Inhibition of α-glucosidase by flavonoids of Cymbopogon citratus (DC) Stapf.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves extracts from Cymbopogon citratus (DC) Stapf. are widely used in traditional medicine exhibiting several in vivo biological activities, including antidiabetic. Several flavonoids, including aglycones and glycosides, were reported in this plant and previous studies suggested that flavonoids may interact with targets related to diabetes. AIM OF THE STUDY: Evaluated the hypoglycemic activity of C. citratus flavonoids through α-glucosidase inhibition and assess the structure-activity relationship using molecular docking studies. MATERIAL AND METHODS: An infusion of C. citratus leaves and its flavonoid-rich fraction were prepared. Five flavonoids from this fraction were isolated and structurally characterized by UV spectral analysis with shift reagents, HPLC-PDA-ESI/MSn and 1H NMR. The antidiabetic potential of C. citratus infusion, its flavonoid-rich fraction, glycosylated flavonoids and aglycones was evaluated trough the in vitro inhibition of yeast α-glucosidase. Posteriorly, molecular docking of the tested flavonoids was performed to investigate its possible interactions with the α-glucosidase pocket. RESULTS: The infusion of C. citratus, its flavonoid-rich fraction, luteolin and five flavone glycosides namely, luteolin 6-C-ß-glucopyranoside (isoorientin), luteolin 7-O-neohesperidoside (ionicerin), luteolin 7-O-ß-glucopyranoside (cynaroside), Luteolin 2″-O-rhamnosyl-C-(6-deoxy-ribo-hexos-3-ulosyl) (cassiaoccidentalin B), luteolin 6-C-α-arabinofuranosil-(1→2)-α-L-rhamnopyranoside (kurilesin A) showed higher inhibitory activity than the reference drug. This activity increased by the addition of a sugar moiety. However, the di-glycosides were less active than mono-glycosides. The docking studies showed interactions of sugar moieties and A or B rings with the catalytic pocket mainly through hydrogen bonds. CONCLUSIONS: Our results corroborate the potential of C. citratus as a medicinal plant for the treatment of diabetes and revealed that its flavonoid glycosides has hypoglycemic effect and can be explored as drug candidates to act as α-glucosidase inhibitors in the treatment of diabetes.

In Vitro Hypocholesterolemic Effect of Coffee Compounds.

(1) Background: Cholesterol bioaccessibility is an indicator of cholesterol that is available for absorption and therefore can be a measure of hypocholesterolemic potential. In this work, the effect of commercial espresso coffee and coffee extracts on cholesterol solubility are studied in an in vitro model composed by glycodeoxycholic bile salt, as a measure of its bioaccessibility. (2) Methods: Polysaccharide extracts from coffees obtained with different extraction conditions were purified by selective precipitation with ethanol, and their sugars content were characterized by GC-FID. Hexane extraction allowed us to obtain the coffee lipids. Espresso coffee samples and extracts were tested regarding their concentration dependence on the solubility of labeled 13C-4 cholesterol by bile salt micelles, using quantitative 13C NMR. (3) Results and Discussion: Espresso coffee and coffee extracts were rich in polysaccharides, mainly arabinogalactans and galactomannans. These polysaccharides decrease cholesterol solubility and, simultaneously, the bile salts' concentration. Coffee lipid extracts were also found to decrease cholesterol solubility, although not affecting bile salt concentration. (4) Conclusions: Coffee soluble fiber, composed by the arabinogalactans and galactomannans, showed to sequester bile salts from the solution, leading to a decrease in cholesterol bioaccessibility. Coffee lipids also decrease cholesterol bioaccessibility, although the mechanism of action identified is the co-solubilization in the bile salt micelles. The effect of both polysaccharides and lipids showed to be additive, representing the overall effect observed in a typical espresso coffee. The effect of polysaccharides and lipids on cholesterol bioaccessibility should be accounted on the formulation of hypocholesterolemic food ingredients.

Development of a bioreactor system for cytotoxic evaluation of pharmacological compounds in living cells using NMR spectroscopy.

INTRODUCTION: The evaluation of drug's cytotoxicity is a crucial step in the development of new pharmacological compounds. 31P NMR can be a tool for toxicological screening, as it enables the study of drugs' cytotoxicity and their effect on cell energy metabolism in a real-time, in a non- invasive and non-destructive way. This paper details a step-by-step protocol to implement a bioreactor system able to maintain cell viability during NMR acquisitions, at high cell densities and for several hours, enabling toxicological evaluation of pharmacological compounds in living cells. METHOD: HeLa cells were immobilized in agarose gel threads and continuously perfused with oxygenated medium inside a 5 mm NMR tube. Signals corresponding to intracellular high-energy phosphorous compounds were continuously monitored by 31P NMR to assess cell energy levels, intracellular pH and intracellular free Mg2+ concentrations ([Mg2+]f) under control and in the presence of two different cytotoxic drugs, calix-NH2 or 5-fluorouracil (5-FU). RESULTS: The bioreactor system was effective in maintaining cell energy levels as well as intracellular pH and [Mg2+]f along time, with a good 31P NMR signal to noise ratio. Calix-NH2 and 5-FU decreased cell energy levels by 35% and 39%, respectively, with a negligible increase in intracellular [Mg2+]f, and without affecting intracellular pH. DISCUSSION: The immobilization and perfusion system here detailed, along with 31P NMR, is useful in toxicological evaluation of new pharmacological compounds, enabling the continuous assessment of drugs' effect on energy levels, intracellular pH and [Mg2+]f in intact cells, for several hours without compromising cell viability.

In vitro study of the insulin-mimetic behaviour of vanadium(IV, V) coordination compounds.

A representative set of vanadium(IV and V) compounds in varying coordination environments has been tested in the concentration range 1 to 10(-6) mM, using transformed mice fibroblasts (cell line SV 3T3), with respect to their short-term cell toxicity (up to 36 hours) and their ability to stimulate glucose uptake by cells. These insulin-mimetic tests have also been carried out with non-transformed human fibroblasts (cell line F26). The compounds under investigation comprise established insulin-mimetic species such as vanadate ([H(2)VO(4)](-)), [VO(acetylacetonate)(2)], [VO(2)(dipicolinate)](-) and [VO(maltolate)(2)], and new systems and coordination compounds containing OO, ON, OS, NS and ONS donor atom sets. A vitality test assay, measuring the reduction equivalents released in the mitochondrial respiratory chain by intracellular glucose degradation, is introduced and the results are counter-checked with (3)H-labelled glucose. Most compounds are toxic at the 1 mM concentration level, and most compounds are essentially non-toxic and about as effective as or more potent than insulin at concentrations of 0.01 mM and below. V(V) compounds tend to be less toxic than V(IV)compounds, and complexes containing thio functional ligands are somewhat more toxic than others. Generally, ON ligation is superior in insulin-mimetic efficacy to OO or O/ NS coordination, irrespective of the vanadium oxidation state. There is, however, no striking correlation between the nature of the ligand systems and the insulin-mimetic potency in these cell culture tests, encompassing 41 vanadium compounds, the results on 22 of which are reported in detail here. The syntheses and characteristics of various new compounds are provided together with selected speciation results. The crystal and molecular structures of [[VO(naph-tris)](2)] [where naph-tris is the Schiff base formed between o-hydroxynaphthaldehyde and tris(hydroxymethyl)amine] are reported. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0311-5.