Integrating amino acid oxidase with photoresponsive probe: A fast quantitative readout platform of amino acid enantiomers.

Low-cost, high-throughput, broadly useful photoresponsive enantiomeric excess (ee) sensing of amino acids remains challenging to date. Herein, based on the selective oxidation reaction of amino acid oxidase (AAO) to amino acid enantiomers (D/L-AA) and the oxidation reaction of substrate (H2O2) with aromatic boronic ester, we put forward a photoresponsive strategy for the determination of D/L-AA at a certain concentration. In this scheme, the substrate H2O2 produced by the enzyme-catalyzed reaction was determined by sensitive fluorescent and colorimetric response of ethyl-3-(3-(benzothiazol-2-yl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)-2-cyanoacrylate (HBT-PB) to reflect the enantiomeric content at a certain concentration. The photoresponsive probe HBT-PB was readily available and inexpensive with sensitive long-wavelength red fluorescence and colorimetric light response to H2O2, the detection limit (LOD) was estimated as 2.91 µM. The operation of the sensing method was simple and data collection and processing are straightforward. The practicability of the scheme was favorably confirmed by accurate and scientific analysis of methionine and Dopa samples. As a result, the scheme was not only suitable for high-throughput screening but also adaptable to low-cost and sensitive RGB colorimetric analysis platform (LOD of methionine and Dopa was calculated as 9.23 µM and 8.34 µM respectively) with modern plate readers, and possessed extremely high enantioselectivity and wide applicability which benefited from the specificity and efficiency of enzyme catalytic reaction.

Rapid and comprehensive profiling of α-glucosidase inhibitors in Buddleja Flos by ultrafiltration HPLC-QTOF-MS/MS with diagnostic ions filtering strategy.

Buddleja Flos is used as yellow rice colorant and a well-known traditional Chinese medicine. But its biochemical profiling is still lack due to complex matrix. Here, ultrafiltration high-performance liquid chromatograph-quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS) with diagnostic ions filtering strategy was proposed for rapid and comprehensive investigation of its α-glucosidase inhibitors. As a result, 33 bioactive compounds (13 phenylethanoid glycosides and 20 flavonoids) were successfully screened and identified. In addition, α-glucosidase inhibitory activities of twenty-two references were verified. Six flavonoid aglycones (4, 28, and 30-33) showed excellent α-glucosidase inhibitory activities (IC50, from 5.11 ± 0.85 to 32.49 ± 9.76 µg/mL), much higher than that of acarbose (IC50, 195.49 ± 10.05 µg/mL). Five flavonoid-monoglycosides (7, 12, 13, 20, and 22) presented moderate inhibitory activities with IC50 from 160.98 ± 23.19 to 249.37 ± 35.83 µg/mL. Results showcased the high efficiency of proposed strategy in profiling of bioactive compounds from natural products.

Profiling of tyrosinase inhibitors in mango leaves for a sustainable agro-industry.

Although mango leaves are the main ingredients in some traditional Chinese medicine preparations and folk tea, they with considerable quantities are usually discarded as agricultural waste. Thus, to extend their potential, reverse ultrafiltration-HPLC-DAD-QTOF-MS/MS combining with key ion filtering strategy was proposed to efficiently fish and systematically identify tyrosinase inhibitors in ethyl acetate fraction of mango leaves, which has the highest total phenolic content (40.00 ± 0.84 mg GAE/g DW) and tyrosinase inhibition activity (IC50, 17.62 ± 1.26 µg/mL). Finally, 36 polyphenolic tyrosinase inhibitors were unambiguously characterized or tentatively identified, and three of them were found in mango leaves for the first time. Results suggested that the proposed strategy was powerful for effective identification of bioactive compounds in complex mixtures (e.g. food, agricultural and sideline products), and the findings would lay a foundation for potential applications of mango leaves in pharmaceutical, cosmetic, and food industrial fields.

The biotransformation of Bupleuri Radix by human gut microbiota.

1. Bupleuri Radix (BR) is a herbal medicine traditionally used orally in oriental countries, which inevitably comes into contact with the intestinal microbiota. However, whether gut microbiota contribute to the biotransformation of BR, and/or the formation of pharmacologically active compounds remains unknown.2. In this study, the main saikosaponins (SAPs) of Bupleurum (including saikosaponin a, b1, b2, c, d, f, h) and BR extract (BRE) were individually incubated with human fecal suspensions (HFS), and metabolic time courses of SAPs and their metabolites by human gut bacteria were systematically characterized.3. Deglycosylation and dehydration were the main metabolic pathways identified for SAPs including newly investigated saikosaponin f (SSf) and saikosaponin h (SSh); dehydration had not been reported previously. A total of 19 dehydrated and deglycosylated metabolites of SAPs were detected and characterized, and 10 of them were newly identified. Moreover, SAPs of BRE were found to be deglycosylated to prosaikogenins. In addition, 13 metabolic pathways related to human gut microbiota were identified for phytochemicals of BRE except for SAPs. Gut microbiota may play a significant role in the biotransformation of BR in humans.

Flavonoid aglycone-oriented data-mining in high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry: efficient and targeted profiling of flavonoids in Scutellaria barbata.

The high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS) technique is a powerful tool for compound identification in complex natural products. However, untargeted MS/MS data analysis needs skillful experience and sometimes neglects minor compounds, which are co-eluted with major ones or overshadowed by the matrix. Flavonoids are the main bioactive components in Scutellaria barbata, and the total flavonoid content is 47.02 ± 3.23 mg QE/g DW. Although some flavonoid aglycones and their O-glycosides have been found in S. barbata, comprehensive profiling of flavonoids is unknown. Therefore, we report a flavonoid aglycone-oriented data-mining strategy for efficient and targeted profiling of flavonoids in S. barbata. The strategy includes four steps: (1) HPLC-QTOF-MS analysis of S. barbata; (2) construction of a flavonoid aglycone-based database according to biosynthetic pathway analysis and reported data; (3) extraction of through flavonoid aglycone-based ion chromatography; (4) identification of targeted flavonoids by MS/MS analysis. As a result, 45 flavonoids, including 24 flavones, 1 flavonol, 13 flavanones, and 7 flavanonols, were unambiguously or tentatively identified, while 20 of them were reported in S. barbata for the first time. Moreover, 14 available flavonoids were sensitively, precisely, and accurately determined by standard calibration curves, with limit of detection at 0.06 to 1.55 µg/g, limit of quantification at 0.16 to 3.70 µg/g, relative standard deviation (RSD) less than 9.0% for intra- and inter-day variations, and recovery at 92.6-108.1%. The matrix did not obviously suppress or enhance the ionization of 14 flavonoids, and finally their contents ranging from 0.04 to 4.49 mg/g in S. barbata were successfully achieved. Collectively, our results demonstrate that an efficient, reliable, and valuable strategy has been provided to rapidly and sensitively screen, profile, and quantify chemical components of complex natural products. Graphical abstract.

Identification of minor lignans, alkaloids, and phenylpropanoid glycosides in Magnolia officinalis by HPLC‒DAD‒QTOF-MS/MS.

An effective strategy based on high-speed counter-current chromatography (HSCCC) knockout combination with HPLC-DAD-QTOF-MS/MS analysis were developed to identify minor lignans, alkaloids, and phenylpropanoid glycosides in M. officinalis. Petroleum ether/ethyl acetate/methanol/water (8:4:7:5, v/v/v/v) as solvent system was firstly selected to separate the crude extract of M. officinalis. Two major lignans, honokiol and magnolol were knocked out, and minor components were enriched. Then, five standards (honokiol, magnolol, magnocurarine, magnoflorine and acteoside) were used as examples to discuss their fragmentation patterns for structural identification. By comprehensive screening, sixteen lignans, nine alkaloids, six phenylpropanoid glycosides were unambiguously or tentatively identified by comparing their retention time, UV spectra, accurate mass and fragmentation patterns with standards or reported components. Eight of them, as far as was known, were discovered from M. officinalis for the first time. The proposed method might provide a model for the effective identification of minor components from complex herbs. Additionally, this study laid a foundation for the study of quality control, and clinical applications of M. officinalis.