Combinatorial preparation and structural characterization of anthocyanins and aglycones from Purple-heart Radish for evaluation of physicochemical stability and pancreatic lipase inhibitory activity.

In this study, an efficient approach to preparation of different anthocyanins from Purple-heart Radish was developed by combining microwave-assisted extraction (MAE), macroporous resin purification (MRP) and ultrasound-assisted acid hydrolysis (UAAH) for evaluation of physicochemical stability and pancreatic lipase (PL) inhibitory activity. By optimization of MAE, MRP and UAAH processes, the anthocyanins reached the yield of 6.081 ± 0.106 mg/g, the purity of 78.54 ± 0.62 % (w/w) and the content of 76.29 ± 1.31 % (w/w), respectively. With high-resolution UHPLC-Q-Orbitrap/MS, 15 anthocyanins were identified as pelargonins with diverse glucosides and confirmed by pelargonidin standard. By glycosylation, pelargonins exhibited higher stability in different pH, temperature, light, metal ions environments than that of pelargonidin. However, PL inhibitory assay, kinetic analysis and molecular docking demonstrated that pelargonidin had higher PL inhibitory activity than pelargonins even though with similar binding sites and a dose-effect relationship. The above results revealed that the effect of glycosylation and deglycosylation on PL inhibitory activity and physicochemical stability.

Screening and identification of lipase inhibitors extracted from Dioscorea nipponica Makino by UV-vis and HPLC coupled to UPLC-Q-TOF-MS/MS.

Dioscoreae nipponica Makino (D. nipponica) as the rhizome of dioscoreaceae rich in steroidal saponins, has been reported to have the hypolipidemic effects etc. However, it is still unclear which exact active components are primary responsible for the beneficial effects. This study was conducted to fish out the lipase inhibitors from D. nipponica, and evaluate the inhibitory activity on porcine pancreatic lipase (PPL) through in vitro kinetic assay using p-nitrophenyl palmitate as substrate. Accordingly, the ethanolic extract was subjected to D101 macroporous resin purification for spectrophotometric screening, high performance liquid chromatography (HPLC) separation and structural characterization by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Through orlistat validation, the PPL inhibitory activity and IC50 value of the extract were respectively 68.34 ± 1.47 % and 107.05 µg/mL under the optimized inhibition conditions. From 6 steroidal saponins identified, the inhibitory components named the protodioscin, protogracillin, dioscin and gracillin were fished out by grouping separation and HPLC analysis. Furthermore, dioscin and gracillin with the parent structure of diogenin were confirmed as the major inhibitors by virtue of stability tests based on transformation of protodioscin and protogracillin. Finally, the inhibitory mechanism of the major inhibitors toward PPL was further clarified by kinetic analysis and molecular docking analysis. The proposed method not only revealed the PPL inhibitory components in D. nipponica, but also provided an effective approach to hierarchical screening of PPL inhibitors from natural plants.

A hierarchical screening method for detection of illegal adulterants in Fur seal ginseng pills by profiling analysis of HPLC multi-dimensional fingerprints.

A novel method for hierarchical screening of illegal adulterants in Fur seal ginseng pills products was developed by multi-dimensional fingerprint profiling analysis. Fingerprint feature of the samples was acquired by high-performance liquid chromatography analysis of 11 batches of samples with diode array detector and fluorescence detector, and then potential illegal adulterants including phosphodiesterase type-5 inhibitors, androgens, α receptor antagonists and yohimbine, were further separated at multiple wavelengths to reduce or remove interferences from sample matrix for highlight their chromatographic characteristics. Accordingly, a hierarchical screening strategy was designed by first-order and second-order fingerprints combined with spectral examination to achieve high accuracy and reliability. The method was successfully applied to screening of illegal adulterants in real samples, and it also exhibited good quantification performance through validation tests. From 16 batches of samples, three suspected samples were confirmed to be positive, containing 9.37µg/g of testosterone, 18.8 µg/g of tadalafil, and 48.5 µg/g of sildenafil, respectively. The recoveries and relative standard deviations were in the range of 83.6-103.1% and 4.2-6.8%, respectively. The proposed method provided a simple, efficient and promising alternative to monitoring functional foods.

Scanning electrochemical microscopy of DNA hybridization on DNA microarrays enhanced by HRP-modified SiO2 nanoparticles.

Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

Qualitative and quantitative detection of DNA amplified with HRP-modified SiO2 nanoparticles using scanning electrochemical microscopy.

Qualitative and quantitative detection of DNA was achieved by a "sandwich" DNA sensor through SG/TC (substrate generation and tip collection) mode of scanning electrochemical microscopy (SECM). The "sandwich" DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the gold substrate surface, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2 nanoparticles were linked to the sandwich structure through biotin-streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. For SECM imaging experiment, we structured the microsensor platform through localized desorption of 1-dodecanethiol monolayer. Approach curves were employed for quantitative detection of DNA concentration. The detection limit of complementary DNA was as low as 0.8pM. This technique is promising for the application on electrochemical DNA chip.

[Determination of the eliminating ratio of TCM polysaccharide to hydroxyl radical by kinetic fluorescent spectrophotometry].

OBJECTIVE: To explore reaction dynamic of hydroxyl radical (*OH) and salicylic acid,and to determine the elimination ratios of TCM polysaccharide to *OH by kinetic fluorescent analysis. METHODS: The impact dynamics factors of this reaction were studied by fluorescent, such as the reaction of concentration, reaction time and temperature. The dynamical equation was built, a kinetic fluorescent spectrophotometry based on the reaction was developed to determine the elimination ratio. Repetitiveness and reliability of this method were tested by vitamin C. RESULTS: The dynamical equation of reaction rate to salicylic acid was gamma = 0. 9818x -1. 1801 under the condition of lambda ex = 295 nm, lambda em = 411 nm at room temperature, r approximately 1. The 50% elimination ratio (IC50) of TCM polysaccharide of Tangerine peel and Ganoderma lucidum to *OH was 78.01 mg/L and 232.5 mg/L, respectively. The IC50 of vitamin C was 24.52 microg/L, RSD was 0.23% (n = 5). CONCLUSIONS: The method is sensitive and reliable, it can be used to determine the elimination ratio of TCM polysaccharide to *OH.