Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O--D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

O-glycosylated C-glycosylflavones from Passiflora platyloba

Five C-glycosylflavonoids including two new compounds, isovitexin 7-rhamnosylglucoside and isomollupentin 7-rhamnosylglucoside, were obtained from the leaves of Passiflora platyloba. The known flavonoids were isovitexin, vitexin and isomollupentin (6-C-arabinosylapigenin). In addition, the coumarin esculetin was isolated.     

Les flavonoides des rameaux vegetatifs de Periploca graeca

The flavonoid content of the vegetative stems of Periploca graeca include derivatives of flavonols (quercetin and kaempferol), anthocyanidins (     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Kinetic Characterization of the Oxidation of Esculetin by Polyphenol Oxidase and Peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

Transglucosylases in Cichorium intybus converting cichoriin to esculin

The heads of Cichorium intybus contain two enzymes concerned in the formation of esculin (esculetin 6-glucoside) from cichoriin (esculetin 7-glucoside). Both enzymes can catalyse two reactions, i.e. hydrolysis (HD) of cichoriin to give esculetin, and transglucosylation (TG) from this glucoside to the liberated aglucone forming esculin. One of them, designated enzyme A, is a high molecular weight protein with predominantly TG activity, and dissociates during isolation into the other enzyme having higher HD activity. Enzyme A shows high substrate specificity and different pH optima in HD and TG reactions, as in the case with the transglucosylase from Daphne odora.     

Position specificity of an o-dihydroxycoumarin glucosyltransferase from tobacco cell suspension culture

A partially purified UDP-glucose: o-dihydroxycoumarin glucosyltransferase from tobacco cell culture required an intact coumarin ring system with o-dihydroxy groups for highest activity. The enzyme exhibited strict position specificity towards the 7-OH group of both esculetin and daphnetin with the formation of cichoriin and daphnin, respectively. The apparent K_m values were 111 and 95 μM, while the V_max for esculetin was 26.4 pkat/mg protein.     

A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice

Previous studies have demonstrated that a natural coumarin compound esculetin (Esc) possesses antioxidant, anti-tumor, and anti-inflammation activities and rescues cultured primary neurons from NMDA toxicity. In this study, we investigated the neuroprotective effects of Esc on cerebral ischemia/reperfusion (I/R) injury in a middle cerebral artery occlusion model in mice. Esc (20 μg) was administered intracerebroventricularly at 30 min before ischemia. We found that Esc significantly reduced infarct volume and decreased neurological deficit scores after 75 min of ischemia and 24 h of reperfusion. Post-treatment of Esc still provided neuroprotection even when Esc was administered after 4 h of reperfusion. Our data also indicated that intraperitoneal administration of Esc showed protective effects on cerebral I/R injury in a dose-dependent manner. We further explored the protective mechanisms of Esc on cerebral I/R injury and found that Esc decreased cleaved caspase 3 level, a marker of apoptosis. Finally, our data demonstrated that Esc exerted its anti-apoptotic activity by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, two apoptosis-related proteins. Because of its clinical use as an anticoagulant and its safety profile, Esc may have a therapeutic potential for the treatment of stroke in the future clinical trials.     

6-Hydroxyflavonoids from Pulicaria dysenterica (compositae)

Methyl ethers of 6-hydroxykaempferol and quercetagetin, together with scutellarein, were isolated from the leaves of Pulicaria dysenterica. The pattern of compounds is different from that previously recorded in the flowers.