Deep eutectic solvent as a green solvent for enhanced extraction of narirutin, naringin, hesperidin and neohesperidin from Aurantii Fructus.

INTRODUCTION: In the present study, a green and efficient extraction method using deep eutectic solvents as extraction solvent was developed for extracting the four major active compounds narirutin, naringin, hesperidin and neohesperidin from Aurantii Fructus. METHODOLOGY: A series of tunable deep eutectic solvents were prepared and investigated by mixing choline chloride or betaine to different hydrogen-bond donors, and betaine/ethanediol was found to be the most suitable extraction solvent. To achieve the best extraction yield, the primary factors affecting the extraction efficiency, such as hydrogen-bond acceptor/hydrogen-bond donor ratio, water content in deep eutectic solvents, extraction temperature, solid/liquid ratio and extraction time, were investigated. RESULTS: The optimal extraction conditions were 40% of water in betaine/ethanediol (1:4) at 60°C for heated extraction of 30 min and solid/liquid ratio 1:100 g/mL. Under the optimum extraction condition, the extraction yields of narirutin, naringin, hesperidin, and neohesperidin were 8.39 ± 0.61, 83.98 ± 1.92, 3.03 ± 0.35 and 35.94 ± 0.63 mg/g, respectively, which were much higher than those of methanol as extraction solvent (5.5 ± 0.48, 64.23 ± 1.51, 2.16 ± 0.15 and 30.14 ± 0.62 mg/g). CONCLUSION: The present results showed that deep eutectic solvents could be promising green and efficient solvents for extraction of the bioactive ingredients from traditional Chinese medicine.

[Preparation of flavonoid reference standards from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis].

Flavonoid reference standards were targeted-prepared from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. With HPLC-MS analysis of Scutellariae Radix, 19 flavonoid components were identified by analyzing and comparing their retention times, ultraviolet spectra, and mass spectrometry data with literature. The separation and purification protocols of all targeted flavonoid reference standards were optimally designed according to the results of HPLC-MS analysis and related literature. The ethanol extract of Scutellariae Radix was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol successively. The ethyl acetate extract and n-butanol extract were separately subjected to primary separation by low pressure reverse phase preparative chromatography. Then the fractions containing targeted compounds were further purified by low pressure reverse and normal phases preparative chromatography. Finally, baicalin and wogonoside reference standards were obtained from n-butanol extract; baicaelin, wogonin, and oroxylin A reference standards were obtained from ethyl acetate extract. The structures of the 5 reference standards were identified by mass spectrometry (MS) and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The HPLC analytical results showed that the purities of the 5 reference standards were all above 98%. It is demonstrated that the rapid targeted-preparation method under the guidance of the HPLC-MS analysis is applicable for the isolation and preparation of chemical components in traditional Chinese medicines.

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics.

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C(18) column with isocratic elution at a flow rate of 1mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10ng/mL within a linear range of 10-1000ng/mL (R=0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi.

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 µg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 µg/ml. The half maximal inhibitory concentration (IC(50)) of naringenin for motility of adult females was 2.5 µg/ml. Flavone immobilized female adult worms at 31.2 µg/ml (MTT>80%) and microfilariae at 62.5 µg/ml. Rutin killed microfilariae at 125 µg/ml and inhibited MTT-reduction in female worms for >65% at 500 µg/ml. Naringin had adulticidal effects at 125 µg/ml while chrysin killed microfilariae at 250 µg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin>flavone=hesperetin>rutin>naringin>chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.