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.

Enzyme and microwave co-assisted extraction, structural characterization and antioxidant activity of polysaccharides from Purple-heart Radish.

A novel method of simultaneous extraction and separation of diverse polysaccharides from Purple-heart Radish was developed by integrating EAE with MAATPE. The effects of different enzymes, the ATPS composition, extraction temperature, time etc. were investigated by single-factor experiments and RSM. Under the optimum conditions, the extraction yields of PTP, PBP and total polysaccharides were 9.107 ± 0.391%, 32.506 ± 0.046% and 41.613 ± 0.437%, respectively. By means of HPGPC and PMP-HPLC, Mw of PTP and Mw of PBP were 15935 Da and 27962 Da, respectively. PTP and PBP were mainly composed of mannose, glucuronic acid, aminogalactose, glucose, galactose and arabinose. Moreover, both polysaccharides exhibited stronger antioxidant activities for scavenging multiple radicals and anti-lipid peroxidation. Compared to the conventional extraction methods, EAE-MAATPE achieved higher extraction efficiency due to the synergistic effect between EAE and MAATPE leading to rupture and enzymolysis of cell. Thus, EAE-MAATPE provided an efficient alternative to simultaneous extraction of different polysaccharides from natural products.

Microwave-assisted extraction followed by salting-out phase separation for hierarchical screening of illegal adulterants in aphrodisiac health products by multi-dimensional fingerprint profiling analysis.

A novel method for hierarchical screening of illegal adulterants in Fur seal ginseng pills (FSGP) products was developed by microwave-assisted extraction (MAE) coupled to salting-out assisted liquid-liquid extraction (SALLE) with multi-dimensional fingerprint profiling analysis. Using a homogeneous system formed by dimethyl carbonate (DMC) and water as the extractant, the MAE conditions were investigated to maximize extraction recoveries, followed by addition of ammonium sulfate to induce DMC phase separation for SALLE enrichment of 16 potentially illegal adulterants such as phosphodiesterase type-5 inhibitors, androgens, α receptor antagonists and yohimbine etc. By means of high-performance liquid chromatography (HPLC) with diode array detection (DAD) and fluorescence detection (FLD), multi-dimensional fingerprints were acquired by multi-wavelength detection to highlight the signals of the potentially illegal adulterants and reduce or remove interferences from the sample matrix. For high accuracy and reliability, a hierarchical screening strategy was designed by multi-dimensional fingerprinting profiling analysis (MDFPA). The method exhibited proper identification and quantification performance, and it was successfully applied to screening of illegal adulterants in 18 batches of the samples through the step-by-step MDFPA. Also, the results were further confirmed by ultra high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap/MS). The proposed method was proved to be a green, efficient and reliable alternative to monitoring aphrodisiac health products.

Multi-dimensional fingerprint profiling analysis for screening and quantification of illegal adulterated antidiabetics in hypoglycemic health products by aqueous two-phase extraction and multi-wavelength detection.

A novel method for screening and quantification of illegal adulterated antidiabetics in hypoglycemic health products was developed by multi-dimensional fingerprint profiling analysis (MDFPA). By means of aqueous two-phase extraction (ATPE), using aqueous two-phase system (ATPS) of butanol-water as the extractant, 11 common antidiabetics could be effectively extracted to the upper and lower phases, respectively. HPLC separation conditions for the extracts from two phases were investigated by multi-wavelength detection before and after p-nitrobenzoyl chloride (p-NBC) and 2,4-dinitrofluorobenzene (DNBF) derivatizations to establish multi-dimensional fingerprints. For high accuracy and reliability, a hierarchical screening approach to screening illegal adulterated antidiabetics in samples was established by MDFPA and spectral purity examination. Meanwhile, detection limits of identification for illegal adulterants were defined by detection limits of spectra (SLOD). The proposed method exhibited good identification and quantification performances. SLODs, LODs and LOQs of 11 antidiabetics were 1.22-8.37 µg/g, 0.225-4.23 µg/g and 0.755-14.10 µg/g, respectively. They had good linearity ranged from 2.0 µg/g to 300.0 µg/g (R2 ≥ 0.9978). The recoveries and RSDs were 76.83-109.6% and 0.50-6.5%, respectively. The method was successfully applied to screening of 15 batches of samples in different forms. Among them, four samples were detected to contain 5.47 µg/g of metformin, 6.50 µg/g of phenformin, 3.69 µg/g of glibenclamide and 9.11 µg/g of glimepiride, respectively. The results proved that it was an efficient and feasible alternative to screening and detection of illegal adulterated antidiabetics in hypoglycemic health products.