Antifungal Activities of Dimeric Sesquiterpenes, Shizukaols C and F, Isolated from Chloranthus japonicus Sieb.

Two dimeric sesquiterpenes were separated from Chloranthus japonicus Sieb. and identified as shizukaols C and F. They exhibited potent antifungal activities (MICs = 4-16 µg/ml) in vitro against various plant pathogenic fungi (Pythium ultimum, Phytophthora infestans, Botrytis cinerea, Colletotrichum lagenarium, Alternaria kikuchiana, and Magnaporthe grisea). Shizukaol C showed 88% and 91% protective activities in the greenhouse against Puccinia recondita (wheat leaf rust) and Phytophthora infestans (tomato late blight), respectively, at 100 µg/ml; shizukaol F exhibited 93% antifungal activity against Puccinia recondita at the same concentration. Therefore, these compounds might serve as interesting candidates for effective antifungal agents.

Inhibition of the Calcineurin Pathway by Two Flavonoids Isolated from Miliusa sinensis Finet & Gagnep.

In order to discover plant-derived signaling pathway inhibitors with antifungal properties, a two-component screening system utilizing the calcineurin and Hog1 mitogen-activated protein kinase pathways responsible for the virulence networks of Cryptococcus neoformans was employed, owing to the counter-regulatory actions of these pathways. Of the 1,000 plant extracts tested, two bioactive compounds from Miliusa sinensis were found to act specifically on the calcineurin pathway of C. neoformans. These compounds, identified as pashanone and 5-hydroxy-6,7-dimethoxyflavanone, exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentration values ranging from 4.0 to >128 µg/ml.

A Phenylpropanoid Glycoside as a Calcineurin Inhibitor Isolated from Magnolia obovata Thunb.

To identify plant-derived cell signaling inhibitors with antifungal properties, a twocomponent screening system using both wild-type Cryptococcus neoformans and a calcineurin mutant was employed owing to their counter-regulatory actions on the Hog1 mitogenactivated protein kinase and calcineurin pathways. Of the 2,000 plant extracts evaluated, a single bioactive compound from M. obovata Thunb. was found to act specifically on the calcineurin pathway of C. neoformans. This compound was identified as magnoloside A, and had potent antifungal activities against various Cryptococcus strains with minimum inhibitory concentration values ranging from 1.0 to 4.0 µg/ml.

Inhibition of the calcineurin pathway by two tannins, chebulagic acid and chebulanin, isolated from Harrisonia abyssinica Oliv.

In order to discover and develop novel signaling inhibitors from plants, a screening system was established targeting the two-component system of Cryptococcus neoformans by using the wild type and a calcineurin mutant of C. neoformans, based on the counter-regulatory action of high-osmolarity glycerol (Hog1) mitogen-activated protein kinase and the calcineurin pathways in C. neoformans. Among 10,000 plant extracts, that from Harrisonia abyssinica Oliv. exhibited the most potent inhibitory activity against C. neoformans var. grubii H99 with fludioxonil. Bioassay-guided fractionation was used to isolate two bioactive compounds from H. abyssinica, and these compounds were identified as chebulagic acid and chebulanin using spectroscopic methods. These compounds specifically inhibited the calcineurin pathway in C. neoformans. Moreover, they exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentrations ranging from 0.25 to over 64 µg/ml.

Pedobacter luteus sp. nov., isolated from soil.

Two strains of Gram-staining-negative, rod-shaped bacteria that were motile by gliding, N7d-4(T) and B4a-b5, were isolated during a study of culturable bacteria in soil cultivated with potatoes. These isolates grew at 15-37 °C and at pH 6.5-7.0. The major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. The major polar lipids were phosphatidyl-N-methylethanolamine and phosphatidylethanolamine. The strains contained d-18 : 0 and d-19 : 0 sphingosines. The DNA G+C contents of strains N7d-4(T) and B4a-b5 were 48.5 and 46.9 mol% (HPLC), respectively. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains N7d-4(T) and B4a-b5 were affiliated with Pedobacter species in the family Sphingobacteriaceae. Strains N7d-4(T) and B4a-b5 shared 99.9 % sequence similarity, and the most closely related Pedobacter type strains were Pedobacter composti TR6-06(T) (96.5 and 96.7 % sequence similarity, respectively), P. oryzae N7(T) (95.4 and 95.6 %) and P. caeni LMG 22862(T) (94.0 and 94.4 %). Phenotypic data and phylogenetic inference clearly distinguished the two isolates from other Pedobacter species. Based on these data, the isolates are considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter luteus sp. nov. is proposed. The type strain is N7d-4(T) ( = KCTC 22699(T)  = DSM 22385(T)).

Phycicoccus ochangensis sp. nov., isolated from soil of a potato cultivation field.

Two novel, Gram-positive, motile, coccal bacteria, strains L1b-b9(T) and B5a-b5, were isolated from a potato cultivation field in Ochang, Korea. These isolates grew at 10-45°C, pH 5.0-10.0, and in the presence of 8% (w/v) NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major menaquinone was MK-8(H(4)) and the main cellular fatty acids were iso-C(14:0), iso-C(15:0), and anteiso-C(15:0). Polar lipids in strain L1b-b9(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and an unknown glyco-amino lipid. The G+C content of genomic DNA was 73.6 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains L1b-b9(T) and B5a-b5 shared 99.36% similarity and formed a robust clade with the type species of the genus Phycicoccus. Strain L1b-b9(T) is related most closely to Phycicoccus cremeus V2M29(T) (97.52% 16S rRNA gene sequence similarity). On the basis of phylogenetic characteristics, the name Phycicoccus ochangensis sp. nov. is proposed for strain LIb-b9(T) (=KCTC 19695(T) [corrected] =JCM 17595(T)).

Monitoring of possible horizontal gene transfer from transgenic potatoes to soil microorganisms in the potato fields and the emergence of variants in Phytophthora infestans.

To examine the possibility of horizontal gene transfer between transgenic potatoes and microorganisms in potato fields, the gene flow from transgenic potatoes containing nucleoside diphosphate kinase 2 (NDPK2) gene to microorganisms in soils was investigated. The soil samples collected from the potato fields from March to October in 2007 were examined by PCR, Southern hybridization, and AFLP fingerprinting. The NDPK2 gene from soil genomic DNAs was not detected by both PCR and Southern hybridization, indicating that gene-transfer did not occur in the potato fields. In addition, no discrepancy was found in pathogenicity and noticeable changes for the appearance of variants of Phytophthora infestans in each generation when serial inoculations and the analysis of genomic DNAs by AFLP was conducted. Thus, these data suggest that transgenic potatoes do not give significant impacts on the communities of soil microorganisms and the emergence of variants although continued research efforts may be necessary to make a decisive conclusion.

Chitin synthase 2 inhibitory activity of O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one, isolated from Chamaecyparis pisifera.

In the course of search for potent chitin synthase inhibitors from plant extracts, the chitin synthase 2 inhibitors, O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one which have diterpene skeleton, were isolated from the leaves of Chamaecyparis pisifera. These compounds inhibited chitin synthase 2 of Saccharomyces cerevisiae with the IC50 values of 5.8 and 226.4 microM, respectively. Especially, O-methyl pisiferic acid showed 15.3-fold stronger inhibitory activity than polyoxin D (IC50=88.6 microM), a well-known chitin synthase inhibitor. These compounds exhibited weaker inhibitory activities against chitin synthase 1 than chitin synthase 2, whereas it showed no inhibitory activity for chitin synthase 3. The compound exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate (Ki=5 microM). These results indicated that O-methyl pisiferic acid is a specific inhibitor of chitin synthase 2. The compound also inhibited chitin synthase 1 of Candida albicans, which represents analogues to chitin synthase 2 of S. cerevisiae, with an IC50 of 75.6 microM, which represents 1.8-fold weaker activity than that of polyoxin D. Although O-methyl pisiferic acid has been reported for antibacterial and insecticidal activities, the present study is the first report on its inhibitory activity against chitin synthase 2.

Anti-tumor activity of Gastrodia elata Blume is closely associated with a GTP-Ras-dependent pathway.

Gastrodia elata Blume (GEB) is an important medicinal plant in Korea. In order to confirm the anti-tumor activities of GEB extracts, we carried out various in vitro anti-tumor assays, including a wound assay and an invasion assay using an ethyl ether extract of GEB. The results showed that the GEB extract exhibits potent anti-tumor activity in vitro in a dose-dependent manner. The expression of CD44, cdc42, Timp-2 or RhoA mRNA did not change by GEB treatment, compared to that of the control. GTP-Ras, an active form of a G-coupled protein family, however, is associated with the anti-tumor activity of GEB extracts. We examined various molecular markers related to metastasis by reverse transcriptase-polymerase chain reaction with the extract of GEB-treated B16 cells. There was an increase in GTP-Ras expression by the Gastrodia elata Blume extract. Together, these results suggest that the Gastrodia elata Blume extract could have potential in alleviating tumorigenesis, by a GTP-Ras-dependent pathway; although the precise molecular mechanisms are still being examined.

Ceftezole, a cephem antibiotic, is an alpha-glucosidase inhibitor with in vivo anti-diabetic activity.

Using a high throughput-compatible assay to screen for potential alpha-glucosidase inhibitors, we found that the beta-lactam antibiotic ceftezole exhibited potent alpha-glucosidase inhibitory activity. In in vitro alpha-glucosidase assays, ceftezole was shown to be a reversible, non-competitive inhibitor of yeast alpha-glucosidase with a Ki value of 5.78 x 10(-7) M when the enzyme mixture was pretreated with ceftezole. Using an in vivo streptozotocin-induced mouse model, we confirmed that blood glucose levels decreased by 30% 20 min after ceftezole treatment (10 mg/kg/day). Expression levels of glycogen synthase kinase-3, peroxisome proliferator-activated receptor-gamma, and uncoupling protein-3 mRNA were also slightly decreased compared to controls following ceftezole treatment. Taken together, these in vivo and in vitro results suggest that ceftezole may be a clinically useful anti-diabetic compound.

Inhibition of chitin synthase 2 and antifungal activity of lignans from the stem bark of Lindera erythrocarpa.

Potent chitin synthase 2 inhibitors, methyllinderone (1), linderone (2) and kanakugiol (3) were isolated from the stem bark of L. erythrocarpa Makino (Lauraceae). These compounds inhibited chitin synthase 2 with IC(50) values of 23.3, 21.4 and 23.8 microg/mL, respectively. Methyllinderone (1) and linderone (2) exhibited no inhibitory activities for chitin synthases 1 and 3 from S. cerevisiae, and chitin synthase 1 from Candida albicans up to the concentration of 280 microg/mL, while kanakugiol (3) exhibited very weak activity against chitin synthase 1 of C. albicans with an IC(50) of 160 microg/mL. All of the compounds showed moderate to weak antifungal activities against various pathogenic fungi (MIC: 8 - >128 microg/mL) including Cryptococcus neoformans, Aspergillus fumigatus, and Colletotrichum lagenarium. The results indicate that these compounds are specific inhibitors of chitin synthase 2 and can potentially serve as antifungal agents.

Inhibition of chitin synthases and antifungal activities by 2'-benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and its derivatives.

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2'-benzoyloxycinnamaldehyde (2'-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2'-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 microg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 microg/ml. Its derivatives, 2'-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2'-BCA, with the IC50s of 37.2 and 26.6 microg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC = 16 microg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2'-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2'-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.

Phellinsin A from Phellinus sp. PL3 exhibits antioxidant activities.

Phellinsin A, which was isolated from the culture broth of Phellinus sp. PL3, exhibited significant low-density lipoproteins (LDL)-antioxidant activity. It inhibited the Cu2+-mediated oxidation of LDL (IC50: 5.3 microM) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH)-mediated oxidation of LDL (IC50: 2.8 microM) in the thiobarbituric acid-reactive substances (TBARS) assay as well as the macrophage-mediated LDL oxidation (73% inhibition at 5 microM). In addition, it delayed LDL oxidation with a prolonged lag time (192 min at 2 microM, control: 44 min). This compound also showed a 10-fold more potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity (IC50: 1.7 microM) than trolox (IC50: 18.6 microM), a known DPPH inhibitor. In addition, phellinsin A inhibited xanthine oxidase activity with an IC50 value of 31.0 microM, whereas allopurinol, a xanthine oxidase inhibitor, showed an IC50 value of 40.7 microM.

Wisteria floribunda gall extract inhibits cell migration in mouse B16F1 melanoma cells by regulating CD44 expression and GTP-RhoA activity.

Extracts from galls grown on Wisteria floribunda are used as an anti-tumoral preparation in oriental traditional medicine. Here, we investigated the molecular mechanism of this anti-tumoral effect by first examining whether the extract inhibited cell migration in a B16 cell-based wound healing assay. The gall extract delayed wound healing in a dose- and time-dependent manner, indicating that one or more components of the fraction inhibited cell migration. Examination of two molecules known to be involved in metastasis, CD44, and RhoA-GTP, revealed that the gall extract decreased CD44 expression in a concentration-dependent manner, and also increased RhoA-GTP activity in comparison to untreated controls. Taken together, these results suggest that the Wisteria gall extract may inhibit cancer cell migration via inhibition of CD44 mRNA expression and activation of the GTP-RhoA protein.

Anticancer activity of 3-O-acyl and alkyl-(-)-epicatechin derivatives.

By changing the structure or replacing the gallate group of (-)-ECG, 3-O-acyl and alkyl-(-)-epicatechin derivatives were synthesized to be screen as anticancer agents using the MTT assay in vitro against cancer cell lines (PC3, SKOV3, U373MG). 3-O-Acyl and alkyl-(-)-epicatechin derivatives (4-25) exhibited better anticancer activity than (-)-ECG and specially, compounds 6-8, 17-19, which were modified aliphatic chains with moderate sizes (C8-C12) showed strong anticancer activity (IC50=6.4-31.2 microM). The introduction of an alkyloxy group on 3-O-hydroxyl instead of an acyloxy group significantly enhanced inhibitory activity. Consequently, the compound that showed the most potency as anticancer agents were 3-O-decyl-(-)-epicatechin (18) (IC50=8.9, 7.9, 6.4 microM against PC3, SKOV3, U373MG, respectively), which modified the appropriate lipophilic group on the C-3 hydroxyl as an alkyloxy group.

Obovatols, new chitin synthase 2 inhibitors of Saccharomyces cerevisiae from Magnolia obovata.

In the course of the search for inhibitors of ScCHS2 from natural sources, we have isolated a new type of chitin synthase 2 inhibitor, obovatol, which has a biphenol skeleton, from Magnolia obovata. Obovatol inhibited chitin synthase 2 activity of Saccharomyces cerevisiae with an IC(50) of 38 microM. Its derivative, tetrahydroobovatol, inhibited chitin synthase 2 activity under the same conditions with an IC(50) of 59 microM. These compounds exhibited no inhibitory activity for ScCHS3, and showed less inhibitory activity for chitin synthase 1 than for chitin synthase 2 (IC(50) > 1 mM). These results indicated that obovatol and tetrahydroobovatol are specific inhibitors of ScCHS2. They also inhibited CaCHS1, which is structurally and functionally analogous to ScCHS2, with similar IC(50)s to ScCHS2 (IC(50) 28 and 51 microM, respectively). The compounds exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate [inhibition constant (K(i)) 21.8 microM for obovatol and 23.1 microM for tetrahydroobovatol]. Furthermore, they showed antifungal activities against various pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC 7.8 mg/L). The results indicate that obovatol and tetrahydroobovatol can potentially serve as antifungal agents.