Isomers are widely distributed in Chinese herbal medicines,and can be discriminated by energy-resolved mass spectrometry( ER-MS). However,ER-MS was performed through direct injection of reference compounds with syringe pump,which encountered a significant technical barrier for high-throughput and automated measurements. Herein,online ER-MS was conducted using LC-MS platform,and a pair of isomers,kaempferol vs luteolin,were employed as a case study to illustrate and assess the utility of online ER-MS for isomeric discrimination. High-resolution tandem mass spectrometry data of both flavonoids were acquired on LC-QE-Orbitrap-MS,and the fragmentation pathways responsible for the primary fragment ions were proposed. The primary signal in MS1 occurred at m/z 285( [M-H]-),and the primary signals of either compound generated by retro-Diels-Alder fragmentation were observed at m/z 151 and 133. The spectral information was subsequently transferred onto LC-Qtrap-MS platform to carry out online ER-MS. Two precursor-to-product ion transition candidates were constructed as m/z 285>151 and 285>133,and either afterward derived a set of pseudo-ion transitions( PITs) and so forth,exactly corresponding to a series of progressive collision energies( eg-5,-8,-11 e V,and so on). All PITs were typed into the monitoring list of multiple reaction monitoring program to generate the peak area datasets. Either dataset was normalized using the highest values in the set and imported into Graph Pad Prism software to plot the Gaus-sian-shaped curve that was termed as the break-down graph. The apex of the regressive curve was termed as optimal collision energy( OCE). The OCE values corresponding to m/z 285>151 were calculated as-29. 06 e V and-35. 71 e V for kaempferol and luteolin,respectively. In the case of m/z 285>133,the OCEs were yielded as-44. 15 e V for kaempferol and-49. 01 e V for luteolin. With re-ference to their chemical structures,the location of hydroxyl group was regarded to be responsible for the differences of either m/z 285>151 or 285>133 between the isomers,attributing to their different bond properties. Above all,online ER-MS offers an eligible tool for isomeric discrimination,and provides meaningful information for the accurate chemical composition characterization based on LC-MS,which is not limited to Chinese herbal medicines.
Chromatography, Liquid ; Flavonoids ; Kaempferols ; Luteolin ; Tandem Mass Spectrometry

OBJECTIVETo find out the bioactive principles of Alpinia tonkinensis, by investigating its chemical constituents.

METHODIt was extracted with MeOH, distributed by different solvents, and isolated via column chromatography on silica gel.

RESULTSix compounds were elucidated through spectral analysis, which were as follows: 4',7-dimethylkaempferol(I), 5-hydroxy-3',4',7-trimethoxyflavanone(II), kumatakenin(III), 4',5,7-trimethoxyflavonol(IV), ombuine(V), and kaempferol(VI).

CONCLUSIONSix flavonoids were isolated from this plant for the first time, and so were four compounds from genus Alpinia.

Alpinia ; chemistry ; Flavonoids ; chemistry ; isolation & purification ; Kaempferols ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Rhizome ; chemistry

OBJECTIVETo study the chemical constituents isolated from the whole plant of Aconitum tanguticum.

METHODChemical constituents were isolated and purified from the title plant by using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, ODS and preparative HPLC. Their structures were elucidated by spectroscopic techniques including 1H-NMR, 13C-NMR, 2D-NMR, and ESI-MS.

RESULTSeven compounds were isolated from this plant and their structures were identified as kaempferol-3-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside]-7-O-alpha-L-rhamnopyrano-side (1), kaempferol-3-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside]-7-O-alpha-L-rhamnopyranoside (2), kaempferol 7-O-alpha-L-rhamnopyranoside (3), gentiopieroside (4), vomifoliol-9-O-beta-D-glucopyranoside (5), dihydrovomifoliol-9-O-beta-D-glucopyranoside (6) and 3,4-dihydroxyphenyl alcohol-beta-D-glucopyranoside (7).

CONCLUSIONAll the compounds were isolated from this plant for the first time.

Aconitum ; chemistry ; Glucosides ; chemistry ; Iridoid Glucosides ; chemistry ; Kaempferols ; chemistry ; Magnetic Resonance Spectroscopy ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo develop a LC-MS/MS method for determination of astragalin in rat plasma and study its pharmacokinetics in rats.

METHODSThe analytical column was packed with ZORBAX SB-C18, and the mobile phase was methanol-water containing 10 mmol/L ammonium acetate-formic acid (80:20:0.15, V/V/V). Quercetin was used as the internal standard (IS). Multiple reaction monitoring (MRM) mode was employed, and the transition of m/z was m/z 449.1→m/z 287.1 for astragalin, and m/z 301.1→m/z 151.1 for IS.

RESULTSA good liner relationship was obtained within the range of 1.00-1000 ng/ml (r(2)=0.9929), and the lower limit of quantification of astragalin was 1.00 ng/ml in rat plasma. The extraction recoveries were all above 93%. After oral administration of astragalin, the maximum plasma concentration of astragalin was 231.1∓67.3 ng/ml and the time to reach this value was 0.5∓0.1 h, with a half-life of 3.9∓1.3 h and an AUC of 782.6∓152.8 ng·h/ml.

CONCLUSIONThe method is highly sensitive, selective and rapid for determination of the concentration of astragalin in rat plasma to facilitates the study of its pharmacokinetics.

Animals ; Chromatography, High Pressure Liquid ; Kaempferols ; blood ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

The flavonoids in Panax notoginseng were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), and the content of three main flavonoids in P. notoginseng of different specifications and grades collected from different habitats was determined by HPLC-DAD. Flavonoids and anthocyanins were analyzed by UPLC-Q-TOF-MS/MS in the positive and negative ion modes, respectively. Twelve flavonoid glycosides and one anthocyanin glycoside in P. notoginseng were identified, but no flavonoid aglycones were detected. Among them, 12 compounds were identified in the underground part of P. notoginseng for the first time and eight compounds were first reported in this plant. Moreover, six and four compounds were identified in the Panax genus and the Araliaceae family for the first time, respectively. A method for simultaneous determination of three flavonoids in P. notoginseng was established by HPLC-DAD. The content of flavonoids in 721 P. notoginseng samples of 124 specifications and grades collected from 20 different habitats was simultaneously determined. Among three flavonoids determined, the content of quercetin-3-O-(2″-β-D-xylosyl)-β-D-galactoside was the highest with the average content in the tested samples of 161.0 μg·g~(-1). The content of compounds quercetin-3-O-hexosyl-hexoside and kaempferol-3-O-pentosyl-hexoside was relatively low, with the average content of 18.5 μg·g~(-1)(calculated as quercetin-3-O-sophoroside) and 49.4 μg·g~(-1)(calculated as kaempferol-3-O-sangbu diglycoside). There were significant differences in flavonoids content of samples from different production area. The content of flavonoids in spring P. notoginseng was significantly lower than that in winter P. notoginseng when the other influencing factors such as production areas, germplasm resources, and cultivation conditions were fixed. As for P. notoginseng of different specifications, the flavonoid content in the part connecting the taproot and the aboveground stem was significantly higher than that in other parts. The results of large-scale data showed that the flavonoid content gradually increased with the increase in the number of heads. There were significant differences between the flavonoid content in most specifications and grades, especially the 20-head P. notoginseng and countless head P. notoginseng, whose content was significantly lower and significantly higher than that of other specifications and grades, respectively. This study provides a scientific basis for the study of the effective components and quality control of P. notoginseng from the perspective of flavonoids.
Flavonoids/analysis* ; Anthocyanins/analysis* ; Quercetin ; Chromatography, High Pressure Liquid/methods* ; Kaempferols ; Tandem Mass Spectrometry/methods* ; Glycosides

The aim of present study was to investigate the effects of kaempferol on cellular proliferation and cell cycle arrest and explore the mechanism for these effects in human breast carcinoma MDA-MB-453 cells. Cells were treated with kaempferol at various concentrations (ranging from 1 to 200 microM) for 24 and 48 hrs. Kaempferol significantly inhibited cancer cell growth in cells exposed to 50 and 10 microM of kaempferol and incubated for 24 and 48 hrs, respectively. Exposure to kaempferol resulted in cell cycle arrest at the G2/M phase. Of the G2/M-phase related proteins, kaempferol down-regulated CDK1 and cyclin A and B in cells exposed to kaempferol. In addition, small DNA fragments at the sub-G0 phase were increased by up to 23.12 and 31.90% at 10 and 50 microM incubated for 24 and 48 hrs, respectively. The kaempferol-induced apoptosis was associated with the up-regulation of p53. In addition, the phosphorylation of p53 at the Ser-15 residue was observed with kaempferol. Kaempferol inhibits cell proliferation by disrupting the cell cycle, which is strongly associated with the induction of arrest at G2/M phase and may induce apoptosis via p53 phosphorylation in human breast carcinoma MDA-MB-453 cells.
Apoptosis ; Breast ; Breast Neoplasms ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cyclin A ; DNA ; Humans ; Kaempferols ; Phosphorylation ; Proteins ; Up-Regulation

OBJECTIVETo determine quercetin and kaempferol in the plant of genus Dysosma that come from different species, different plant parts or different growing areas, which provide the basis of rational utilization of Dysosma plants.

METHODThe analysis was performed on a Diamonsil C18 column (4.6 mm x 150 mm, 5 microm) eluted with the mobile phase of methanol-water containing 0.1% phosphoric acid (60:40). The flow rate was 1 mL x min(-1), the detection wavelength was 360 nm; and the column temperature was set at 25 degrees C.

RESULTThe linear ranges of quercetin and kaempferol are 0.22-1.1 microg and 0.42-2.1 microg. The average recoveries of quercetin and kaempferol are 97.1% (RSD 1.4%) and 99.6% (RSD 2.4%); respectively.

CONCLUSIONThe contents of flavones in different species of Dysosma are significantly different.

Berberidaceae ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; methods ; Drugs, Chinese Herbal ; analysis ; Kaempferols ; analysis ; Quercetin ; analysis

OBJECTIVETo study chemical constituents contained in Phymatopteris hastate and their antioxidant activity.

METHODChemical constituents were separated and purified from P. hastate by using such methods as silica gel, Toyopearl HW-40C and HPLC preparative chromatography. Their structures were identified by spectroscopic methods such as NMR. Furthermore, 1, 1-diphenyl-2-picryl-hydrazyl(DPPH) method was used to assess the antioxidant activity of each compound.

RESULTFourteen compounds were separated and identified as 4-O-beta-D-glucopyranosyl-ethyl-trans-caffeicate (1), kaempferlo-7-O-alpha-L-rhamnopyranside (2), kaempferol-3, 7-di-O-alpha-L-rhamnopyranoside (3), kaempferol-3-O-alpha-L-arabinofuranosyl-7-O-alpha-L-rhamnopyranoside (4), juglanin (5), naringin (6), naringenin-7-O-beta-D-glucopyranoside (7), trans-caffeic acid (8), trans-caffeic acid-3-O-beta-D-glucopyranoside (9), trans-cinnamic acid-4-O-beta-D- glucopyranoside (10), trans-melilotoside (11), cis-melilotoside (12), ethyl chlorogenate (13), protocatechuic acid (14). The antioxidation experiment showed an obvious antioxidant activity in compounds 1-9, 13-14.

CONCLUSIONAll of the compounds were separated from this genus for the first time. Among them, compound 1 was not seen in literature reports and assumed to be a new artifact derived from compound 9 and ethanol. Compounds 1-9, 13-14 showed a remarkable antioxidant activity.

Antioxidants ; pharmacology ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Flavanones ; analysis ; Kaempferols ; analysis ; Magnetic Resonance Spectroscopy

OBJECTIVETo study the chemical constituents of the twigs of Morus atropurpurea.

METHODThe compounds of the EtOAc fraction were isolated and purified by column chromatography on silica gel, polyamide, Sephadex LH -20, and their structures were elucidated on the basis of spectroscopic evidence (MS, NMR).

RESULTEleven compounds were identified as mulberrin (1), cyclomulberrin (2), morusin (3), cyclomorusin (4), 2', 4',4, 2"-tetrahydroxy-3'-{3"-methylbut-3"-enyl-}-chalcone (5), mulberrofran G (6), scopoletin (7), moruchalcone A (8), kaempferol (9), ursolic acid (10), beta-daucosterol (11).

CONCLUSIONExcept compounds 9 and 11, all the other compounds were obtained from M. atropurpurea for the first time.

Flavonoids ; chemistry ; Kaempferols ; chemistry ; Magnetic Resonance Spectroscopy ; Morus ; chemistry ; Phytosterols ; chemistry ; Plant Stems ; chemistry ; Scopoletin ; chemistry ; Triterpenes ; chemistry

Eight phenolic compounds (1-7) were isolated from the methanol extract of the root of Rhodiola dumulosa. Their structures were identified as kaemperol (1), Quercetin (2), Gallic acid (3), (+) -Isolariciresinol-3alpha-O-beta-D-glucopyranoside (4), (-)-Isolariciresinol-3alpha-O-beta-D-glucopyra-noside (5), kaemperol-3-O-beta-D-glucopyranoside-7-alpha-O-L-rhamnoside (6), rutin (7) respectively on the basis of chemical and spectroscopic evidence. The compounds 3-7 were isolated from R. dumulosa for the first time.
Kaempferols ; chemistry ; isolation & purification ; Phenols ; chemistry ; isolation & purification ; Quercetin ; chemistry ; isolation & purification ; Rhodiola ; chemistry ; Rutin ; chemistry ; isolation & purification