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In light of recent experiments suggesting high-spin (HS) Ni(II) species in the catalytic cycle of [NiFe] hydrogenase, a series of models of the Ni(II) forms Ni-SI(I,II), SI-CO and Ni-R(I,II,III) were examined in their high-spin states via density functional calculations. Because of its importance in the catalytic cycle, the Ni-C form was also included in this study. Unlike the Ni(II) forms in previous studies, in which a low-spin (LS) state was assumed and a square-planar structure found, the optimized geometries of these HS Ni(II) forms resemble those observed in the crystal structures: a distorted tetrahedral to distorted pyramidal coordination for the NiS4. This resemblance is particularly significant because the LS state is 20-30 kcal/mol less stable than the HS state for the geometry of the crystal structure. If these Ni(II) forms in the enzyme are not high spin, a large change in geometry at the active site is required during the catalytic cycle. Furthermore, only the HS state for the CO-inhibited form SI-CO has CO stretching frequencies that match the experimental results. As in the previous work, these new results show that the heterolytic cleavage reaction of dihydrogen (where H2 is cleaved with the metal acting as a hydride acceptor and a cysteine as the proton acceptor) has a lower energy barrier and is more exothermic when the active site is oxidized to Ni(III). The enzyme models described here are supported by a calibrated correlation of the calculated and measured CO stretching frequencies of the forms of the enzyme. The correlation coefficient for the final set of models of the forms of [NiFe] hydrogenase is 0.8.

A novel and rapid method for simultaneous extraction and separation of the different polysaccharides from Semen Cassiae (SC) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/ammonium sulfate system as a multiphase solvent, the effects of MAATPE on the extraction of polysaccharides from SC such as the composition of the ATPS, extraction time, temperature and solvent-to-material ratio were investigated by UV-vis analysis. Under the optimum conditions, the yields of polysaccharides were 4.49% for the top phase, 8.80% for the bottom phase and 13.29% for total polysaccharides, respectively. Compared with heating solvent extraction and ultrasonic assisted extraction, MAATPE exhibited the higher extraction yields in shorter time. Fourier-transform infrared spectra showed that two polysaccharides extracted from SC to the top and bottom phases by MAATPE were different from each other in their chemical structures. Through acid hydrolysis and PMP derivatization prior to HPLC, analytical results by indicated that a polysaccharide of the top phases was a relatively homogeneous homepolysaccharide composed of dominant gucose glucose while that of the bottom phase was a water-soluble heteropolysaccharide with multiple components of glucose, xylose, arabinose, galactose, mannose and glucuronic acid. Molar ratios of monosaccharides were 95.13:4.27:0.60 of glucose: arabinose: galactose for the polysaccharide from the top phase and 62.96:14.07:6.67: 6.67:5.19:4.44 of glucose: xylose: arabinose: galactose: mannose: glucuronic acid for that from the bottom phase, respectively. The mechanism for MAATPE process was also discussed in detail. MAATPE with the aid of microwave and the selectivity of the ATPS not only improved yields of the extraction, but also obtained a variety of polysaccharides. Hence, it was proved as a green, efficient and promising alternative to simultaneous extraction of polysaccharides from SC.

A rapid and effective method of integrating extraction and purification for alkaloids from Sophora flavescens Ait. was developed by microwave-assisted aqueous two-phase extraction (MAATPE) based on the high efficiency of microwave-assisted extraction (MAE) and the demixing effect of aqueous two-phase extraction (ATPE). The aqueous two-phase system (ATPS), ethanol/ammonia sulfate was chosen from seven combinations of ethanol/salt systems, and its extraction properties were investigated in detail. Key factors, namely, the compositions of ATPS, solvent-to-materials ratio, and the extraction temperature were selected for optimization of the experimental conditions using response surface methodology (RSM) on the basis of the results of the single-factor experiment. The final optimized conditions were, the compositions of ATPS: ethanol 28% (w/w) and (NH 4 ) 2 SO 4 18% (w/w), solvent-to-material ratio 60:1, temperature 90°C, extraction time 5 min, and microwave power 780 W. MAATPE was superior to MAE, the latter using a single solvent, not only in extraction yield but also in impurity content. Moreover, compared with the combination of MAE and ATPE in the two-step mode, MAATP demonstrated fewer impurities, a better yield (63.78 ± 0.45 mg/g) and a higher recovery (92.09 ± 0.14%) in the extraction and purification of alkaloids. A continuous multiphase-extraction model of MAATPE was proposed to explicate the extraction mechanism. MAATPE revealed that the interaction between microwave and ATPS cannot only cause plant cell rupture but also accelerate demixing, improving mass-transfer from solid-liquid extraction to liquidliquid purification. MAATPE simplified procedures also contributed to the lower loss occurrence, better extraction efficiency, and reduced impurity to target constituents.

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