Screening and characterization of natural antioxidants in four Glycyrrhiza species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Licorice, derived from the dried roots and rhizomes of several species of genus Glycyrrhiza L. (Leguminosae family), has been traditionally used in herbal medicine for over 4000 years. In recent years, the interest in antioxidative constituents in licorice has greatly increased. In this work, a new method based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking test combined with HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis was developed to screen and identify the antioxidants in licorice. The results of the method validation indicated that the developed method was reliable and repeatable. Compared with DPPH on-line method, the HPLC-Q-TOF MS/MS method combined with DPPH spiking test offered much higher sensitivity and resolution. Using this method, 35 radical scavengers were screened from four Glycyrrhiza species (G. inflata, G. glabra, G. pallidiflora and G. uralensis), and 21 of them were unambiguously or tentatively identified by HPLC-Q-TOF MS/MS. Among the 21 identified flavonoids, 10 compounds had been reported to possess antioxidative activities in the previous studies, and the radical scavenging activities of the other 11 compounds were reported for the first time. The effects of six purified flavonoids on DPPH radical and lipid peroxidation were evaluated for validation of the developed method. The results indicated that HPLC-Q-TOF MS/MS coupled with DPPH treatment is an efficient and powerful method to discover the potential antioxidative compounds from the complex natural product mixtures. In this study, the identified components with free radical scavenging activity, would help to explain the therapeutic benefit of licorice in the treatment of human disease associated with oxidative stress.

Li YJ ，Chen J ，Li Y ，Li Q ，Zheng YF ，Fu Y ，Li P ... -  《-》

Chemical profiling and antioxidant evaluation of Yangxinshi Tablet by HPLC-ESI-Q-TOF-MS/MS combined with DPPH assay.

Yangxinshi Tablet (YXST) is a Chinese patent medicine commonly used to treat cardiovascular diseases. However, its detailed chemical basis and mechanisms of action have not been clarified. In this study, high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) was applied for comprehensive analysis of the chemical constituents in YXST. A total of 127 compounds, including 19 phenolic acids, 12 alkaloids, 51 flavanoids, 32 triterpenoids, 2 lignans, 2 phenylethanoid glycosides, 2 anthraquinones, 1 coumarin, and 6 other compounds, were identified or tentatively deduced by comparing their retention times and MS spectra with those of authentic standards or literature data. To further prove the antioxidant activity of YXST, its free radical scavenging capacity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) spectrophotometric assay and the antioxidants in YXST were rapidly screened by DPPH-HPLC experiment. Especially, salvianolic acid A and salvianolic acid B showed excellent DPPH scavenging activities with the IC50 of 151.9 and 275.6μg/mL, respectively, which were stronger than that of l-ascorbic acid (positive control) with the IC50 of 297.1μg/mL. Additionally, these two most potent antioxidants were detectable in rat plasma after oral administration. In conclusion, this study reported important clues for the further pharmacological and clinical studies of YXST. Meanwhile, it provided a practical strategy for rapid screening and identifying of in vivo antioxidant in traditional Chinese medicine preparations.

Zhu J ，Yi X ，Zhang J ，Chen S ，Wu Y ... -  《-》

Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.

Li YJ ，Chen J ，Li Y ，Li P ... -  《-》

Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum by selective solid phase extraction and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry.

The complexity of natural products always leads to the co-elution of interfering compounds with bioactive compounds, which then has a detrimental effect on structural elucidation. Here, a new method, based on selective solid phase extraction combined with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS), is described for sensitive screening, selective extraction and identification of polyphenolic antioxidants in Polygonatum odoratum. First, 25 polyphenolic antioxidants (1-25) were screened by DPPH spiking with HPLC. Second, polydopamine coated Fe3O4 microspheres (Fe3O4@PDA) were prepared to selectively extract target antioxidants with extraction efficiency from 55% to 100% when the amount of Fe3O4@PDA, extraction time, desorption solvent and time were 10mg, 20 min, acetonitrile, and 5 min. Third, 25 antioxidants (10 cinnamides and 15 homoisoflavanones) were identified by HPLC-DAD-QTOF-MS/MS. Furthermore, the DPPH scavenging activities of purified compounds (IC50, 1.6-32.8 μg/mL) validated the method. Among the identified antioxidants, four of them (12, 13, 18 and 19) were new compounds, four of them (2, 4, 8 and 14) were first obtained from family Liliaceae, five of them (1, 3, 5, 7 and 9) were first reported in genus Polygonatum, while one compound (24) was first identified in this species. The results indicated that the proposed method was an efficient and sensitive approach to explore polyphenolic antioxidants from complex natural products.

Hu X ，Zhao H ，Shi S ，Li H ，Zhou X ，Jiao F ，Jiang X ，Peng D ，Chen X ... -  《-》

Indirect identification of antioxidants in Polygalae Radix through their reaction with 2,2-diphenyl-1-picrylhydrazyl and subsequent HPLC-ESI-Q-TOF-MS/MS.

A rapid and efficient method for the identification of antioxidants in the traditional Chinese medicine Polygalae Radix (PR) by HPLC-ESI-Q-TOF-MS/MS is described. The method is based on the hypothesis that upon reaction of antioxidants with 1,1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas of compounds with potential antioxidant activities in the HPLC chromatogram will be significantly reduced in comparison to the untreated sample. The identity confirmation was achieved by Q-TOF-MS/MS technique. With this method, eight components were proposed possessing potent antioxidant activity. They were identified as sibiricose A5, sibiricose A6, sucrose monoester, polygalaxanthone III, tenuifoliside B, 3',6-disinapoylsucrose (DISS), sucrose diester, tenuifoliside C, respectively. DISS was proposed to be the most potent one. The antioxidant activity of DISS was evaluated by DPPH, ABTS radical scavenging assay and ferric-reducing antioxidant power (FRAP) assay in vitro. Vitamin C (Vc) was used as positive control substance. DISS showed moderate DPPH (DISS's IC50 value was 1024.17 μg/mL, Vc's was 294.68 μg/mL) and ABTS (IC50 324.13 μg/mL, Vc's was 117.50 μg/mL) free radical scavenging capacity and ferric-reducing antioxidant power. DISS can be used as a new source of natural antioxidant in foods and cosmetics.

Shi Q ，Chen J ，Zhou Q ，Lei H ，Luan L ，Liu X ，Wu Y ... -  《-》