18.1) with an IC50 of 15 microM. The aphid would likely detoxify the ingested toxic tannic acid to relatively nontoxic gallic acid, whereas the non-adapted pink bollworm Pectinophora gossypiella larvae are sensitive to the ingested tannic acid. We have isolated a large number of phytochemicals possessing activity against insects by using the premise that plant sources of medicinals may also serve as plant sources of insecticidal compounds. It is a popular belief that the origin of secondary metabolites in plants was a response to the pressures of natural selection during the coevolution of
plants and insects. The efficacy of present day plant medicinals in affecting man may in part be rooted in the similarity of the primary metabolism of insects and higher animals. Hence, the often documented sources of plant medicinals may also serve as sources of insecticidal compounds. We recently reported the isolation of the previously unknown limonoid dumsin from an East African medicinal plant "Msinduzi" (Swahili, tentatively identified as Croton jatrophoides Pax). This compound exhibited growth inhibitory properties in our in-house artificial diet feeding assays using two lepidopteran larvae. In our continuing effort to explore the chemistry of naturally occurring pesticides we describe here the isolation of an additional limonoid from C. jatrophoides exhibiting similar insect antifeedant activity.
Impacts
The results obtained may provide more scientifically sound and environmentally acceptable species specific insect control agents.
Publications
- Calderron, J. S., Cespedes, C. L., Rosas, R., Gomez-Garibay, F., Federico, S., Salazar, J. R., Lina, L. E., Aranda, E. and Kubo, I. 2001. Acetylcholinesterase and insect growth inhibitory activities of Gutierrerda microcephala on fall armyworm Spodoptera frugiperda. Z. Naturforsch. C: J. Biosci. 56:382-394.
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Progress 01/01/00 to 12/31/00
Outputs
In our continuing search for alternate insect control agents on the basis of rational approach, we have focused on the modes of mosquito larvicidal activity of alkanols using Culex tarsalis larvae as a model. Primary alcohols, from hexanol to pentadecanol were tested for their effects on the succinate-supported respiration of intact mitochondria isolated from rat liver. Potency increased with number of carbons in the chain until reaching a peak, in this case at undecanol, then tapered off to tridecanol before reaching a cutoff, at tetradecanol. Alkanols, especially undecanol, might be used for selective pest control. Alkanols are stable, colorless, inexpensive, biodegradable and essentially non-toxic to humans, making them promising candidates for pest management programs. On the basis of the same surface activity concept found with alkanols, a variety of naturally occurring aldehydes have been evaluated for their insecticidal activity. The three aldehydes, propanol,
(E)-2-pentenal and 2-methyl-(E)-2-butenal, have been noted to have excellent potential as affordable postharvest insect control agents, killing 100% of the aphids with little or no detectable harm to a majority of the commodities tested. A sesquiterpene, polygodial was previously characterized from plants as insecticide. This sesquiterpene was found to inhibit mitochondrial ATP synthase. It appears that polygodial initially acts as a nonionic surfactant. For example, polygodial induces leakage by disrupting the membrane surface. More specifically, it inhibits the plasma membrane proteins by disrupting and disorganizing the hydrogen bonds at the lipid bilayer-protein interface. It should be kept in mind, however, that polygodial does not act by a single defined process, but, rather, has multiple functions. In contrast, biological activities exerted by alkanols are due entirely to their surfactant properties.
Impacts
(N/A)
Publications
- Hammolnd, D. G. and Kubo, I. 2000. Alkanols inhibit respiration of intact mitochondria and display cutoff similar to that measured in vivo. J. Pharm. Exp. Therap. 293:822-828.
- Hammond, D. G., Rangel, S., and Kubo, I. 2000. Volatile aldehydes are promising broad-spectrum postharvest insecticide. J. Agric. Food Chem. 48:4410-4417.
- Wood, W. F., Hanke, F. J., Kubo, I., Carroll, J. A. and Crews, P. 2000. Buzonamine, a new alkaloid from the defensive secretion of the millipede, Buzonium crassipes. Biochem. System. Ecol. 28:305-312.
- Lunde, C. S. and Kubo, I. 2000. Effect of polygodial on the mitochondrial ATPase of Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 44:1943-1953.
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Progress 01/01/99 to 12/31/99
Outputs
Our overall project objective is to develop ecologically friendly and environmentally acceptable insect control agents from botanical sources. Emphasis has been placed on mosquito control agents using Culex tarsalis larvae as a model because the mosquito still continues to pose serious public health problems. The study was focused on the modes of mosquito larvicidal activity of aliphatic primary alkanols. The larvicidal activity increased steadily in relation to chain length until it peaked with dodecanol. The maximum larvicidal activity depends on the hydrophobic alkyl (tail) chain length from the hydrophilic hydroxyl group (head). The study has now been further extended to understand their mode of larvicidal action. These mosquito larvicidal alcohols were tested for their effects on the succinate-supported respiration of intact mitochondria isolated from rat liver as well as mosquitoes. Alkanols were found to inhibit State 3 and uncoupled respiration. The ADP/O
ratios, a measure of the efficiency of oxidative phosphorylation, were also lowered, but to a lesser degree when calculated on the basis of percent of controls. Given each alkanol's nearly identical effect on State 3 and uncoupled respiration, action is not directly on ATP synthase, but earlier in the respiratory process. In agreement with many other studies of the homologous series of alkanols, potency increased with number of carbons in the chain until reaching a peak, in this case at undecanol, then tapered off to tridecanol before reaching a cutoff, at tetradecanol. If tetradecanol or longer homologues have activity, it is only after a lag phase of more than 15 minutes preincubation. All alkanols up to tridecanol also acted as uncouplers. At higher doses, hexanol inhibited State 4 rates, whereas longer chain alkanols did not, even at doses that completely eliminated respiratory control. Hexanol and decanol were also assayed against freeze-thawed (broken) mitochondria to
distinguish effects on the mitochondrial substrate carrier from that on the electron transport chain. Both compounds were only weak inhibitors of respiration in broken mitochondria, suggesting that inhibition originates from interference with the dicarboxylate carrier - which must transport succinate across the mitochondrial membranes before it can be fed into Complex II - rather than affecting the electron transport chain itself. It is well known that inhibition of the respiratory chain in mitochondria results in cell toxicities. If mosquitoes larvae were prevented from making ATP, approximately 90% of which is produced in mitochondria, then certainly they would die. However, it is uncertain if the respiratory inhibition mechanism is the primary mode of larvicidal action of alkanols against mosquitoes larvae because we do not know if alkanols can reach the mitochondria in vivo. If alkanol molecules permeate and reach the mitochondria inner membrane, this needs to be confirmed
quantitatively. Nevertheless, alkanols are stable, inexpensive, biodegradable and essentially non-toxic to humans, making them promising candidates for pest management programs.
Impacts
(N/A)
Publications
- Hammond, D. G. and Kubo, I. 1999. Structure-activity relationship of alkanols as mosquito larvicides with novel findings regarding their mode of action. Bioorg. Med. Chem., 7:271-278.
- Hammond, D. G. and Kubo, I. 2000. Alkanols inhibit respiration of intact mitochondria and display cutoff similar to that measured in vivo. J. Pharm. Exp. Therap. IN PRESS.
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Progress 01/01/98 to 12/01/98
Outputs
In our continuing search for alternate insect control agents on the basis of rational approach, we have been focusing on the modes of mosquito larvicidal activity of alkanols using CULEX TARSALIS larvae as a model. Primary alcohols, from methanol to eicosanol, were applied to water for control of larval stage mosquitoes. By applying the alkanols as soluble solutions rather than as insoluble monolayers, and by trapping larvae under glass in assays that isolated them from surface phenomena believed to be responsible for death by suffocation, we have shown that the action of alkanols against mosquito larvae is biochemical in nature, not just physical. We found that activity levels off after undecanol C(11) but does not disappear until after pentadecanol C(15), that it is reversible, and that chain length plays a role not only in potency, but also in the time needed to manifest toxic effects. Mosquitoes appear to be the first animal for which cutoff has been demonstrated
to occur at a chain length beyond C(12), suggesting the possibility that alkanols might be used for selective pest control. Alkanols are stable, colorless, inexpensive, biodegradable and essential non-toxic to humans, making them promising candidates for pest management programs.
Impacts
(N/A)
Publications
- KUBO, I. and SMITH, L. R. 1998. Crinitol, an acyclic diterpene diol from marine algae. In: Studies in Natural Products Chemistry, Vol. 20, Structure and Chemistry Part D (A. U. Rahman, ed.), Elsevier,
- LEE, J. R., LEE, S. H., KUBO, I. and HONG, S. D. 1998. Antifungal activity of medium-chain [C(6)-C(13)] alkenals against, and their inhibitory effect on the plasma membrane proton-ATPase of SACCHAROMYCES CEREVISIAE. J. Microbial. Biotech.
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Progress 01/01/97 to 12/01/97
Outputs
Based on our previous finding of insect growth inhibitory activity of a simple acyclic diterpene alcohol, crinitol isolated from a marine algae, we attempted to gain its structural criteria for activity. The study focused on the modes of mosquito larvicidal activity of alkanols using CULEX TARSALIS larvae as a model. The larvicidal activity increased steadily in relation to chain length until it peaked with 1-dodecanol (C12). Potency tapered off as chain length was increased further to C13. The maximum larvicidal activity depends on the hydrophobic alkyl (tail) chain length from the hydrophilic hydroxyl group (head).
Impacts
(N/A)
Publications
- KAMIKAWA, T., HANAOKA, Y., FUJIE, S., SAITO, K., YAMAGIWA, Y., FUKUHARA, K. and KUBO, I. 1996. SRS-A antagonist pyranoquinolone alkaloids from East African Fagara plants and their synthesis. Bioorg. Med. Chem. 4:1317-1320.
- KUBO, I., JAMALAMADAKA, V., KAMIKAWA, T., TAKAHASHI, K., TABATA, K., and KUSUMI,T. 1996. Absolute stereochemistry of tanabalin, an insect antifeedant clerodane from TANACETUM BALSAMITA. Chem. Lett. 441-442.
- HARAGUCHI, H., OHMI, I., and KUBO, I. 1996. Inhibition of aldose reductase by maesanin and related p-benzoquinones and effects on other enzymes. Bioorg. Med. Chem. 4:49-53.
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Progress 01/01/96 to 12/30/96
Outputs
A new clerodane diterpenoid, isolated from a Brazilian medicinal plant, shows insect antifeedant activity against several lepidopterous larvae. Since the compounds we reported as insect control agents were isolated from medicinal plants, their various pharmacological activities were also tested by collaborators. Based on our previous study on an acyclic diterpenoid insect growth inhibitor isolated from a marine alga, it has been determined that certain chemical properties are essential to elicit biological activity. One primary area of investigation is a study of the balance between hydrophilic and hydrophobic portions of the molecule. This has been continued using mosquito larvae in order to gain new insights into their structural functions.
Impacts
(N/A)
Publications
- KUBO, I., JAMALAMADAKA, V., KAMIKAWA, T., TAKAHASHI, K., TABATA, K., and KUSUMI,T. 1996. Absolute stereochemistry of tanabalin, an insect antifeedant clerodane from TANACETUM BALSAMITA. Chem. Lett. 441-442.
- HARAGUCHI, H., OHMI, I., and KUBO, I. 1996. Inhibition of aldose reductase by maesanin and related p-benzoquinones and effects on other enzymes. Bioorg. Med. Chem. 4:49-53.
- KAMIKAWA, T., HANAOKA, Y., FUJIE, S., SAITO, K., YAMAGIWA, Y., FUKUHARA, K. and KUBO, I. 1996. SRS-A antagonist pyranoquinolone alkaloids from East African Fagara plants and their synthesis. Bioorg. Med. Chem. 4:1317-1320.
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Progress 01/01/95 to 12/30/95
Outputs
Natural products such as crinitol, isolated from a marine alga, show activity against insects such as mosquitos and several lepidopterous larvae. It has been determined that certain chemical properties are key to elicit biological activity. One primary area of investigation is a study of the balance between hydrophilic and hydrophobic portions of the molecule.
Impacts
(N/A)
Publications
- No publications reported this period.
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