Evaluation of digestibility differences for apple polyphenolics using in vitro elderly and adult digestion models.

We evaluated the in vitro digestibility of apple polyphenols mimicking elderly and adult digestion models (dynamic and static systems). The digestibility of total apple polyphenols in small intestine was much higher in the adult dynamic system (62 µg/100 g fresh apple) compared to the static system (20 µg/100 g fresh apple) and elderly dynamic digestion conditions (33 µg/100 g fresh apple). Elderly in vitro static digestion showed better antioxidant activity than the adult system (OH and ABTS+ methods). Thus, the in vitro dynamic digestion system can more truly reflect the digestion of apple polyphenols than static digestion system. Moreover, elderly digestion conditions negatively influenced the digestibility of apple polyphenols including chlorogenic acid, epicatechin, phlorizin, rutin, phloretin, hyperoside, proanthocyanidin B2, and quercetin. Hence, appropriate selection of in vitro digestion models for elderly is a prerequisite to exploring the digestibility of phytochemicals for the development of functional food products for elderly.

Solvent effect on phenolics and antioxidant activity of Huangshan Gongju (Dendranthema morifolium (Ramat) Tzvel. cv. Gongju) extract.

Huangshan Gongju was extracted with organic solvents (ethanol, methanol and acetone) of different concentrations (0-90%), and the extracts' phenolic content and antioxidant activity, as well as the correlations between them were examined. With the increasing concentration of organic solvent, the total phenolic compound (TPC) increased continuously and met its maximum at 70% acetone, whereas the total flavonoid compound (TFC) and most individual phenolics met their maximums at 70% ethanol. Similar changes occurred to the antioxidant activity, including DPPH and ABTS scavenging activities, and their maximums were respectively found at 50% acetone and 70% ethanol. The antioxidant activity correlated strongly with TPC/TFC (r > 0.954, p < 0.01) and individual phenolics (r > 0.886, p < 0.05), and the strongest correlations between them were mainly given by luteolin-7-O-glucoside (r > 0.975, p < 0.001). These results suggested that high content organic solvent (50-70%) was beneficial to obtain Huangshan Gongju extracts of higher phenolic content and antioxidant activity, and 70% ethanol may be the promising solvent. Besides, phenolics were found to be the main antioxidants of Huangshan Gongju extracts, and flavonoids especially luteolin-7-O-glucoside may play more important roles in the antioxidant activity.

The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunaliella salina.

A 5'-flanking region of an actin gene from the green unicellular alga Dunaliella salina (D. salina) was cloned using a genome-walking method by PCR and its structural features were characterized. Two repetitive sequences found, over 75 bp in length each, were located at position -573 and -424 bp,respectively, relative to the AUG codon. The actin gene promoter region of D. salina displayed a consensus sequence of GCTC (G/C) AAGGC, a CCAAT motif and two TATA-like motifs that did not have a canonical sequence of a TATA box. The 5' flanking region of the actin gene was exploited to direct expression of the bialaphos resistance gene (bar) from Streptomyces hygroscopicus as a dominant marker in the nuclear transformation of D. salina. Direct selection of bar resistant transformants was achieved by allowing a 24 h period of recovery of cells transformed by biolistic procedure, followed by growth of the cells for one week under standard condition prior to harvesting and plating on the solid medium containing 0.5 microg/mL of phosphinothricin (PPT). Five colonies picked from the plate were analyzed, of which the integration of the bar gene was demonstrated in the nuclear genome. Southern blotting revealed that only one of five transformants contained a single copy of the bar gene whereas others contained multiple copies,suggesting that nuclear transformation of D. salina mainly occurred through illegitimate recombination events,resulting in ectopic integration of the introduced DNA. The integration patterns of the foreign DNA in this experiment appeared not to influence the bar gene expression in the transformants containing single or multiple inserts. The bar gene expression in the five transformants was verified by RT-PCR, confirming transcription of the chimeric DNA. These transformants were maintained on agar plates in the absence of PPT for more than seven months and retained resistance to the herbicide at 1 microg/mL. This work demonstrates that the actin gene promoter-driven expression of the bar gene may be used as a dominant selectable marker for nuclear transformation of D. salina.

[Cloning and functional analyses of promoters of two carbonic anhydrase genes from Dunaliella salina].

The cloning vectors pMD-DCA1 and pMD-CA containing the promoters of duplicated carbonic anhydrase 1 (DCA1) and carbonic anhydrase (CA) genes, respectively, from Dunaliella salina, and expression vector pDM307 containing bar-NOS polyA fragment were digested with EcoR I. The bar-NOS polyA fragment was fused, respectively, to the fragments of the vectors pMD-DCA1 and pMD-CA to form transgenic D. salina expression vectors pMDDC-B and pMDC-B. The micro-shots were prepared by coating two constructs (pMDDC-B and pMDC-B) with gold particle. Each sample was bombarded once, twice, and thrice, respectively, with micro-projectile gun at a rupture pressure of 690 kPa in helium gas. The screening culture of the bombarded alga cells was performed in PKS liquid and solid medium containing 3 mg/L phosphinothricin (PPT) to develop the transformed cells of D. salina. Analyses of the transformed cells were carried out through PCR, Southern blotting, and Northern blotting. The results of screening culture showed that the expression of the external bar gene of vectors pMDDC-B and pMDC-B was stable and transient, respectively, in the transformed D. salina cells. In the meantime, the transformed efficiency of particle bombardment twice was higher than that of once or thrice particle bombardment at a rupture pressure of 690 kPa in helium gas. PCR and Southern blotting analyses indicated that the external bar gene was integrated into the genome of the cells. Northern analysis indicated that expression efficiency of the bar gene driven by DCA1 promoter was regulated by the gradient concentration of sodium chloride, and the positive blotting signal intensity of the bar mRNA was highest in the medium containing 2 mol/L of sodium chloride. The findings of the present study suggest that promoter of the DCA1 gene may be an inducible promoter following a hyperosmotic shock with high activity and safety in the research of transgenic D. salina. The tandem GT sequences of the promoter region of DCA1 and CA genes may be related to the molecular mechanisms of the extreme halo-toleration of the unicellular green alga, D. salina.