In addition to membrane injury, an affinity for melanin might be involved in the high sensitivity of human melanoma cells to hederacolchiside A1.

We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells.

In vitro anti-inflammatory and anti-proliferative activity of sulfolipids from the red alga Porphyridium cruentum.

A sulfoglycolipidic fraction (SF) isolated from the red microalga Porphyridium cruentum was analyzed for fatty acid composition and assayed for ability to inhibit, in vitro, the generation of superoxide anion in primed leucocytes and the proliferation of a panel of human cancer cell-lines. Results demonstrated that SF contained large amounts of palmitic acid (26.1%), arachidonic acid (C20: 4 omega-6, 36.8%), and eicopentaenoic (C20:5 omega-3, 16.6%) acids, and noticeable amounts of 16:1n-9 fatty acid (10.5%). It strongly inhibited both the production of superoxide anion generated by peritoneal leukocytes primed with phorbol myristate acetate (IC(50): 29.5 microg/mL), and the growth of human colon adenocarcinoma DLD-1 and to a lesser extent of human breast adenocarcinoma MCF-7, human prostate adenocarcinoma PC-3, and human malignant melanoma M4 Beu cell-lines, and therefore might have a chemopreventive or chemotherapeutic potential, or both. It was found markedly more cytotoxic than sulfoquinovosyldiacylglycerols from plant used as a standard (STD), due to a stronger ability to inhibit DNA alpha-polymerase (IC(50): 378 microg/mL, vs 1784 microg/mL for STD). After a 48-h continuous treatment, IC(50) values for growth inhibition were in the range of 20-46 microg/mL instead of 94 to >250 microg/mL for STD, and those for inhibition of metabolic activity were in the range of 34-87 microg/mL instead of >250 microg/mL for STD. The higher anti-proliferative effect was observed on colon adenocarcinoma DLD-1 cells, and the weaker effect was observed on breast adenocarcinoma MCF-7.

Evaluation of the dietetic and therapeutic potential of a high molecular weight hydroxycinnamate-derived polymer from Symphytum asperum Lepech. Regarding its antioxidant, antilipoperoxidant, antiinflammatory, and cytotoxic properties.

A water-soluble hydroxycinnamate-derived polymer (>1000 kDa) from Symphytum asperum Lepech. (Boraginaceae) strongly reduced the diphenylpicrylhydrazyl radical (IC(50) approximately 0.7 microg/mL) and inhibited the nonenzymatic lipid peroxidation of bovine brain extracts (IC(50) approximately 10 ng). This polymer exhibited only a low hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50) > 100 microg/mL) but strongly decreased superoxide anion generation in either the reaction of phenazine methosulfate with NADH and molecular oxygen (IC(50) approximately 13.4 microg/mL) or in rat PMA-activated leukocytes (IC(50) approximately 5 microg/mL). The ability to inhibit both degranulation of azurophil granules and superoxide generation in primed leukocytes indicates that the NADPH oxidase responsible for this later effect is inhibited, pointing to the Symphytum asperum polymer as a potent antiinflammatory and vasoprotective agent. At all concentrations tested (0-200 microg/mL), we observed no cytotoxicity on normal human fibroblasts and neither antiproliferative effects nor proliferation activation on neoplastic cells.

The production of pyrethrins by plant cell and tissue cultures of Chrysanthemum cinerariaefolium and Tagetes species.

Pyrethrins, the most economically important natural insecticide, comprise a group of six closely related monoterpene esters. The industrial production is based on their extraction from Chrysanthemum cinerariaefolium (Pyrethrum) capitula. The world production of natural pyrethrins still falls short of global market demand stimulating the research in in vitro production as an alternative to conventional cultivation methods. The different biotechnological alternatives such as callus cultures, shoot and root cultures, plant cell suspension cultures, and bioconversion of precursors by means of enzymatic synthesis or genetically engineered microorganisms, as well as the progress achieved in methods for the identification and quantitation of insecticidal compounds have been reviewed. Although technology for plant cell culture exists, industrial applications have, to date, been limited due to both the low economical viability and technological feasibility at large scale. Bioconversion of readily available precursors looks more attractive, but more research is needed before this technology is used for the industrial production of pyrethrins.

Non-digestible oligosaccharides used as prebiotic agents: mode of production and beneficial effects on animal and human health.

Prebiotic agents are food ingredients that are potentially beneficial to the health of consumers. The main commercial prebiotic agents consist of oligosaccharides and dietary fibres (mainly inulin). They are essentially obtained by one of three processes: 1) the direct extraction of natural polysaccharides from plants; 2) the controlled hydrolysis of such natural polysaccharides; 3) enzymatic synthesis, using hydrolases and/or glycosyl transferases. Both of these enzyme types catalyse transglycosylation reactions, allowing synthesis of small molecular weight synthetic oligosaccharides from mono- and disaccharides. Presently, in Europe, inulin-type fructans, characterised by the presence of fructosyl units bound to the beta-2,1 position of sucrose, are considered as one of the carbohydrate prebiotic references. Prebiotics escape enzymatic digestion in the upper gastrointestinal tract and enter the caecum without change to their structure. None are excreted in the stools, indicating that they are fermented by colonic flora so as to give a mixture of short-chain fatty acids (acetate, propionate and butyrate), L-lactate, carbon dioxide and hydrogen. By stimulating bifidobacteria, they may have the following implications for health: 1) potential protective effects against colorectal cancer and infectious bowel diseases by inhibiting putrefactive bacteria (Clostridium perfringens ) and pathogen bacteria (Escherichia coli, Salmonella, Listeria and Shigella ), respectively; 2) improvement of glucid and lipid metabolisms; 3) fibre-like properties by decreasing the renal nitrogen excretion; 4) improvement in the bioavailability of essential minerals; and 5) low cariogenic factor. These potential beneficial effects have been largely studied in animals but have not really been proven in humans. The development of a second generation of oligosaccharides and the putative implication of a complex bacterial trophic chain in the intestinal prebiotic fermentation process are also discussed.

Rapid mass propagation of Chrysanthemum cinerariaefolium Vis. by callus culture and ability to synthesise pyrethrins.

Rapid mass propagation of Chrysanthemum cinerariaefolium from young flower heads was developed to compare the ability of callus, in vitro shoots and rooted plants, and original plants to synthesize pyrethrins. The ability to synthesise all six pyrethrin components increased with differentiation. Jasmolin II and cinerin II were the main products present in mother plant shoots, whereas pyrethrin I was the greater component present in callus and in vitro plants. Clonal propagation increased the pyrethrin I content compared to that of plant shoots and young flowers. Total pyrethrin content was the same in in vitro and plant shoots, but lower in these shoots than in young flowers. The pyrethrin I/pyrethrin II ratio, which is directly related to insecticidal activity, varied from 3.4 in in vitro shoots to 0.87 in mother plant shoots and young flowers.

[Synthesis of novel fructo-oligosaccharides (FOS) by enzymatic reaction]. / Synthèse de noveaux fructo-oligosaccharides (FOS) par voie enzymatique.

Fructo-oligosaccharides (FOS) are new food ingredients that are able to beneficially affect the host by selectively stimulating the growth and/or activity of colonic bifidobacteria (concept of prebiotics). A commercial enzyme preparation was found to possess a high fructosyltransferase activity and could be used as a biocatalyst for the industrial production of FOS from sucrose. Under optimum conditions (pH: 5.5, temperature: 55 degrees C and 7 units of fructosyltransferase activity per gram sucrose), in presence of glucose (competitive inhibitor) the actual yield reached the theoretical value (up to 50%). Actually, FOS that are commercially available for their prebiotic properties belong to inulin type with low degree of polymerisation (DP:3 to 10). Our FOS were identified by both HPLC and 13C-NMR spectrometry as neo-FOS type (neo-kestose, neo-nystose and neo-fructofuranosylnystose), a new structure which is very close to inulin type (same linkage between fructosyl units). The neo-FOS may act as a prebiotic factor due to their structural similarity with inulin type.

Cryopreservation of Chrysanthemum cinerariaefolium Vis. cells and its impact on their pyrethrin biosynthesis ability.

An efficient protocol for cryogenic storage of high-pyrethrin-producing cell lines of Chrysanthemum cinerariaefolium is described. Optimal survival (92%) was obtained with cells precultured in 1/2 Murashige and Skoog nutrient medium containing 180 g ⋅ l-1 sucrose for 30 days, then incubated in the same medium in the presence of 5% DMSO for 1 h in an ice bath, cooled slowly to -20°C and immersed for 30 min in liquid nitrogen. After cryopreservation, the cells conserved the same growth pattern, but displayed different biochemical properties. The subculture derived from the thawed cells was characterized by a lower chlorophyll content and a higher pyrethrin biosynthesis ability.

Stabilization of Octastatin, a somatostatin analogue: comparative accelerated stability studies of two formulations for freeze-dried products.

Octastatin (vapreotide INN) is a somatostatin analogue being developed in gastro-enterologic, neuroendocrine and oncologic applications. The pharmaceutical form is a freeze-dried preparation for parental injection use. This study was intended to evaluate the stability of the freeze-dried products and to determine the optimally stable formulation. Two types of stabilizing agents (lactose and glutamic acid/sodium glutamate buffer) and three dosage forms (0.5, 1.5, 15 mg of vapreotide base) were investigated. The peptide content and chemical stability of cakes stored at 50 degrees C and 70% relative humidity were determined by HPLC and the regression analysis parameters calculated. Results indicate that the formulation with glutamate buffer is appropriate for long term storage.

Stabilization of Octastatin, a somatostatin analogue. Preparation of freeze-dried products for parenteral injection.

Octastatin (RC-160, vapreotide INN) is a somatostatin analogue being developed for use in oncological, enterologic and neuroendocrine applications. The pharmaceutical form is a freeze-dried preparation for parenteral injection use. Three dosage forms containing 0.5, 1.5 and 15 mg of vapreotide base have been investigated. Various freeze-drying conditions, stabilizing agents, membranes for sterile filtration and heating procedures have been examined. The formulation with glutamic acid-sodium glutamate buffer as a stabilizing agent, the type of membrane for filtration and the freeze-drying procedure have been found appropriate for subsequent industrial production. No evident degradation was observed either after manufacturing, or after a three-week accelerated stability study at 50 degrees C and 70% relative humidity.

Microbiological conversions of solamargine.

A strain of Aspergillus ochraceus, a microorganism classically used for bioconversions of the steroid nucleus, was tested on solasodine glucosides. It induced specific modifications to the oside chain of the solamargine molecule, without modifying the genin nucleus. These modifications depended on the composition of the culture medium and the aeration, and led under certain conditions to a single isomer of the secondary glucoside beta 2-solamargine or Khasianine: o-alpha-L-rhamnopyranosyl-(1-->4 glu)-O-(3)-beta-D-glucopyranosyl solasodine.

Densitometry for analysis of protein and peptide hydrolysates: application to luteinizing hormone releasing hormone.

Densitometric analysis has been performed on various standard protein and peptide solutions and on luteinizing hormone releasing hormone hydrolysate. After thin-layer chromatographic separation using two-dimensional separation on cellulose plates, the plates were mapped and each amino acid was assayed individually in order to obtain a specific integration profile of the hydrolysate. Densitometry proved to be a useful method for the screening of protein hydrolysates and is accurate enough for the assay of peptide hydrolysates. The hormone was hydrolysed with hydrochloric acid-trifluoroacetic acid (1:1, v/v) in the presence of thioglycolic acid. This limits the loss of tryptophan and does not interfere with the analysis. Results were sufficiently reliable, accurate and reproducible for routine analysis.

High-yield ethanol production from Jerusalem artichoke tubers.

Fermentation conditions were optimized for the production of ethanol from Jerusalem artichoke with a strain of Saccharomyces cerevisiae able to use high-concentration juice and undiluted pulp. Yields (95 to 125 g ethanol/l=85 to 98% of the theoretical value) exceeded those obtained with strain of Kluyveromyces used classically.

Properties of a new alkaline proteinase from Aspergillus niger.

A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).

[Antienzymatic activities of two Leguminosae: bean pods and bean bulls. Antiamylase activity]. / Activités antienzymatiques chez deux Légumineuses: la cosse de Haricot et la cuticule de Fève. Activité antiamylasique.

Amylase inhibitory activities have been sought in the powders of Phaseolus vulgaris pods and of Vicia faba bean hulls. Unlike to the bean, Phaseolus vulgaris pod has no amylase inhibitory activity and the presence of tannin could not be determined. Contrary, Vicia faba bean hull shows an obvious amylase inhibitory activity. It contains an appreciable content of tannins which may be responsible of this activity. The quantities of amylase whose are inhibited increase in some measure with the quantities of enzyme added. They are not in direct ratio to the quantities of extract, then, inhibition may be of no competitive type.

[Enzyme inhibitory activities of two Leguminosae: Phaseolus vulgaris L. pods and Vicia faba L. hulls. Trypsin inhibitory activity]. / Activités antienzymatiques chez deux Légumineuses: la cosse de Haricot et la cuticule de Fève. Activité antitrypsine.

Trypsin inhibitory activities of Phaseolus vulgaris L. pods and Vicia faba L. bean hulls have been studied according to the extraction conditions of the powder: solvent pattern, temperature and pH. The two vegetal powders have a trypsin inhibitory activity quite similar, weak but appreciable. The trypsin inhibitor of Phaseolus vulgaris L. pods is thermosensitive but stable at low pH. Inversely the Vicia faba L. bean hulls one is thermostable but it feels low pH.