Iloneoside, an antimalarial pregnane glycoside isolated from Gongronema latifolium leaf, potentiates the activity of chloroquine against multidrug resistant Plasmodium falciparum.

The rapid spread of drug resistant malaria parasites has necessitated the search for novel antimalarials and chemosensitizers capable of reversing drug resistance in the parasites. A number of studies have revealed the resistance reversal activities of pregnane glycosides and the antimalarial activity of a pregnane glycoside obtained from Gongronema species. However, the pregnane (2) and pregnane glycosides (1, 3-4) isolated from Gongronema latifolium leaf have not been evaluated for these activities. This study was therefore carried out to evaluate the antiplasmodial and chloroquine resistance reversal activities of a pregnane and three pregnane glycosides isolated from G. latifolium leaf in vitro. The compounds were evaluated for their inhibitory activities against P. falciparum 3D7 (a chloroquine-sensitive strain) and P. falciparum W2 (a chloroquine-resistant clone) in vitro. The activities of chloroquine in separate combination with each of the compounds against P. falciparum W2 were also evaluated. Moreover, the interaction of the active compounds (1 and 4) with selected P. falciparum proteins (PfProteins) were evaluated in silico. The results revealed that only 1 and 4 were active against P. falciparum 3D7 and P. falciparum W2. Also, 2 and 3 did not exhibit chloroquine resistance reversal activity. Activity of chloroquine against P. falciparum W2 was potentiated by 1 by 3200% at concentrations higher than 0.625 µg/mL. Also, 1 and 4 demonstrated similar binding patterns and higher binding tendencies to the selected PfProteins compared to chloroquine. Thus, 1 (iloneoside) is an antimalarial pregnane glycoside which can potentiate the activity of chloroquine against multidrug resistant P. falciparum.

Acute and Subchronic Oral Toxicity Study of Polyherbal Formulation Containing Allium sativum L., Terminalia bellirica (Gaertn.) Roxb., Curcuma aeruginosa Roxb., and Amomum compactum Sol. ex. Maton in Rats.

The polyherbal formulation containing Allium sativum L., Terminalia bellirica (Gaertn.) Roxb., Curcuma aeruginosa Roxb., and Amomum compactum Sol ex. Maton has been used for hypertension treatment empirically. Our previous study showed its blood pressure-lowering effect on a rat model of hypertension. However, toxicity data were not available for this polyherbal formulation. This study is aimed at evaluating the acute and subchronic oral toxicity of the polyherbal formulation in rats. The acute toxicity study was conducted on 6 female Wistar rats using the fixed-dose method for the treatment group and 5 female Wistar rats for the control. The single dose of 2,000 mg/kg of the polyherbal formulation was given orally. There were no significant toxic effects and no death observed until the end of the study, and it was showed that the lethal dose 50% (LD50) of the polyherbal formulation was estimated to be more than 2,000 mg/kg. The macroscopic and microscopic examination of vital organs showed no symptoms of toxicity. At the subchronic toxicity study, the polyherbal formulation with 3 dose variations of 252 mg/kg, 1,008 mg/kg, and 4,032 mg/kg was administered for 91 days orally. The lowest dose of 252 mg/kg is equivalent to the daily recommended dose for a human. There were no significant toxic effects observed at all doses on physical sign and symptoms, weight gain, food intake, hematological parameters, biochemical parameters, and macroscopic and microscopic examination of organs. These findings showed that the short- and long-term oral administration of the polyherbal formulation is safe to use within its dose recommendation.

Antioxidant effects of the water-soluble fraction of baked sponge cake made with silky fowl egg: comparison with White Leghorn egg.

1. The antioxidant effects of the water-soluble fraction of baked sponge cakes made with silky fowl eggs and White Leghorn eggs were studied. The mechanism of the antioxidant effect was also investigated. 2. The antioxidant effect on the oxidation of linoleic acid increased in the water-soluble fraction of cake made with silky eggs. In contrast, Leghorn eggs significantly decreased the rate of antioxidant activity. The browning index of the water-soluble fraction of baked sponge cake made with silky fowl eggs changed from 0.052 to 1.240 after 20 min at 180 degrees C, while that made with Leghorn eggs changed from 0.037 to 0.710. 3. There are correlations between the rate of browning index and antioxidant activity. Superoxide anion (O2(-)) and hydrogen peroxide (H(2)O(2)) in water-soluble fractions of baked sponge cakes made with silky fowl eggs and hen's eggs were formed during light exposure for 20 min at 10,000 lux, and their formation could be significantly inhibited by the addition of tryptophan or mannitol, scavengers of hydroxyl radicals (*OH). These results were strong evidence of direct participation of *OH, formed by the Haber-Weiss reaction, in the water-soluble fraction of baked sponge cakes. The rate of decrease in active oxygen by scavengers decreased in Leghorn eggs more efficiently than in silky eggs. 4. The present experiments suggested that the use of silky fowl eggs could improve the quality and oxidative stability of baked cakes.

Distribution of N-acetylneuraminic acid and sialylglycan in eggs of the Silky fowl.

1. The distribution of sialic acid in the eggs of original Silky fowl was investigated. The sialic acid contents of the yolk, albumen and the chalaza of a single egg were 205.2, 11.96 and 0.83 mg, respectively. 2. The sialic acid content of the yolk of Silky eggs was 11.5-fold higher than that of a conventional domestic fowl yolk. 3. Sialic acid isolated from Silky yolk was entirely N-acetylneuraminic acid (NeuAc). No N-glycolylneuraminic acid or O-acetyl containing sialic acid was observed. 4. The structure of the major sialylglycan in Silky egg yolk was determined to be a disialyl-biantennary chain in which the NeuAc residues were alpha2-6 linked to glucose. No alpha2-3 linkage was observed. 5. Thus, the Silky fowl's egg provides an excellent source of NeuAc and sialylglycan.

Preparation of 1,4-oxaselenin from agNO(3)/LDA-assisted reaction of 3-selena-4-pentyn-1-one as potential antitumor agents.

1,4-Oxaselenins were synthesized from 3-selena-4-pentyn-1-ones by the use of AgNO(3) and LDA. One of the obtained oxaselenins, 2-(4-chlorophenyl)-6-phenyl-1,4-oxaselenin 5c, showed an inhibitory effect against the proliferation of human cancer cells and inducing effects on the early stage of apoptosis.

X-ray diffraction and high-resolution NMR spectroscopic analysis of 2,4,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosono-1,5-lactone.

The X-ray diffraction and high-resolution 1H and 13C NMR spectral data are reported for 2,4,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosono-1,5-lactone.

Stereoselective synthesis of the alpha-glycoside of a KDO "C"-disaccharide.

The reaction of tert-butyl (4,5,7, 8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl chloride)onate donor 7 with the 6-formylgalactopyranoside acceptor 4 in the presence of SmI(2) provided only the KDO alpha-C-disaccharide 8. The bulky tert-butyl ester in the donor was used to reverse the stereochemical outcome of C-glycosylation, stereoselectively forming the alpha-"C"-disaccharide of KDO.

Novel compounds, '1,3-selenazine derivatives' as specific inhibitors of eukaryotic elongation factor-2 kinase.

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1, 3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC(50)=0.36 microM) and TS-4 (IC(50)=0.31 microM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC(50)=4.7 microM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.

Identification of L-inositol and scyllitol and their distribution in various organs in chrysanthemum.

Two unidentified soluble carbohydrates were isolated from chrysanthemum (Dendranthema x grandiflorum (Ramat.) Kitamura) leaves using HPLC. The compounds were identified as 1 L-chiro-inositol, called L-inositol (1) and scyllo-inositol, called scyllitol (2) from the results of 1H-NMR, 13C-NMR, and CI-MS spectra. L-Inositol and scyllitol were distributed in four cultivars tested. L-Inositol concentration of petals gradually decreased during the flower bud development, but the L-inositol content increased by about 7 times. Scyllitol was detected only at an early stage of flower bud.

Induction of apoptosis in human gastric adenocarcinoma cells by two novel organoselenium compounds, TS-2 and TS-6.

Two new organoselenium compounds, 4-ethyl-4-hydroxy-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine (TS-2) and 4-hydroxy-4-methyl-6-propyl-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine++ + (TS-6) were investigated for their inhibitory effect on the growth of 8 human tumor cell lines, including stomach, lung, prostate and colon cancer cell lines, in vitro. Both TS-2 and TS-6 exhibited the strongest cytotoxicity against a gastric adenocarcinoma (TMK-1) among 8 human tumor cell lines, and their IC50 were 2.38 microM and 2.78 microM, respectively. Twenty-four-h exposure of TMK-1 cells to TS-2 or TS-6 (0.1-100 microM) in the absence of serum led to a concentration-dependent increase of cytotoxicity. Exposure of TMK-1 cells to TS-2 or TS-6 (5, 10 or 20 microM) in the presence of 5% serum resulted in significant inhibition of TMK-1 cell proliferation in a concentration- and time-dependent manner. Morphological changes including shrinkage of the nucleus, and DNA ladder fragmentation, which are considered to be the typical features of apoptosis, were observed in TMK-1 cells in response to TS-2 or TS-6. Furthermore, the caspase-3 activity in TMK-1 cells treated with TS-2 or TS-6 was also found to be increased in a time-dependent manner, in parallel with the induction of DNA fragmentation. Taken together, the results of the present study suggest that the inhibition of growth of TMK-1 cells by TS-2 and TS-6 is mediated by the induction of apoptosis.

1,3-Selenazine derivatives induce cytotoxicity and DNA fragmentation in human HT-1080 fibrosarcoma cells.

The inhibitory effects of a series of 5,6-dihydro-4H-1,3-selenazine derivatives, 1,3-selenazole, and 5,6-dihydro-4H-1,3-thiazine derivatives on the proliferation of human HT-1080 fibrosarcoma cells were investigated. The compounds 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-4-methyl-6-propyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine++ + (TS-6) exhibited the strongest inhibitory effect among 1, 3-selenazine derivatives, and the EC(50) of TS-2 and TS-6 was 7.76 and 8.40 microM, respectively. On the other hand, 1,3-selenazole and 5,6-dihydro-4H-1,3-thiazines had no inhibitory effects. TS-2 and TS-6 inhibited the proliferation of HT-1080 cells time- and dose-dependently. They induced dose-dependent DNA fragmentation in HT-1080 cells, revealing a typical apoptosis characteristics. The present study demonstrated that TS-2 and TS-6 inhibited HT-1080 proliferation through the induction of DNA fragmentation.

Novel compounds, 1,3-selenazine derivatives, as antibacterial agents against Escherichia coli and Staphylococcus aureus.

The antibacterial activities of several kinds of novel 4H-5,6-dihydro-1,3-selenazine derivatives against Escherichia coli and Staphylococcus aureus were investigated. 4-Hydroxy-4-methyl-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine (TS-1), 4-ethyl-4-hydroxy-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine (TS-2), and 4-hydroxy-4-methyl-2-pentyl-4H-5,6-dihydro-1,3-selenazine (PS-1) exhibited strong inhibitory activity against E. coli, and TS-1, TS-2, PS-1, 4-hydroxy-4-methyl-2-pentyl-6-propyl-4H-5,6-dihydro-1,3-selenazine (PS-6) and 4-hydroxy-4-methyl-2-pentyl-6-phenyl-4H-5,6-dihydro-1,3-selenazine (PS-8) also showed inhibitory activity against S. aureus. 4H-5,6-dihydro-1,3-thiazines and 1,3-selenazole had no inhibitory activities against both bacteria. TS-1, TS-2, and PS-1 exhibited marked antibacterial activities against both Gram-negative and Gram-positive bacteria.

Comparison of egg-yolk protein hydrolysate and soyabean protein hydrolysate in terms of nitrogen utilization.

Egg-yolk protein hydrolysate (YPp) is an alternative protein source in formulas for infants with intolerance to cow's milk or soyabean protein, or for patients with intestinal disorders. However, the nutritional value of YPp has never been investigated. YPp was prepared by enzymic hydrolysis of delipidated yolk protein, which led to an average peptide length of 2.6 residues. Three experiments were performed. In Expt 1, we compared the intestinal absorption rate of YPp and soyabean protein hydrolysate (SPp) in rats. YPp and SPp solutions were injected into the duodenum of anaesthetized rats and blood samples were taken from the portal vein at 7, 15, 30, 60, and 120 min. A higher amino acid concentration in the serum of the YPp group demonstrated that YPp was absorbed faster than SPp. In Expt 2, the effects of dietary YPp and SPp on body-weight gain, protein efficiency ratio (PER) and feed efficiency ratio (FER) were determined. At the end of the experiment, body weight had increased in both groups, while PER and FER were significantly higher in rats fed on YPp. In Expt 3, to investigate the effects of dietary YPp and SPp on N metabolism, we determined the biological value and net protein utilization. Yolk protein was the reference protein. Biological value and net protein utilization values were very similar between animals fed on yolk protein and YPp diets, and significantly higher than in rats fed on the SPp diet. The present findings demonstrate that there is no adverse effect of hydrolysis of yolk protein on N utilization, and that the nutritive value of YPp is similar to that of yolk protein and superior to that of SPp.

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent.

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.

Occurence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.

Identification of Methyl ß-Glucopyranoside and Xylose as Soluble Sugar Constituents in Roses (Rosa hybrida L.).

Two unidentified sugars were isolated from rose petals using HPLC. The isolated compounds were identified as methyl ß-glucopyranoside and xylose using (1)H-NMR, (13)C-NMR, and GC-MS. Methyl ß-glucopyranoside and xylose were distributed in three cultivars tested relatively in large amounts. These results indicate that methyl ß-glucopyranoside and xylose occur universally as soluble sugar constituents in roses.