Hibicuslide C-induced cell death in Candida albicans involves apoptosis mechanism.

AIM: To provide the observation that hibicuslide C-induced cell death in yeast Candida albicans involves apoptosis mechanism. METHODS AND RESULTS: Hibicuslide C was isolated from Abutilon theophrasti by column chromatography. In reactive oxygen species (ROS) assay, C. albicans treated with hibicuslide C showed increase in ROS, and its accumulation induced fungal cell death. In particular, hydroxyl radicals were a large part of the ROS. Mitochondrial dysfunction including mitochondrial depolarization and release of cytochrome c, which is a pro-apoptotic factor, was detected by JC-1 assay and Western blot. CaspACE FITC-VAD-FMK staining using caspase inhibitor showed metacaspase activation. Also, the increase in intracellular Ca(2+), which is a signal molecule of apoptosis, was detected by Fura-2AM and Rhod-2AM assays. Finally, annexin V-FITC and PI double staining and TUNEL assay confirmed that hibicuslide C induces early apoptosis followed by secondary necrosis in C. albicans. CONCLUSIONS: Hibicuslide C exerts antifungal activity against C. albicans through new mechanism inducing apoptosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida albicans is the common cause of nosocomial infections with high mortality. Our findings provide that hibicuslide C can be a model molecule that induces apoptosis in C. albicans.

Effects of myricetin on the bioavailability of carvedilol in rats.

CONTEXT: As an inhibitor of CYP2C9, CYP2D6 and P-gp, myricetin might affect the bioavailability of carvedilol when myricetin and carvedilol are used concomitantly for the prevention or therapy of cardiovascular diseases as a combination therapy. However, the effect of myricetin on the pharmacokinetics of carvedilol has not been reported in vivo. OBJECTIVE: This study investigated the effects of myricetin on the pharmacokinetics of carvedilol after oral or intravenous administration of carvedilol in rats. MATERIALS AND METHODS: Carvedilol was administered orally or intravenously with or without oral administration of myricetin to rats. RESULTS: The effects of myricetin on P-gp, CYP2C9 and 2D6 activity were evaluated. Myricetin inhibited CYP2C9 and CYP2D6 enzyme activity with IC50 of 13 and 57 µM, respectively. In addition, myricetin significantly enhanced the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. Compared with the control group, the AUC was significantly increased by 52.0-85.1%, and the C(max) was significantly increased by 93.1-133.4% in the presence of myricetin after oral administration of carvedilol. Consequently, the relative bioavailability of carvedilol was increased by 1.17- to 1.85-fold and the absolute bioavailability of carvedilol in the presence of myricetin was increased by 18.1-86.4%. T(max) was significantly decreased. DISCUSSION AND CONCLUSION: The enhanced oral bioavailability of carvedilol may result from both inhibition of CYP2C9 or CYP2D6-mediated metabolism and P-gp-mediated efflux of carvedilol in small intestine and/or in liver by myricetin rather than reducing renal elimination. Concomitant use of myricetin or myricetin-containing dietary supplements with carvedilol will require close monitoring for potential drug interactions.

Development of an in vitro assay system for screening of gp41 inhibitory compounds.

The transmembrane protein of HIV-1, gp41, mediates fusion between membranes of the virus and target cell. Strong interaction between the helical regions in the ectodomain of gp41 has been exploited to develop a method that can detect a potential inhibitor against gp41. The N-terminus coiled-coil or the C-terminus helical sequences within the ectodomain of gp41 were inserted into the C-terminus of thioredoxin (Trx) or glutathione S-transferase (GST) to generate the fusion proteins, Trx-N and GST-C, respectively. The inserted sequences of GST-C and Trx-N cause the two proteins to interact with each other and to form a complex. Furthermore, GST-C binds specifically to the surface-coated Trx-N, and the amount of attached GST-C is detected by an ELISA assay using anti-GST antibodies. Peptides derived from the helical regions of gp41 compete with GST-C for binding to Trx-N as well as prevent the gp41-mediated cell fusion. This in vitro assay system can be applied to screening compounds that have an inhibitory activity against gp41.

A new sulfated beta-galactan from clams with anti-HIV activity.

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.

Virus-cell fusion inhibitory activity for the polysaccharides from various Korean edible clams.

In order to find potent virus-cell fusion inhibitory components from Korean edible clams, thirteen prepared polysaccharides were introduced to syncytia formation inhibition assay, which is based on the interaction between the HIV-1 envelope protein gp120/41 and the cellular membrane protein CD4 of T lymphocytes. Among them, Meretrix petechialis showed a potent virus-cell fusion inhibitory activity. Fusion index (FI) and percent (%) fusion inhibition of the polysaccharide of this clam were 0.21 +/- 0.02, and 67.52 +/- 4.09 at 100 microg/ml, respectively. It exhibited almost equivalent virus-cell fusion inhibitory activity to that of dextran sulfate which was used as a standard control.

Anti-herpetic activity of various medicinal plant extracts.

In order to find antiviral compounds againstHerpes simplex virus type I (HSV-1) and II (HSV-2) from natural products, a convenient virus-induced cytopathic effect (CPE) inhibition assay was introduced. More than 300 fractions were prepared by solvent fractionation from sixty collected plants or purchased herbal medicines, and their anti-herpetic activities were evaluated. Among them, several medicinal plants showed potent anti-herpetic activity. Selective indexes (SI) of the EtOAc extract of Caraganae Radix (Caragana sinica) against HSV-1 and HSV-2 were more than 8.06 and 24.79, SI of the MeOH extract ofAcer okamotoanum leaves were 3.92 and 3.51, SI of the CH(2)Cl(2) extract of Veratri Rhizoma et Radix (Veratrum patulum) were 5.49 and 1.31 and SI of the MeOH extract of aerial part of Osmundae Rhizoma (Osmunda japonica) were more than 3.45 and 1.25, respectively.

HIV-1 integrase inhibitory phenylpropanoid glycosides from Clerodendron trichotomum.

Seven phenylpropanoid glycosides named acteoside (1), acteoside isomer (2), leucosceptoside A (3), plantainoside C (4), jionoside D (5), martynoside (6), and isomartynoside (7) were isolated from Clerodendron trichotomum. Compounds 1 and 2 showed potent inhibitory activities against HIV-1 integrase with IC50 values of 7.8 +/- 3.6 and 13.7 +/- 6.0 microM, respectively.

Amentoflavone inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB activation in macrophages.

Amentoflavone is a bi-flavonoid compound with anti-fungal and anti-inflammatory activities. We isolated amentoflavone from Selaginella tamariscina (Selaginellaceae) and studied its effects on nuclear factor-kappaB (NF-kappaB)-mediated inducible nitric oxide synthase (iNOS) gene expression in RAW 264.7 cells. Amentoflavone inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To clarify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of amentoflavone on the transactivation of iNOS gene by luciferase reporter activity using -1.59 kb flanking region. Amentoflavone potently suppressed the reporter gene activity. The LPS-induced activation of NF-kappaB was also found to be significantly blocked by amentoflavone, but AP-1 activation was unaffected. Furthermore, the nuclear translocation of p65 by LPS was inhibited by amentoflavone. NF-kappaB activation is controlled by the phosphorylation and subsequent degradation of I-kappaBalpha, and the cytosolic degradation of I-kappaBalpha was found to be inhibited by amentoflavone. These findings suggest that the inhibition of LPS-induced NO formation by amentoflavone is due to its inhibition of NF-kappaB by blocking I-kappaBalpha degradation, which may be the mechanistic basis of the anti-inflammatory effects of amentoflavone.

A new prenylated flavonol from the roots of Sophora flavescens.

A new prenylated flavonol, sophoflavescenol (1), together with five known flavonoids, kurarinol, kushenol K, kushenol H, trifolirhizin, and kuraidin, were isolated from the roots of Sophora flavescens. The structure of 1 was determined by spectroscopic analysis. Among the five known flavonoids, kurarinol, kushenol K, and kushenol H showed weak antiviral activity against Herpes simplex virus types I and II.

A cytotoxic pennogenin glycoside from Majanthemum dilatatum.

A cytotoxic pennogenin glycoside, pennogenin 3-O-alpha-L-rhamnopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->4)-[ alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (1) was isolated from the whole herb of Majanthemum dilatatum. Compound 1 showed potent cytotoxic activity against human cancer cell lines.

A new flavonol glycoside gallate ester from Acer okamotoanum and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase.

Bioassay-directed chromatographic fractionation of an ethyl acetate extract of the leaves of Acer okamotoanum using HIV-1 integrase afforded a new acylated flavonol glycoside, quercetin 3-O-(2",6"-O-digalloyl)-beta-D-galactopyranoside (1), together with six known flavonol glycosides and three known phenolic compounds. The structure of the new compound was determined by spectroscopic methods. The most active compounds were quercetin 3-O-(2"-galloyl)-alpha-L-arabinopyranoside (6) and 1, which exhibited IC50 values of 18.1 +/- 1.3 and 24.2 +/- 6.6 micrograms/mL, respectively, against HIV-1 integrase.

Inhibition of gp 120-CD4 interaction by various plant extracts.

Various assay methods have been developed to evaluate the effectiveness of substances against human immunodeficiency virus (HIV). One of them is the Syncytia formation inhibition assay, which is based on the inhibition of the interaction between the HIV-1 envelope protein gp 120 and the cellular membrane protein CD4. A variation of this assay using recombinant virus vPE 16 and CD4(+) HeLa cell was developed to find anti-HIV compounds in natural products that inhibit gp 120-CD4 binding. VPE 16 expresses glycoprotein gp 160, which is glycosylated then processed into gp 120 and gp 41 on its envelope. A total of 50 plant extracts were screened with this system. Extracts from Calicarpa japonica and Sedum sarmentosum were among those that showed strong inhibition of the gp 120-CD4 interaction.

7-feruloylloganin: an iridoid glucoside from stems of Lonicera insularis.

A new iridoid glucoside, 7-feruloylloganin (1), was isolated from stems of Lonicera insularis, along with six known lignans including (-)-pinoresinol, 9alpha-hydroxypinoresinol, balanophonin, erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy]-propane-1,3-diol, threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy]-propane-1,3-diol and buddlenol A. The structure of 1 was determined by analyses of 2D NMR (1H-1H COSY, HMQC and HMBC) and HRFABMS.

Synthesis and biological activities of C-2, N-9 substituted 6-benzylaminopurine derivatives as cyclin-dependent kinase inhibitor.

In this study, C-2, N-9 substituted 6-benzylaminopurine derivatives were synthesized and their inhibitory effects on cyclin-dependent kinase (CDK2) were evaluated. The effect of substituents at the C-2 and N-9 positions of substituted purine was investigated. Among the compounds tested, compound 7b-iii (6-benzylamino-2-thiomorpholinyl-9-isopropylpurine) was the most active inhibitor (IC50 = 0.9 microM). Compound 7b-iii showed 10-fold higher activity compared to olomoucine and almost the same activity as roscovitine. Results from structure-activity relationship studies should allow the design of more potent and selective CDK inhibitors, which may provide an effective therapy for cancer or other CDK dependent diseases.

Two interaction modes of the gp41-derived peptides with gp41 and their correlation with antimembrane fusion activity.

Peptides derived from gp41 effectively block the gp41-mediated cell fusion or HIV infection. A 36-mer (naDP178), 51-mer (C51) and 27-mer peptide (C27) from the membrane proximal region of gp41 have been examined their interaction modes with the coiled-coil motif of gp41 presented in thioredoxin (Trx-N) or the bacterially expressed ectodomain of gp41 (Ec-gp41ec). All of these peptides effectively inhibited the gp41-mediated membrane fusion, however, they showed distinct interaction modes with Ec-gp41ec or Trx-N. C51 peptide bound tightly to Trx-N, and it increased the solubility of Ec-gp41ec. naDP178 showed very weak binding affinity to Trx-N, however, it effectively solubilized Ec-gp41ec. In contrast, C27 peptide showed significant binding to Trx-N; however, it did not affect the solubility of Ec-gp41ec. These interaction modes of C-peptides were assumed to be related to their different inhibitory mechanism against gp41-mediated cell fusion.

Isolation of virus-cell fusion inhibitory components from Eugenia caryophyllata.

The fractionation of Eugenia caryophyllata (Myrtaceae) guided by the syncytia formation inhibition assay led to the isolation of four tannins (eugeniin, casuarictin, 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-beta-D-glucopyranose, and tellimagrandin I), and two chromones (biflorin and isobiflorin). Among the isolated compounds, tellimagrandin (4) showed a significantly high inhibitory activity on the syncytia formation with an IC50 value of 16.12 +/- 1.98 micrograms/ml.

Respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells.

Increased gelatinolytic activity was observed in respiratory syncytial virus (RSV)-infected HEp-2 cells by using zymography. The anti-matrix metalloproteinase-9 (MMP-9) antibody specifically reduced the gelatinolytic activity suggesting that the increased gelatinolytic activity was due to the MMP-9. It was also supported by the results from immunofluorescent staining, treatment of MMP inhibitors, and RSV infection of the cell clones that were transfected with plasmids to express more MMP-9 and tissue type inhibitor of metalloproteinase-1 (TIMP-1). The gelatinolytic activity of extracellular MMP-9 in RSV-infected HEp-2 cells increased 1.5 +/- 0.2 fold compared with the control (p < 0.01). Cell surface MMP-9 expression was also clearly detected by immunofluorescent staining. Treatment with 1,10-phenanthroline (0.05 mM), ethylenediamine-tetraacetate (EDTA) (1.5 mM), and penta-O-galloyl-beta-D-glucose (PGG) (3.3 microM) inhibited RSV multiplication as well as syncytia formation. Furthermore, the average syncytia size increased when the cells expressing more MMP-9 were infected by RSV. In contrast, syncytia formation was inhibited in the cells manipulated to express TIMP-1. Thus, this study concludes that although RSV infection induces MMP-9, which can enhance the syncytia formation leading to RSV multiplication and spread it can be inhibited by MMP inhibitors.