Synthesis and antiviral activity of 7-O-(omega-substituted)-alkyl-3-O-methylquercetin derivatives.

3',4'-Di-O-benzyl-3-O-methylquercetin (2), the precursor in the synthesis of an important antivirally active flavone 3-O-methylquercetin (1), was regioselectively alkylated at the 7-OH position by a series of 1,omega-dihaloalkanes and omega-bromoalkanols. Dimerization of the flavone had to be avoided by applying strict reaction conditions. Subsequent debenzylation was carried out by catalytic transfer hydrogenolysis, affording quantitatively the 7-O-(omega-haloalkyl)-3-O-methylquercetin (11-14) and 7-O-(omega-hydroxyalkyl)-3-O-methylquercetin derivatives (15, 16). All compounds were tested for their antiviral activity against poliomyelitis- and HIV-viruses.

Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata.

From a bioassay-guided fractionation procedure using Herpes simplex virus type 1 as the target model, a virucidal saponin mixture (maesasaponin mixture B) was isolated from the MeOH extract of leaves of Maesa lanceolata. The maesasaponin mixture B consisted of six homologous oleanane-type triterpenoid saponins 1-6, identified by 1H, 13C, and 2D NMR spectroscopy and mass spectrometry. The maesasaponin mixture B (1-6) showed several biological activities expected for saponins. It exhibited a moderate virucidal activity against enveloped viruses. The maesasaponin mixture B (1-6) was tested for further properties. The saponin mixture was found to be highly hemolytic and molluscicidal: it hemolyzed 50% of human erythrocytes (1% suspension) at a concentration estimated at 1.6 microg/mL and exerted against Biomphalaria glabrata snails a severe toxic effect with LD95 and LD50 values of 4.1 and 2.3 ppm, respectively. In addition, the maesasaponin mixture B (1-6) showed moderate fungistatic and antimutagenic properties. The evaluation of these diverse activities is described.

Supplementation of calves with stabilized orthosilicic acid. Effect on the Si, Ca, Mg, and P concentrations in serum and the collagen concentration in skin and cartilage.

The bioavailability of silicon in stabilized orthosilicic acid was investigated in a double blind, placebo controlled supplementation study of calves maintained on a normal diet. The total dietary Si intake was increased by 4.9% in the form of stabilized orthosilicic acid. After 23 wk of Si supplementation, the serum Si concentration increased (p = 0.0001, n = 29) by 70% compared to control animals in spite of the low Si dose administered and the Si adequate diet. The individually administered Si dose was significantly associated with the serum Si concentration (r = 0.44, p = 0.016, n = 29). The collagen concentration in dermis was significantly higher (p = 0.019, n = 4) in the Si group and a positive correlation (r = 0.72, p = 0.018, n = 9) was found between the Si concentration in serum and the collagen concentration in cartilage. The calcium (Ca) and phosphorus (P) concentrations in serum were marginally higher for animals supplemented with Si compared to control animals. In serum, a significant linear relationship was found between the Si and the Ca concentration (r = 0.31, p = 0.019, n = 59), whereas the magnesium concentration correlated marginally with the Si concentration (r = 0.25, p = 0.068, n = 59). In summary, increasing the total dietary Si intake by 4.9% in the form of stabilized orthosilicic acid resulted in a 70% higher Si concentration in serum indicating a high bioavailability of Si in this supplement. The positive correlation between the serum Si concentration and the collagen concentration in cartilage and the serum Ca concentration, respectively, suggest the involvement of Si both in the formation of extracellular matrix components and in Ca metabolism.

Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects.

Phenylketonuric (PKU) subjects have a limited supply of selenium (Se) in their phenylalanine-restricted diet. A Se repletion (1 microgram Se/kg/day)/depletion study was conducted in PKU children to determine the effect of Se on thyroid function parameters. The initial plasma Se concentration (mean +/- SD: 0.26 +/- 0.12 mumol/L, p < 0.00003, n = 10) and glutathione peroxidase (GSH-Px) activity (140 +/- 58 U/L, p < 0.00003, n = 10) were significantly lower compared to age-matched controls. After 14 weeks of supplementation, the plasma Se concentration (mean +/- SD: 0.74 +/- 0.20 mumol/L) normalized (normal range: 0.57-1.15 mumol/L, mean +/- SD: 0.76 +/- 0.13 mumol/L, n = 32) and remained stable thereafter during repletion. Plasma GSH-Px activity reached normal values after 18 weeks of supplementation (312 +/- 57 U/L; normal range: 238-492 U/L, mean +/- SD: 345 +/- 54 U/L, n = 32) and increased significantly for up to eight weeks thereafter (332 +/- 52 U/L). Individual and mean thyroid parameters were initially normal in all cases. The mean concentrations of plasma thyroxine (T4: p < 0.025), free T4 (FT4: p < 0.01) and reverse triiodothyronine (rT3: p < 0.005) decreased to 75% of their initial value within three weeks of Se supplementation and remained stable thereafter, within a normal physiological range during selenium supplementation. They increased back to their initial values three weeks (T4: p < 0.05, FT4: p < 0.05) and six weeks (rT3: p < 0.025) respectively, after the end of the supplementation. In conclusion, Se supplementation modifies thyroid function parameters in Se-deficient PKU subjects most likely by an increase in activity of type I 5'-deiodinase (5'-DIase I).

Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants.

Selected plants having a history of use in Polynesian traditional medicine for the treatment of infectious disease were investigated for anti-viral, anti-fungal and anti-bacterial activity in vitro. Extracts from Scaevola sericea, Psychotria hawaiiensis, Pipturus albidus and Eugenia malaccensis showed selective anti-viral activity against Herpes Simplex Virus-1 and 2 and Vesicular Stomatitis Virus. Aleurites moluccana extracts showed anti-bacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, while Pipturus albidus and Eugenia malaccensis extracts showed growth inhibition of Staphylococcus aureus and Streptococcus pyogenes. Psychotria hawaiiensis and Solanum niger inhibited growth of the fungi Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum, while Ipomoea sp., Pipturus albidus, Scaevola sericea, Eugenia malaccensis, Piper methysticum, Barringtonia asiatica and Adansonia digitata extracts showed anti-fungal activity to a lesser extent. Eugenia malaccensis was also found to inhibit the classical pathway of complement suggesting that an immunological basis for its in vivo activity was identified. This study has confirmed some of the ethnobotanical reports of Hawaiian medicinal plants having curative properties against infections using biological assays in vitro.

Isoenzymic patterns of tyrosinase in the rabbit choroid and retina/retinal pigment epithelium.

Tyrosinase in crude extracts from the adult rabbit choroid and retina/retinal pigment epithelium was found to be differently affected by the inhibitors sodium metabisulfite, cyanide, diethyldithiocarbamate and 2,2'-dipyridyl:the latter inhibited the activity in the retina/retinal pigment epithelium extract after 24 hr of incubation, but not that in the choroid. The first three inhibitors, on the other hand, inhibited the activity in the choroid extract, but not in the retina/retinal pigment epithelium extract, after 3 hr of incubation. Both extracts contained one insoluble and two soluble isoenzymes. The mobilities and the apparent molecular weights of the soluble isoenzymes (more markedly so for the most rapidly migrating) differed, as revealed by polyacrylamide gel electrophoresis, resulting in distinct isoenzymic patterns. We suggest that such patterns may be related to the characteristic melanogenic features of the retina/retinal pigment epithelium and choroid.

Structure-activity relationship of flavonoids with superoxide scavenging activity.

The superoxide scavenging activities of 12 flavonoids were measured. The superoxide anions were generated by a hypoxanthine-xanthine oxidase system and measured by the nitrite method. The results showed that the scavenging ability enhanced with an increasing number of hydroxyl groups in ring B. Substitution at C3 position with a hydroxyl group increased the activity. Compared to a methoxyl group or a glycoside in this position, a free hydroxyl group showed the highest activity. A saturated C2-C3 bond showed a higher activity than a unsaturated bond. The absence of a carbonyl group at C4 position increased the activity.

Seleno-lactobacillus. An organic selenium source.

Lactic acid bacteria are nonpathogenic bacteria commonly used in food processing. An evaluation was made of the capacity to concentrate selenium in species of Lactobacillus. A selenium concentration of 1 microgram/mL in the culture medium yielded in a bacterial content of 400 micrograms/g dry biomass. Dialysis and TCA precipitation experiments of a native intracellular extract proved that at least 80% of the total selenium is associated with organic molecules. Seleno-cysteine was identified as the only seleno-amino acid present in the intracellular selenoproteins. This study shows that species of the lactic acid bacteria are able to concentrate selenium intracellular as seleno-cysteine, which could be applied in supplementation studies.

Can ethnopharmacology contribute to the development of antiviral drugs?

In recent years, many compounds having potent antiviral activity in cell cultures and in experimental animals have been detected, but only a few have been approved by Western health authorities for clinical use. Nevertheless, some of these compounds are currently undergoing either preclinical or clinical evaluation, and perspectives for finding new interesting antiviral drugs are promising. Among these antiviral substances are several natural compounds isolated from plants used in traditional medicine including polysaccharides, flavonoids, terpenes, alkaloids, phenolics and amino acids. Some of these plant compounds exhibit a unique antiviral mechanism of action and are good candidates for further clinical research. What follows is a brief summary of the selection methods of plants for antiviral screening and in vitro and in vivo assays, which are currently used for detecting this activity in plant extracts. The importance of the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on plant-derived antirhinovirus and anti-HIV agents.

4'-Hydroxy-3-methoxyflavones with potent antipicornavirus activity.

4'-Hydroxy-3-methoxyflavones are natural compounds with known antiviral activities against picornaviruses such as poliomyelitis and rhinoviruses. In order to establish a structure-activity relationship a series of analogues were synthesized, and their antiviral activities and cytotoxicities were compared with those of flavones from natural origin. The 4'-hydroxyl and 3-methoxyl groups, a substitution in the 5 position and a polysubstituted A ring appeared to be essential requirements for a high activity. The most interesting compound was 4',7-dihydroxy-3-methoxy-5,6-dimethylflavone possessing in vitro TI99 values of greater than 1000 and greater than 200 against poliovirus type 1 and rhinovirus type 15, respectively. This compound was also active against other rhinovirus serotypes (2, 9, 14, 29, 39, 41, 59, 63, 70, 85, and 89) tested, having MIC50 values ranging from 0.016 to 0.5 micrograms/mL. Finally in contrast to quercetin it showed to be not mutagenic in concentrations up to 2.5 mg in the Ames test.

Plant antiviral agents, VI. Isolation of antiviral phenolic glucosides from Populus cultivar Beaupre by droplet counter-current chromatography.

Bioassay-guided isolation of the 80% EtOH extract of the leaves of Populus cultivar Beaupré by means of dccc afforded four known phenolic glucosides. Salicin and salireposide were shown to be, at least partially, responsible for the antiviral activity of the extract against poliomyelitis and Semliki forest viruses.

Inhibition of the multiplication of enveloped and non-enveloped viruses by glucosamine.

Glucosamine can inhibit the development of viral cytopathogenic effect and the production of infective viral particles of both enveloped and non-enveloped viruses. The extent of antiviral activity is dependent on drug concentration, composition of the culture medium and type of cell host.

Selenium and the growth of Haemophilus ducreyi.

One of the growth media in current use for Haemophilus ducreyi comprises Mueller Hinton agar, chocolatised horse blood, serum and IsoVitalex (BBL). For a better understanding of growth factors, attempts were made to simplify this complex medium. The horse blood was replaced by haemin (200 micrograms/ml), the serum by albumin (0.2%), and IsoVitalex was substituted only by L-glutamine 0.01%. Most of the strains grew, but when selenium ions were added in a concentration of 3.25 x 10(-3) micrograms/ml, growth was stimulated and became more luxuriant than growth on conventional media.

The poliovirus-induced shut-off of cellular protein synthesis persists in the presence of 3-methylquercetin, a flavonoid which blocks viral protein and RNA synthesis.

In poliovirus-infected cells, the viral protein and RNA synthesis were severely reduced, provided 3-methylquercetin was present between 1 and 2 h post-infection. Under these conditions, the virally induced host shut-off remained in effect. On the other hand, in uninfected HeLa cells, protein and RNA synthesis was inhibited only slightly by 3-methylquercetin. The inhibition of poliovirus cytopathogenicity in Vero cells by 3-methylquercetin exhibited a similar time dependence.

Lycorine: a eukaryotic termination inhibitor?

The effect of the alkaloid lycorine on viral protein synthesis was studied in poliovirus-infected HeLa cells. The incorporation of [3H]leucine was inhibited by lycorine in a dose-dependent way, although lycorine never completely abolished translation. Using polyacrylamide gel electrophoresis, the viral proteins were identified as derived from the P1 (5' terminal), P2 (middle), or P3 (3' terminal) region of the poliovirus translation unit. The residual labeling of viral proteins in the presence of lycorine was mainly due to synthesis of P1 proteins and slightly less to P2 proteins, while virtually no P3-derived proteins were made. It is suggested that lycorine may act at the level of termination.

Typing Haemophilus ducreyi by indirect immunofluorescence assay.

Rabbits were inoculated with untreated whole cells of 16 strains of Haemophilus ducreyi. Homologous titres in an indirect immunofluorescence test ranged from 1/320 to 1/1280. No cross reactions, or very few, were observed between antisera to these strains and 22 other (possible taxonomically related) species, strains, and genera. A large diversity in types of antigen was observed among the strains of H ducreyi. Antiserum from two antigens (35000 and 3138), however, reacted with all 16 H ducreyi antigens tested. At least nine of the strains of H ducreyi examined could be identified as determining the type of antigen.