Radical Scavenging Detection Research Articles

Hypoglycemic activities of flowers of Xanthoceras sorbifolia and identification of anti-oxidant components by off-line UPLC-QTOF-MS/MS-free radical scavenging detection

ObjectiveTo identify phytochemical constituents present in the extract of flowers of Xanthoceras sorbifolia and evaluate their anti-oxidant and anti-hyperglycemic capacities. MethodsThe AlCl3 colorimetric method and Prussian Blue assay were used to determine the contents of total flavonoids and total phenolic acids in extraction layers, and the bioactive layers was screened through anti − oxidative activity in vitro. The Waters ACQUITY UPLC system and a Waters ACQUITY UPLC BEH C18 column (2.0 mm × 150 mm, 5 μm) were used to identify the ingredients. And anti-oxidative ingredients were screened by off-line UPLC-QTOF-MS/MS-free radical scavenging. The ameliorative role of it was further evaluated in a high-fat, streptozotocin-induced type 2 diabetic rat model and the study was carried out on NADPH oxidase (PDB ID: 2CDU) by molecular docking. ResultsCombined with the results of activity screening in vitro, the anti − oxidative part was identified as the ethyl acetate layer. A total of 24 chemical constituents were identified by liquid chromatography-mass spectrometry in the ethyl acetate layer and 13 main anti-oxidative active constituents were preliminarily screened out through off-line UPLC-QTOF-MS/MS-free radical scavenging. In vivo experiments showed that flowers of X. sorbifolia could significantly reduce the blood glucose level of diabetic mice and alleviate liver cell damage. Based on the results of docking analysis related to the identified phytocompounds and oxidase which involved in type 2 diabetes, quercetin 3-O-rutinoside, kaempferol-3-O-rhamnoside, isorhamnetin-3-O-glucoside, and isoquercitrin showed a better inhibitory profile. ConclusionThe ethyl acetate layer was rich in flavonoids and phenolic acids and had significant anti-oxidant activity, which could prevent hyperglycemia. This observed activity profile suggested X. sorbifolia flowers as a promising new source of tea to develop alternative natural anti-diabetic products with a high safety margin.

Sensitive detection of butyrylcholinesterase activity based on a stimuli-responsive fluorescence reaction

A fluorogenic reaction between the chelate of Mn(II)-citric acid and terephthalic acid (PTA) was discovered, which was carried out through heating the aqueous mixture of Mn2+, citric acid and PTA. Detailed investigations indicated the reaction products were 2-hydroxyterephthalic acid (PTA-OH), which was attributed to the reaction between PTA and OH, formed by the triggering of Mn(II)-citric acid in the presence of dissolved O2. PTA-OH showed a strong blue fluorescence, peaked at 420 nm, and the fluorescence intensity presented a sensitive response to pH of the reaction system. Based on these mechanisms, the fluorogenic reaction was used for the detection of butyrylcholinesterase activity, achieving a detection limit of 0.15 U/L. The detection strategy was successfully applied in human serum samples, and it was also extended for the detection of organophosphorus pesticides and radical scavengers. Such a facile fluorogenic reaction and its stimuli-responsive properties offered an effective tool for designing detection pathways in the fields of clinical diagnosis, environmental monitoring and bioimaging.

Facile Detection of Light-Controlled Radical Scavengers from Natural Products Using In Situ UV-LED NMR Spectroscopy.

With the recent development of chemical analysis technology, attention has been placed on natural light-sensitive compounds that exhibit photoreactivity to expand the structural diversity of natural product chemistry. Photochemical reactions that proceed via a free radical mechanism could be used to modulate the radical-scavenging ability of natural products as well as involve structural change. As the health benefits of radicals are also presented, there is a need for a controllable radical scavenging method for topical and selective application. In this study, we developed a novel acquisition and processing method to identify light-controlled radical scavengers in plant extracts and evaluate their antioxidant activity under light irradiation based on in situ UV-LED NMR spectroscopy. Using the developed method, licochalcones A and B, in which the trans and cis isomers undergo reversible photoisomerization, were selectively identified from licorice root extract, and their light-induced free radical scavenging activity was confirmed.

Hydrothermal synthesis of novel 1-aminoperylene diimide/TiO2/MoS2 composite with enhanced photocatalytic activity

1-aminoperylene diimide/TiO2/MoS2 composite (NH2-PDI/TiO2/MoS2) with ordered structure was prepared by hydrothermal synthesis method. The composite was characterized by XRD, SEM, FTIR, XPS, BET, DRS, PL, EIS, Raman, photocurrent, and Mott-Schottky plots spectroscopy. The potential positions of the conduction and valence bands, and the band gap energy of the semiconductors were estimated. The composite exhibited higher photocatalytic activity compared with the mono-component systems. The apparent rate constants (k) were determined as 0.00616, 0.00352, 0.00738, 0.00517, 0.00752, and 0.00806 min−1 for TiO2, NH2-PDI, NH2-PDI/TiO2, MoS2, MoS2/TiO2, and NH2-PDI/TiO2/MoS2, respectively. The detection of radical scavengers confirmed that superoxide radicals, photogenerated holes, and photogenerated electrons were the main active substances for MB degradation. Between type II- heterojunction mechanism and Z-scheme mechanism, the latter could explain the enhanced photocatalytic activity of the composite better. The Z-scheme mechanism accumulates more electrons at CB level of NH2-PDI and hence generates more super oxide radicals.

Perylene Tetracarboxylic Tetraester Sensitized Titanium Dioxide Nanocomposite for Enhanced Photodegradation

The construction of organic–inorganic hybrids is one of the important ways to improve performances of photocatalysts. In this study, perylene tetracarboxylic tetra (n-butyl) ester loaded titanium dioxide (PTTE-B/TiO2) nanocomposite was successfully synthesized by hydrothermal method. The catalyst was characterized by XRD, DRS, XPS, Photoluminescence spectroscopy (PL) and BET. FT-IR and XPS analyses revealed the presence of Ti[Formula: see text]O bond in the interfaces between TiO2 and PTTE-B. The photoluminescence intensity of PTTE-B/TiO2 was decreased compared to TiO2, which indicated a suppression of electron–hole recombination by the loaded PTTE-B nanoparticles. The photocatalytic activity of PTTE-B/TiO2 nanocomposite for degradation of EBT using visible light was higher than that of prepared TiO2 or TiO2-P25. The detection of radical scavengers confirmed that h[Formula: see text] and [Formula: see text] were the main active substances for Eriochrome Black T (EBT, an azo dye) degradation. The Ti[Formula: see text]O bond acted as a short and fast channel for photogenerated charge carriers to migrate from PTTE-B to TiO2. Furthermore, PTTE-B/TiO2 was found to be stable and reusable.

Isolation of homoisoflavonoids from the fibrous roots of Ophiopogon japonicus by recycling high-speed counter-current chromatography and online antioxidant activity assay

Two pairs of homoisoflavonoid analogues, 6-aldehydo-isoophiopogonanone A (1) / 6-aldehydo-isoophiopogonanone B (2) and methylophiopogonanone A (3) / methylophiopogonanone B (4), were obtained from the fibrous roots of Ophiopogon japonicus (L. f.) Ker-Gawl. (FROJ) by silica gel column chromatography (SGCC) and recycling high-speed counter-current chromatography (rHSCCC). First, the ethyl acetate fraction from the 70% ethanol extract was pre-separated by SGCC with a petroleum ether–ethyl acetate gradient (50:1–2:1, v/v). Then, the two sub-fractions containing homoisoflavonoid analogues were further separated by rHSCCC with n-hexane–ethyl acetate–methanol–acetonitrile–water (3:2:3.5:1:0.5, v/v) and n-hexane–ethyl acetate–methanol–acetonitrile–water (3:2:2.5:1:1.5, v/v). Finally, 6-aldehydo-isoophiopogonone A (16 mg), 6-aldehydo-isoophiopogonanone B (26 mg), methylophiopogonanone A (46 mg), and methylophiopogonanone B (148 mg) were obtained with purities of 97.82%, 96.70%, 97.76%, and 94.62%. Their structures were identified by high-resolution quadrupole-time-of-flight mass spectrometry (HR-QTOF-MS), ultraviolet (UV), and nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR). These results demonstrated that rHSCCC could be used for the large scale preparation of homoisoflavonoid analogues from FROJ, which provides scientific support for utilization of untraditional medicinal part of O. japonicus and also for reduction in waste of plant resources. Additionally, an online antioxidant activity assay was investigated with hyphenated HSCCC-DPPH (1-diphenyl-2-picrylhydrazyl) radical scavenging detection.

PHENOLIC COMPOUNDS IN LAUREL WOOD: A NEW SOURCE OF PROANTHOCYANIDINS

Laurel tree (Laurus nobilis L.) wood is a byproduct of the pruning of this tree widely cultivated for its aromatic leaves. The phenolic composition of laurel wood has not been reported yet. The aim of this work was to study the phenolic profile of an ethyl acetate extract of laurel wood in order to know its potential as a source of antioxidants and valuable compounds. Three flavan-3-ols, 4 dimeric B-type proanthocyanidins, and 2 trimeric A-type procyanidins were isolated by preparative column chromatography and semi-preparative HPLC, using a fast online post-column radical scavenging detection technique as guidance. All isolated compounds were fully characterized by spectroscopic (NMR, IR, UV) and spectrometric (MS) techniques. In addition, 17 minor proanthocyanidins were tentatively identified by liquid chromatography coupled to mass spectrometry (HPLC–ESI–MS). The extract was dominated by the presence of cinnamtannin B-1, a bioactive trimeric A-type procyanidin reported as a strong antioxidant of commercial value. According to the chemical composition found, it can be concluded that laurel wood is a renewable source of potential bioactive phenolic compounds.

Quickly verifying the antioxidant contribution of the individual composition in natural antioxidants by HPLC-free radical scavenging detection

An HPLC-free radical-scavenging activity detection (HPLC-FRSAD) method was established to evaluate the antioxidant activity of each component in a mixed standard phenolics solution (MSPs). This method quickly displayed the differences in each composition in the MSPs with regard to their scavenging abilities by DPPH radical and ABTS radical cation. The relationship between the structures and the antioxidant activities of the compounds was discussed. Gallic acid, quercetin, and kaempferol in the MSPs’ compositions represented stronger scavenging activities of DPPH radical and ABTS radical cation. Furthermore, the developed method was successfully applied to screen major antioxidants from guava leaf tea extract (GLTE). The results revealed that the gallic acid, procyanidin B3, quercetin, and kaempferol in the extract had higher DPPH radical and ABTS radical cation scavenging activities than other components, which would be responsible for the antioxidant activities of GLTE. The present method provides a useful strategy for rapidly recognizing the key antioxidant components in complex natural products.

Functional Teas from the Leaves of Arbutus unedo: Phenolic Content, Antioxidant Activity, and Detection of Efficient Radical Scavengers.

The phenolic content/composition and antioxidant activity of hot/cold infusion and decoction from the leaves of Arbutus unedo were studied for the first time. 1,1-diphenyl-2-picrylhydrazyl (DPPH●), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS●+), crocin-bleaching, copper-reducing, and liposome accelerated oxidation assays were used for the evaluation of the activity in vitro. In vivo, the extracts were examined for their ability to protect S. cerevisiae cells from H2O2 induced oxidative stress. An on-line high-performance liquid chromatography-DPPH● assay was applied to identify potent radical scavengers and comment on their contribution to the total activity. The addition of leaves to boiling water (decoction) was the most appropriate practice to apply since the highest phenol intake (220.2mg gallic acid/cup served) was obtained. Additionally, its antioxidant activity was equal or superior to that of the other extracts. Flavonols (~51-61mg/g dry extract) were the main phenols in all the extracts, with quercitrin accounting for ~20% of the total phenol amount. The on-line DPPH● method verified the high potency of the decoction and indicated as the most active radical scavengers, two galloylquinic acid derivatives and myricitrin, accounting for ~28-45% and ~11-13% of the total scavenging, respectively. Present data may contribute to the future exploitation of A. unedo leaves by the food industry for health-promoting herbal tea preparations and dietary supplements.

Rapid screening, separation, and detection of hydroxyl radical scavengers from total flavonoids of Ginkgo biloba leaves by chromatography combined with molecular devices.

Hydroxyl radicals are the most reactive free radical of human body, a strong contributor to tissue damage. In this study, liquid chromatography coupled to electrospray ionization mass spectrometry was applied to screen and identify hydroxyl radical scavengers from the total flavonoids of Ginkgo biloba leaves, and high-performance counter current chromatography was used to separate and isolate the active compounds. Furthermore, molecular devices were used to determine hydroxyl radical scavenging activities of the obtained hydroxyl radical scavengers and other flavonoids from G. biloba leaves. As a result, six compounds were screened as hydroxyl radical scavengers, but only three flavonoids, namely, rutin, cosmos glycosides and apigenin-7-O-Glu-4'-O-Rha, were isolated successfully from total flavonoids by high-performance counter current chromatography. The purities of the three obtained compounds were over 90%, respectively, as determined by liquid chromatography. Molecular devices with 96-well microplates evaluation indicated that the 50% scavenging concentration values of screened compounds were lower than that of other flavonoids, they performed greater hydroxyl radical scavenging activity, and the evaluation effects were consistent with the liquid chromatography with mass spectrometry screening results. Therefore, chromatography combined with molecular devices is a feasible and an efficient method for systematic screening, identification, isolation, and evaluation of bioactive components in mixture of botanical medicines.

Evaluation of the contributions of polyphenols in Chinese propolis by on-line HPLC–ABTS method

Propolis, a well-known folk medicine in the world, has drawn growing attention due to the various bioactivities. An on-line method of coupling HPLC with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) cationic radical scavenging detection (HPLC–ABTS) was developed to screen the single polyphenol possessing ABTS cationic radical scavenging activity in ethanol extracts of Chinese propolis (EECP). Ten common polyphenols were selected from the active fingerprints of ten EECP samples obtained from different provinces of China by the on-line HPLC–ABTS method and quantified by Trolox equivalent. Their contributions to the antioxidant property of EECP were compared. Furthermore, Pearson’s correlation analysis was applied to determine whether there was a correlation between the antioxidant activity of individual polyphenol and total antioxidant activity of EECP. The results indicated that the composition of polyphenols in ten EECP samples obtained from China was similar. But the different polyphenols contributed in different way. Caffeic acid, kaempferol, and galangin simultaneously were the most prominent contributors to the antioxidant properties of ten EECP. The contributions of pinobanksin 3-acetate, caffeic acid phenethyl ester (CAPE), and caffeic acid cinnamyl ester varied largely with the different geographical regions. They, except galangin, significantly correlated with the antioxidant property of propolis. However, pinobanksin, pinocembrin, chrysin, p-coumaric acid, and ferulic acid rarely contributed to the antioxidant property of Chinese propolis. Therefore, the on-line HPLC–ABTS method was convenient, effective, and comprehensive to evaluate the contributions of polyphenols to the antioxidant property for Chinese propolis.

An on-line HPLC-FRSD system for rapid evaluation of the total antioxidant capacity of Citrus fruits

An on-line high performance liquid chromatography-free radical scavenging detection (HPLC-FRSD) system was developed for rapidly evaluating the total antioxidant capacity (TAC) of Citrus fruits. With the system, all samples can be analyzed within 5min. The on-line HPLC-FRSD system has low limits of detection (0.001–0.010mgmL−1) and quantification (0.005–0.020mgmL−1), excellent recovery rate (90.44–115.72%), stability (RSD<15.80%), reproducibility (RSD<2%), and precision (RSD<2%). Using a guard column instead of an analytic column, this new on-line HPLC-FRSD system performed better than the existing on-line HPLC methods in the analysis of the TAC of Citrus. Compared with the conventional off-line 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiozoline-6)-sulphonic acid (ABTS) radical scavenging methods, our newly developed system is faster and more robust. The methodology can be a good alternative for analysis TACs of Citrus fruits and potentially for other plants and plant-based products.

Low-temperature thin-layer chromatography preliminary bioautographic tests for detection of free radical scavengers and acetylcholinesterase inhibitors in volatile samples

The aim of the presented study was to develop a simple, high-throughput analytical procedure to indicate volatile samples (essential oils and natural aromas commonly used in pharmaceutical and food industry), as well as individual compounds/group of compounds possessing free radical scavenging and/or acetylcholinesterase (AChE) inhibitory activity (one or both of the aforementioned properties). First, simple “dot-blot” tests were proposed to pinpoint the samples with the aforementioned activities. Some of those which gave positive results were passed to further analyses by means of low-temperature thin-layer chromatography (TLC). The comparison of the results obtained on different adsorbents led to a conclusion that TLC-AChE inhibition diazotization test should be performed on plates with polar coatings. The hypothesis for the observed weak AChE inhibitory activity on RP-18W plates, when compared to other adsorbents, is also proposed for further verification. The application of these procedures revealed t...

Preparative isolation and purification of antioxidative diarylheptanoid derivatives from Alnus japonica by high‐speed counter‐current chromatography

This study employed the online HPLC-2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)(+) bioassay to rapidly determine the antioxidant compounds occurring in the crude extract of Alnus japonica. The negative peaks of the ABTS(+) radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS(+)-based antioxidant activity profile showed that three negative peaks exhibited antioxidant activity. High-speed counter-current chromatography (HSCCC) was used for preparative scale separation of the three active peaks from the extract. The purity of the isolated compounds was analyzed by HPLC and their structures were identified by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC). Two solvent systems composed of n-hexane/ethylacetate/methanol/water (4:6:4:6, v/v) and of ethyl acetate/methanol/water (1:0.1:1, v/v) were performed in high-speed counter-current chromatography. Consequently, a total of 527 mg of hirsutanonol 5-O-β-D-glucopyranoside, 80.04 mg of 3-deoxohirsutenonol 5-O-β-D-glucopyranoside, and 91.0 mg of hirsutenone were obtained with purity of 94.7, 90.5, and 98.6%, respectively.

Identification of antioxidant compounds of Mucuna sempervirens by high-speed counter-current chromatographic separation–DPPH radical scavenging detection and their oestrogenic activity

High-speed counter-current chromatography (HSCCC) was used to separate an ethanolic extract of leaves of Mucuna sempervirens into fractions which were then detected their antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The fractions were grouped into seven larger fractions (components) based on their antioxidant activity. The seven components were isolated by preparative HPLC to yield 12 flavonoids and two phenolic acids identified by electrospray ionisation mass spectrometry and nuclear magnetic resonance analyses. The oestrogenic activity of the 12 isolated flavonoids was evaluated by the luciferase assay based on the MVLN cell line. The results indicate that leaves of M. sempervirens are a flavonoid-rich resource that may supply antioxidant and oestrogenic compounds to the human body. The HSCCC separation–DPPH radical scavenging detection could be widely applied for rapid screening and isolation of antioxidants from complex plant extracts.

Isolation, identification and activity of natural antioxidants from horehound (Marrubium vulgare L.) cultivated in Lithuania

In an earlier screening of Lithuanian plants, horehound (Marrubium vulgare) showed good antioxidant activity and as this species is used in herbal teas and cough pastilles it was selected for further investigation. Some fractions of the aerial parts were strong scavengers of the model free radicals DPPH and ABTS+. Activity in the β-carotene bleaching assay and the rapeseed oil oxidation assay was lower. Several active compounds were observed in the crude methanol–water extract, and in butanol and methyl tert-butyl ether fractions using HPLC with on-line radical scavenging detection. After multi-step fractionation of these fractions five compounds possessing radical scavenging activity were purified and their structures were elucidated by NMR and MS as 5,6-dihydroxy-7,4′-dimethoxyflavone (syn. ladanein), 7-O-β-glucopyranosyl luteolin, 7-O-β-glucuronyl luteolin, verbascoside and forsythoside B. Their activities were tested in off-line DPPH and ABTS+ free radical scavenging assays, and compared with the antioxidants rosmarinic acid and Trolox.

SCREENING AND IDENTIFICATION OF RADICAL SCAVENGERS FROM NEO-TARAXACUM SIPHONANTHUM BY ONLINE RAPID SCREENING METHOD AND NUCLEAR MAGNETIC RESONANCE EXPERIMENTS

An online rapid screening method, the high-performance liquid chromatography (HPLC)–diode array detector (DAD)–radical scavenging detection (RSD)–electrospray ionization (ESI)–mass spectroscopy (MS)/MS system, was developed for the screening and identification of radical scavengers from Neo-Taraxacum siphonanthum, a new species found in China in 1989. For further characterization, the target compounds were isolated by silica column chromatography, preparative high-performance liquid chromatography (HPLC), HSCCC, and Sephadex LH-20 column chromatography and elucidated on the basis of ultraviolet (UV), ESI-MS/MS, and nuclear magnetic resonance (NMR) spectroscopy, as well as the chemical analysis. Eighteen antioxidative polyphenols (5 caffeic acid derivatives and 13 flavonoid derivatives) were characterized from Neo-T. siphonanthum. The distribution of all compounds was discussed in a chemosystematic context, which suggested that the genera Neo-Taraxacum and Taraxacum might relate chemosystematically.

Isolation of antioxidative secoiridoids from olive wood ( Olea europaea L.) guided by on-line HPLC–DAD–radical scavenging detection

The woody portion of olive tree pruning is a source of natural antioxidants of potential interest for the food industry. This work deals with the isolation and identification of further antioxidants present in an ethyl acetate extract of olive ( Olea europaea L.) wood. Thus, a new secoiridoid, oleuropein-3″-methyl ether ( 1), together with six known secoiridoids, 7″ S-hydroxyoleuropein ( 2), jaspolyanoside ( 3), ligustroside 3′- O-β- d-glucoside ( 4), jaspolyoside ( 5), isojaspolyoside A ( 6) and oleuropein 3′- O-β- d-glucoside ( 7) were isolated by combining HPLC with fast on-line post-column radical scavenging activity evaluation. The structures of these compounds were determined by spectroscopic methods. The antioxidant activity of the pure compounds was determined by measuring the radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ). Compounds 2, 5 and 7 displayed a higher antioxidative effect than the synthetic antioxidant BHT and lower than rosmarinic acid, whereas compounds 3 and 4 showed weak DPPH scavenging activity.

Effects of Far Infrared Radiation Drying on Antioxidant and Anticoagulant Activities of Ecklonia cava Extracts

Ecklonia cava is popular in Korea as a marine functional materials. Freeze drying (FD) is generally used for drying before extraction of bioactive compounds from E. cava. But, FD resulted in low productivity due to long drying time and the relatively high operating cost. On the other hand, far infrared radiation drying (FIRD) was known to be significant advantages such as higher drying rate, energy saving, and functional improvement. Hence, the objectives of this study were to evaluate the effectiveness of FIRD, the bioactive compound composition, and antioxidant and anticoagulant activities of water and enzymatic (Celluclast, Protamex) extracts from FIR-dried E. cava as compared to those extracts from FD-dried one. In this drying processing, FIRD was more effective than FD in the drying time, the extract yield, and the polyphenolic content in the extracts. Most FIRD extracts except for a dry condition at 80°C had lower IC50 values than FD extracts. Among them, Celluclast extract from E. cava dried by FIRD at 40°C (FIRD40-CE) was separated by HPLC and then radical scavenging activities of the separated compounds were directly evaluated by on-line coupled 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS+) radical scavenging detection (RSD) system. The lower trace shows the peaks on ABTS+-RSD system which indicated the presence of antioxidant activity. In all the tested samples for anticoagulant activity of the extracts, Celluclast extracts from E. cava dried by FD (FD-CE) and FIRD40-CE were selected and a crude polysaccharide fraction with the molecular weights above 30 kDa was obtained. The crude polysaccharide fraction of FD-CE and FIRD40-CE showed the activated partial thromboplastin time activity. These results suggest that FIRD process would be more effective in releasing bioactive compounds from seaweed such as E. cava at an industrial scale.

Rapid screening and identification of the antioxidants in Hippocampus japonicus Kaup by HPLC‐ESI‐TOF/MS and on‐line ABTS free radical scavenging assay

In this paper, a method based on HPLC coupled with ESI-TOF/MS and on-line to a stable free radical scavenging detection system has been developed to rapidly screen and identify the major antioxidants in the water extract of Hippocampus japonicus Kaup, a kind of marine medicinal organism. The developed on-line stable free radical scavenging detection method of water extract from H. japonicus Kaup revealed the presence of three radical scavenging compounds, which were simultaneously identified as hypoxanthine, phenylalanine and tryptophane by high-resolution TOF-MS. To the best of our knowledge, it is original to demonstrate the rapid and successful use of HPLC-ESI-TOF/MS and on-line 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt free radical scavenging assay to directly and simultaneously screen and identify the antioxidants in the water extracts of H. japonicus Kaup without any purification. The present method provides a useful improvement on the reducing of workload and a powerful tool for rapid screening and identification of free radical scavenging compounds in complex natural products.