• The Korean Journal of Pesticide Science
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• v.5 no.3
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• pp.1-11
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• 2001

New technologies will have a large impact on the discovery of new herbicide site of action. Genomics, combinatorial chemistry, and bioinformatics help take advantage of serendipity through tile sequencing of huge numbers of genes or the synthesis of large numbers of chemical compounds. There are approximately $10^{30}\;to\;10^{50}$ possible molecules in molecular space of which only a fraction have been synthesized. Combining this potential with having access to 50,000 plant genes in the future elevates tile probability of discovering flew herbicidal site of actions. If 0.1, 1.0 or 10% of total genes in a typical plant are valid for herbicide target, a plant with 50,000 genes would provide about 50, 500, and 5,000 targets, respectively. However, only 11 herbicide targets have been identified and commercialized. The successful design of novel herbicides depends on careful consideration of a number of factors including target enzyme selections and validations, inhibitor designs, and the metabolic fates. Biochemical information can be used to identify enzymes which produce lethal phenotypes. The identification of a lethal target site is an important step to this approach. An examination of the characteristics of known targets provides of crucial insight as to the definition of a lethal target. Recently, antisense RNA suppression of an enzyme translation has been used to determine the genes required for toxicity and offers a strategy for identifying lethal target sites. After the identification of a lethal target, detailed knowledge such as the enzyme kinetics and the protein structure may be used to design potent inhibitors. Various types of inhibitors may be designed for a given enzyme. Strategies for the selection of new enzyme targets giving the desired physiological response upon partial inhibition include identification of chemical leads, lethal mutants and the use of antisense technology. Enzyme inhibitors having agrochemical utility can be categorized into six major groups: ground-state analogues, group specific reagents, affinity labels, suicide substrates, reaction intermediate analogues, and extraneous site inhibitors. In this review, examples of each category, and their advantages and disadvantages, will be discussed. The target identification and construction of a potent inhibitor, in itself, may not lead to develop an effective herbicide. The desired in vivo activity, uptake and translocation, and metabolism of the inhibitor should be studied in detail to assess the full potential of the target. Strategies for delivery of the compound to the target enzyme and avoidance of premature detoxification may include a proherbicidal approach, especially when inhibitors are highly charged or when selective detoxification or activation can be exploited. Utilization of differences in detoxification or activation between weeds and crops may lead to enhance selectivity. Without a full appreciation of each of these facets of herbicide design, the chances for success with the target or enzyme-driven approach are reduced.

• The Korean Journal of Pesticide Science
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• v.5 no.1
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• pp.36-45
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• 2001

This study was conducted to search new target enzymes of novel herbicide candidate. Total of 107 biochemical inhibitors reported to inhibit over than 100 different plant enzymes were purchased from commercial chemical companies. 15 inhibitors and 34 enzymes were selected by germination assay, seedling assay, wheat leaf disc assay, and whole plant assay. Among them, seven compounds of purine, phehyl-hydrazine, o-phenanthroline, oleylamine, dicyclohexylcarbodiimide, 7,8-benzoquinoline, and aminooxyacetic acid showed high herbicidal activity in the whole plant assay under greenhouse while 7,8-benzoquinone, 8-hydroxyquinoline, 2,2'-dipyridyl, and o-phenanthroline inhibited seed germination of barnyardgrass, rice, and tomato at concentrations of 1.25 to $5{\mu}M$. The compounds of 7,8-benzoquinoline, chlorpromazine, cyanuric fluoride, 4-methylpyrazole, oleylamine, tranylcypromine, and trifluoperazine inhibited the growth of cyanobacteria at 30 to $100{\mu}M$. The compounds of dicyclohexylcarbodiimide and chlorpromazine exhibited whitening effect on tile wheat leaf disc at $100{\mu}M$. These results suggest that the plant-specific enzyme inhibitors which have biological activities may supply the target enzyme for developing new herbicide candidate.

• The Korean Journal of Pesticide Science
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• v.7 no.2
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• pp.92-99
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• 2003

A 96-well plate was applied to determine the DPPH free radical scavenging activity using 107 plant-specific enzyme inhibitors and 100 unknown plant-originated extracts. The final optimum volume was $250{\mu}L$ containing $100{\mu}M$ DPPH ethanolic solution at pH 7.8. In this condition, the radical scavenging activities were significantly increased by two known antioxidants consisting of ascorbate and a-tocopherol in a concentration-dependent manner. Among the 107 inhibitors, ampicillin and gallic acid showed 90.2% and 92.6% antioxidant activity at $100{\mu}M$, respectively, and these results were consisted with previous findings. In the tested 100 natural materials at $50{\mu}g/mL$, antioxidant activity of AT-407 resulted in the highest of 90.1%, and 10 extracts including AT-388 and AT-443 showed over 70%. Our results suggest that the use of 96-well plate for determining DPPH free radical scavenging activity would be a suitable method to select antioxidant-like substances of both synthetic compounds and natural products.

• BMB Reports
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• v.40 no.5
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• pp.636-643
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• 2007

This study was designed to identify and characterize the mechanism of macrophage activation by AAP, an acidic polysaccharide fraction isolated from the roots of Angelica sinensis (Oliv.) Diels. As a result, AAP significantly enhanced nitric oxide (NO) production and cellular lysosomal enzyme activity in murine peritoneal macrophages in vitro and in vivo. Furthermore, L-NAME, a specific inhibitor of inducible nitric oxide synthase (iNOS), effectively suppressed AAP-induced NO generation in macrophages, indicating that AAP stimulated macrophages to produce NO through the induction of iNOS gene expression and the result was further confirmed by the experiment of the increase of AAP-induced iNOS transcription in a dose-dependent manner. To further investigate, AAP was shown to strongly augment toll-like receptor 4 (TLR4) mRNA expression and the pretreatment of macrophages with anti-TLR4 antibody significantly blocked AAP-induced NO release and the increase of iNOS activity, and tumor necrosis factor-$\alpha$ (TNF-$\alpha$) secretion.

• Food Science and Biotechnology
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• v.15 no.4
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• pp.617-624
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• 2006

Various plant phenolics were assessed for (${\beta}$-secretase (BACE1) inhibitory activity in order to screen for anti-dementia agents. Among 39 phenolics, eight compounds, 1,2,3-trigalloyl glucopyranoside, acetonyl geraniin, euphorscopin, furosine, helioscopinin A, helioscopinin B, jolkinin, and rugosin E exhibited strong inhibition of BACE1 with $IC_{50}$ values of $5.87{\times}10^{-8}-54.93{\times}10^{-6}\;M$. Among them, rugosin E was the most potent ($IC_{50}$ $5.87{\times}10^{-8}\;M$). The active compounds were shown to be non-competitive inhibitors by Dixon plot. All the phenolic BACE1 inhibitors except furosin also suppressed prolyl endopeptidase (PEP) activity. However, these phenolic compounds caused less inhibition of ${\alpha}$-secretase (tumor necrosis factor a converting enzyme; TACE) and no significant inhibition of other serine proteases such as trypsin, chymotrypsin, and elastase was seen, demonstrating that they are relatively specific to both BACE1 and PEP. No significant structure-activity relationships were found.

• Advances in Traditional Medicine
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• v.7 no.4
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• pp.341-347
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• 2007

Red seaweed (Callophyllis japonica) has long formed part of the diet of Asians, but the pharmacological properties of this plant have not been evaluated. In this study, we examined the effect of an ethanol extract of C. japonica on the generation of nitric oxide (NO) in RAW 264.7 cells. The C. japonica extract increased the generation of NO and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), which were detected by the Griess method and an enzyme-linked immunosorbent assay, respectively. The increased production of NO by C. japonica extract was inhibited by $N^G$-monomethyl-L-arginine ($100{\mu}M$), a specific inhibitor of NO production in the L-arginine-dependent pathway, and by the nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) inhibitor, pyrrolidine dithiocarbamate ($10-100{\mu}M$) in a dose-dependent manner. These findings demonstrate that C. japonica extract stimulates the production of NO and $TNF-{\alpha}$ in RAW 264.7 cells through the activation of $NF-{\kappa}B$ and that this extract might also inhibit the growth of the human leukemic cells.

• The Korean Journal of Pesticide Science
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• v.5 no.3
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• pp.41-46
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• 2001

This study was conducted to develop a high throughput system for screening acetolactate synthase(ALS) inhibitors, and to detect basic mother molecules for developing new novel herbicide candidates. The high throughput screening (HTS) method using 96-well plate and microplate reader was developed. This method is 8 times more effective than basic technique in one cycle per person. Futhermore, considering for less than 1/10 volume of materials required for ALS test and enzyme kinetics with 16 times faster speed compared to those of former procedure, this HTS method has more than 100 times higher efficacy than basic system in a consecutive procedure. We discovered 11 new ALS inhibitors such as 2-oxoglutaric acid, aminooxyacetic acid, azelaic acid, citric acid, cyanuric fluoride, itaconic acid, malonic acid, niclosamide, oxalic acid, glyoxylic acid, and suramin from 107 commercial plant-specific inhibitors using this technique. We hope these results might be useful to discover lead compounds for developing new novel herbicide candidate.

• Food Science and Preservation
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• v.17 no.2
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• pp.290-296
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• 2010

To develop anti-acidosis and anti-diabetes agentsfrom natural products, the inhibitory activities of Brazilian plant extracts against microbial $\alpha$-amylase and $\alpha$-glucosidase were evaluated. Among 100 different ethanol extracts tested, those of Acacia jurema Mart., Anacardium humile A. St.-Hil., Cedrela odorata L., and Guazuma ulmifolia Lam showed good inhibitoryactivities toward both enzymes. In addition, an extract of Plumeria drastica Mart. showed specific inhibition of $\alpha$-amylase, whereas that of Eugenia uniflora L. demonstrated strong inhibition of the enzyme. IC50 values of $\alpha$-amylase inhibition suggested that the extract of A. humile A. St.-Hil., which has been used as an anti-diabetes medicine in Brazil, had potent inhibitory activity. The IC50 for the A. humile A. St.-Hil. extract ($91.2{\mu}g/mL$) was similar to that of acarbose ($50.5{\mu}g/mL$). This activity of A. humile A. St.-Hil. was not reduced by heat or acid treatment. Moreover, treatment with HCl (0.01 M) for 1 h increased the inhibitory activity from 57.5% to 81.2%. Also, the extract did not cause hemolysis of human red blood cells at levels up to 1 mg/mL. The results indicate that the extract of A. humile A. St.-Hil. is potentially useful as an anti-acidosis and anti-diabetes agent.

• Journal of Plant Biotechnology
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• v.5 no.1
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• pp.33-41
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• 2003

Microspore-derived cell lines resistant to 5-methyltryptophan (5MT, a tryptophan analog) or S-(2-aminoethyl)-L-cysteine (AEC, a Iysine analog) were selected in rice by in vitro mutagenesis. For selection of 5MT or AEC resistant cell lines, suspension-cultured cells were irradiated with gamma rays. Thirteen 5MT resistant cell lines were selected and they were able to grow stably at 2 times higher 5MT concentration. A feedback insensitive form of anthranilate synthesis, the pathway specific control enzyme for tryptophan synthesis, was detected from the 5MT resistant lines. Contents of the free amino acids in five resistant lines (MR12-1 to MR12-5) showed a 7.4 to 46.6 times greater level than that in the control culture. Tryptophan, phenylalanine, and tyrosine levels in the shikimate pathway were 28.1 and 22.5 times higher in MR12-3 and MR12 4, respectively, than that measured in the control cells. Four AEC resistant cell lines were isolated from cultures grown on medium containing 1 mM AEC, They were able to grow stably with 2 mM AEC, while sensitive calli were inhibited by 0.5 mM AEC. Aspartate kinase activities of the resistant lines were insensitive to the natural inhibitor, Iysine, and accumulated 2.2 to 12.9-fold higher levels of free Iysine than that of the control cells. Especially, the levels of aspartate, asparagine, and methionine in the aspartate pathway showed higher accumulation in the AEC resistant lines than that in the control cells.

• Journal of Environmental Science International
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• v.19 no.12
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• pp.1323-1334
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• 2010

The effect of nitric oxide (NO) on antioxidant system and protective mechanism against oxidative stress under UV-B radiation was investigated in leaves of maize (Zea mays L.) seedlings during 3 days growth period. UV-B irradiation caused a decrease of leaf biomass including leaf length, width and weight during growth. Application of NO donor, sodium nitroprusside (SNP), significantly alleviated UV-B stress induced growth suppression. NO donor permitted the survival of more green leaf tissue preventing chlorophyll content reduction and of higher quantum yield for photosystem II than in non-treated controls under UV-B stress, suggesting that NO has protective effect on chloroplast membrane in maize leaves. Flavonoids and anthocyanin, UV-B absorbing compounds, were significantly accumulated in the maize leaves upon UV-B exposure. Moreover, the increase of these compounds was intensified in the NO treated seedlings. UV-B treatment resulted in lipid peroxidation and induced accumulation of hydrogen peroxide ($H_2O_2$) in maize leaves, while NO donor prevented UV-B induced increase in the contents of malondialdehyde (MDA) and $H_2O_2$. These results demonstrate that NO serves as antioxidant agent able to scavenge $H_2O_2$ to protect plant cells from oxidative damage. The activities of two antioxidant enzymes that scavenge reactive oxygen species, catalase (CAT) and ascorbate peroxidase (APX) in maize leaves in the presence of NO donor under UV-B stress were higher than those under UV-B stress alone. Application of 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3- oxide (PTIO), a specific NO scavenger, to the maize leaves arrested NO donor mediated protective effect on leaf growth, photosynthetic pigment and free radical scavenging activity. However, PTIO had little effect on maize leaves under UV-B stress compared with that of UV-B stress alone. $N^{\omega}$-nitro-L-arginine (LNNA), an inhibitor of nitric oxide synthase (NOS), significantly increased $H_2O_2$ and MDA accumulation and decreased antioxidant enzyme activities in maize leaves under UV-B stress. This demonstrates that NOS inhibitor LNNA has opposite effects on oxidative resistance. From these results it is suggested that NO might act as a signal in activating active oxygen scavenging system that protects plants from oxidative stress induced by UV-B radiation and thus confer UV-B tolerance.