Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Sophora flavescens Ait., a well-known Chinese herbal medicine, is widely used in clinical practice for the treatment of viral hepatitis, cancer, gastrointestinal hemorrhage, and skin diseases. This paper is the first report on a method based on the combined use of high-performance liquid chromatography, photodiode array detection, and electrospray ionization tandem mass spectrometry for the comprehensive and systematic separation and characterization of bioactive alkaloids in Sophora flavescens Ait. A total of 22 constituents were identified on the basis of the extracted ion chromatograms for different [M+H](+) ions of the alkaloids present in S. flavescens Ait. Among these, 5 constituents were unambiguously identified by comparing the experimental data on their retention times and MS(n) spectra with those of the authentic compounds, and 17 other constituents were tentatively identified on the basis of their MS(n) fragmentation behaviors and/or molecular weight information from literatures. Furthermore, some characteristic fragmentation pathways of the alkaloids in S. flavescens Ait. were detected and examined. This information may be useful for characterizing the bioactive alkaloids present in S. flavescens Ait. and for possible applications in formulations.     

Identification and characterization of flavonoids from semen zizyphi spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry

Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS ⁿ (HPLC-PDA/LTQ-MS ⁿ ) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C₁₈ column by gradient elution using CH₃CN/H₂O–CH₃COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.     

Binding of flavonoids from Sophora flavescens to the rat uterine estrogen receptor

Prenylflavonoids and lavandulylflavonoids were isolated from the roots of Sophora flavescens Aiton (Fabaceae). The ability of 8-prenylkaempferol (1), kushenol X (2), norkurarinone (3), leachianone A (4), kushenol C (5), maackiain (6) and a root-extract of S. flavescens to displace 17beta-estradiol (E2) from rat uterine estrogen receptor (ER) was determined. Relative binding affinities (RBA) of prenylated flavonoids were weak with RBA values between 0.004 and 0.072. A lavandulyl or prenyl group at the position 8 enhanced binding to rat uterine ER.     

Characterization of phenolic compounds in the Chinese herbal drug Artemisia annua by liquid chromatography coupled to electrospray ionization mass spectrometry

A simple and rapid method has been established for the screening of the main phenolic compounds in Artemisia annua by LC-DAD-ESI-MS(n). A total of 40 phenolic compounds were identified or tentatively characterized in the methanol extract of A. annua, including 8 C-glycosyl flavonoids, 5 O-glycosyl flavonoids, 3 flavonoid aglycones, 21 quinic acid derivatives, 2 benzoicacid glucosides and 1 coumarin. The C-glycosyl flavonoids were reported from A. annua for the first time and they were found to be a new type of main constituents, and might be responsible for its antioxidant and antiviral activity. Quinic acid derivatives were also found to be the major constituents of A. annua.     

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC-DAD-ESI-MS(n)

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography-diode array detection-electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MSⁿ) combined with liquid chromatography-time of flight-mass spectrometry (HPLC-TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MSⁿ fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC-DAD using a C₁₈ column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.     

Identification of polymethoxylated flavones from green tangerine peel (Pericarpium Citri Reticulatae Viride) by chromatographic and spectroscopic techniques

Polymethoxylated flavones (PMFs) were extracted from Pericarpium Citri Reticulatae Viride using a procedure that obtained a consistent mixture of PMFs both in identity and proportion. The mixture consisted of isosinensetin (0.2%) (1), sinensetin (1.7%) (4), tetramethyl-o-isoscutellarein (0.3%) (5), nobiletin (40.5%) (6), tetramethyl-o-scutellarein (1.2%) (7), tangeretin (45.6%) (10), 5-demethylnobiletin (8.7%) (12), 5-demethyl tangeretin (0.8%) (14) and other flavonoids including heptamethoxyflavone (1.0%) (9), among which, compounds 1, 4, 5, 7 and 9 were identified based on their UV spectra, MS data and elution order described in the literature while compounds 6, 10, 12 and 14 were isolated and identified by UV, IR, MS, (1)H NMR, (13)C NMR and 2D NMR spectral studies. In addition, compound 14 was isolated and identified for the first time from Citrus.     

Monoamine oxidase inhibitory components from the roots ofSophora flavescens

In our search for monoamine oxidase (MAO) inhibitors from natural resources, we found that the methanol extract of the roots of Sophora flavescens showed an inhibitory effect on mouse brain monoamine oxidase (MAO). Bioactivity-guided isolation of the extract yielded two known flavonoids, formononetin (1) and kushenol F (2), as active compounds along with three inactive compounds, oxymatrine (3), trifolirhizin (4), and beta-sitosterol (5). Formononetin (1) and kushenol F (2) showed significant inhibitory effects on MAO in a dose-dependent manner with IC50 values of 13.2 and 69.9 microM, respectively. Formononetin (1) showed a slightly more potent inhibitory effect against MAO-B (IC50: 11.0 microM) than MAO-A (IC50: 21.2 microM). Kushenol F (2) also preferentially inhibited the MAO-B activity than MAO-A activity with the IC50 values of 63.1 and 103.7 microM, respectively.     

Analysis of multiple constituents in a Chinese herbal preparation Shuang-Huang-Lian oral liquid by HPLC-DAD-ESI-MSn

A high-performance liquid chromatography/mass spectrometry (HPLC-MS) method for the quality control of Shuang-Huang-Lian oral liquid, an antimicrobial and antipyretic herbal preparation, has been developed. Pure compounds are subjected to tandem mass spectrometry (MS(n)) analysis to clarify their fragmentation rules. Then, the sample of Shuang-Huang-Lian was analyzed by on-line LC-MS(n). A total of 27 compounds, including seven phenylethanoid glycosides, three lignans, seven quinic acids, six saponins and four flavonoids, in the extract of Shuang-Huang-Lian oral liquid have been identified or tentatively characterized. It is expected to develop a comprehensive quality control method of this commonly used herbal preparation.     

Polymethoxylated flavones metabolites in rat plasma after the consumption of Fructus aurantii extract: analysis by liquid chromatography/electrospray ion trap mass spectrometry

A LC with full scan MS(n) method was developed in order to investigate the in vivo absorption and biotransformation of polymethoxylated flavones (PMFs) by analysis of plasma samples from rats after ingestion of Fructus aurantii extract. Four parent compounds and six metabolites with intact flavonoid structures were tentatively identified. The metabolites were either glucuronides of parent compounds or glucuronides of demethylated products of parent compounds.     

Glycosidase inhibitory flavonoids from Sophora flavescens

The methanol extract of Sophora flavescens showed a potent glycosidase inhibitory activity. Active components were identified as well-known flavonoid antioxidants: kushenol A (1), (-)-kurarinone (2), sophoraflavanone G (3), 2'-methoxykurarinone (4), kurarinol (5), 8-prenylkaempferol (6), isoxanthohumol (7), kuraridin (8) and maackian (9). All flavonoids were effective inhibitors of alpha-glucosidase and beta-amylase. Interestingly, lavandulylated flavanones 1-5 had strong alpha-glucosidase inhibitory activities, with IC(50) values of 45 microM, 68 microM, 37 microM, 155 microM and 179 microM, respectively. Kushenol A (1) which does not bear a 4'-hydroxy group showed selective alpha-glucosidase inhibitory activity. Lavandulylated chalcone, kuraridine (8), exhibited IC(50) value of 57 microM against beta-glucosidase, which is the first report of a chalcone displaying glycosidase inhibition. Results showed that 8-lavandulyl group in B-ring was a key factor of the glycosidase inhibitory activities. The inhibition pattern was noncompetitive for alpha-glucosidase, whereas mixed inhibition was observed for beta-amylase.     

Re-evaluation of the antioxidant prenylated flavonoids from the roots of Sophora flavescens

The objective of this research was to re-evaluate the antioxidant effects of the prenylated flavonoids from Sophora flavescens via in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), peroxynitrite (ONOO(-)), and total reactive oxygen species (ROS) assays. In addition, a further examination of kuraridinol, kurarinol, and kurarinone, also isolated from S. flavescens, was carried out by the inhibition of tert-butylhydroperoxide (t-BHP)-induced intracellular ROS generation and t-BHP-induced activation of nuclear factor-kappaB (NF-kappaB). Upon re-examination of the ethyl acetate (EtOAc) soluble fraction of S. flavescens, two major prenylated chalcones, including kuraridin and kuraridinol, along with a minor prenylated flavonol, kushenol C, were isolated as good DPPH scavengers. This was in contrast to the prenylated flavanones, sophoraflavanone G and kurarinone, which were isolated from the methylene chloride (CH(2)Cl(2)) fraction of the same source. Five flavanones consisting of kushenol E, leachianone G, kurarinol, sophoraflavanone G, and kurarinone exhibited significant antioxidant potentials in the ABTS, ONOO(-), and total ROS assays; however, the prenylated chalcones and prenylated flavonol showed more potent scavenging/inhibitory activities than the prenylated flavanones. Therefore, the prenylated chalcones and prenylated flavonol, rather than the prenylated flavanones, may make important contributions toward the marked antioxidant capacities of S. flavescens. Furthermore, kuraridinol, kurarinol, and kurarinone showed significant inhibitory activities against intracellular ROS levels as well as NF-kappaB activation by t-BHP. Overall, the results indicate that S. flavescens and its prenylated flavonoids may possess good anti-inflammatory activity, which is implicated in their significant antioxidant activity.     

Inhibitory activities of prenylated flavonoids from Sophora flavescens against aldose reductase and generation of advanced glycation endproducts

Important targets for the prevention and treatment of diabetic complications include aldose reductase (AR) inhibitors (ARIs) and inhibitors of advanced glycation endproduct (AGE) formation. Here we evaluate the inhibitory activities of prenylated flavonoids isolated from Sophora flavescens, a traditional herbal medicine, on rat lens AR (RLAR), human recombinant AR (HRAR) and AGE formation. Among the tested compounds, two prenylated chalcones--desmethylanhydroicaritin (1) and 8-lavandulylkaempferol (2)--along with five prenylated flavanones--kurarinol (8), kurarinone (9), (2S)-2'-methoxykurarinone (10), (2S)-3beta,7,4'-trihydroxy-5-methoxy-8-(gamma,gamma-dimethylally)-flavanone (11), and kushenol E (13) were potent inhibitors of RLAR, with IC50 values of 0.95, 3.80, 2.13, 2.99, 3.77, 3.63 and 7.74 microM, respectively, compared with quercetin (IC50 7.73 microM). In the HRAR assay, most of the prenylated flavonoids tested showed marked inhibitory activity compared with quercetin (IC50 2.54 microM). In particular, all tested prenylated flavonols, such as desmethylanhydroicaritin (1, IC50 0.45 microM), 8-lavandulylkaempferol (2, IC50 0.79 microM) and kushenol C (3, IC50 0.85 microM), as well as a prenylated chalcone, kuraridin (5, IC50 0.27 microM), and a prenylated flavanone, (2S)-7,4'-dihydroxy-5-methoxy-8-(gamma,gamma-dimethylally)-flavanone (12, IC50 0.37 microM), showed significant inhibitory activities compared with the potent AR inhibitor epalrestat (IC50 0.28 microM). Interestingly, prenylated flavonoids 1 (IC50 104.3 microg mL(-1)), 2 (IC50 132.1 microg mL(-1)), 3 (IC50 84.6 microg mL(-1)) and 11 (IC50 261.0 microg mL(-1)), which harbour a 3-hydroxyl group, also possessed good inhibitory activity toward AGE formation compared with the positive control aminoguanidine (IC50 115.7 microg mL(-1)). Thus, S. flavescens and its prenylated flavonoids inhibit the processes that underlie diabetic complications and related diseases and may therefore have therapeutic benefit.     

粘毛黄芩黄酮类成分的研究

粘毛黄芩Scutellaria viscidula Bunge为唇形科黄芩属植物,民间以其根或全草入药,谓其性味苦寒、具有清热、解毒、祛湿、止血的作用,最近临床报道该药对菌痢有显著疗效。鉴于粘毛黄芩的化学成分迄今未见报道,为此我们进行了较系统的研究,在生药的乙醚提取部分通过聚酰胺柱层析,获得8个黄酮甙元结晶,在其甲醇提取部分分离获得2个     

Systematic screening and characterization of flavonoid glycosides in Carthamus tinctorius L. by liquid chromatography/UV diode-array detection/electrospray ionization tandem mass spectrometry

The traditional Chinese medicine (TCM) is a complex system, which always consists of numerous compounds with significant difference in the content and physical and chemical properties. In this paper, a screening method based on target molecular weights was developed to characterize the flavonoid glycosides in the flower of Carthamus tinctorius L. The screening tables of aglycone and glycan were designed, respectively, in order to select and combine freely. The multiple reaction monitoring (MRM) scan mode with higher sensitivity and selectivity was adopted in the screening, which benefit the characterization for the minor components. Seventy-seven flavonoid glycosides were screened out finally, and their structures were characterized by tandem mass spectrometric method in both positive and negative ion modes. The glycosylation mode, aglycone, sequence and/or the interglycosidic linkages of the glycan portion and glycosylation position were elucidated by the fragmentation rule in the MS. Numerous compounds screened out with this method showed the structure variety in secondary plant metabolites, and the purposeful screening systemically and subsequent structure characterization offered more information about the chemical constitutions of TCM.     

A novel flavonoid from Sophora flavescens

A new dihydroflavonol named kosamol A (1) has been isolated from the roots of Sophora flavescens (Leguminosae) along with twelve related flavonoids. The structure of 1 was determined to be (2R,3R)-5,7,2'4'-tetrahydroxy-6-(3-hydroxy-3-methylbutyl)-8-lavandulylflavanonol on the basis of spectral analyses.     

韧黄芩中黄酮类成分的研究

黄芩属植物我国有100多种,多为野生,南北均产。药用黄芩为唇形科黄芩属多种植物干燥的根,不是单一品种,主产于北方的有3种,主产于南方的有4、5种。而正品黄芩     

Fingerprint analysis of the fruits of Cnidium monnieri extract by high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization (ESI) and tandem mass spectrometry (MS), with parallel analysis by HPLC with UV detection using a diode-array detector (DAD) was developed for the qualitative characterization of coumarin and chromone constituents in the fruits of Cnidium monnieri. The chromatographic separations were performed on a Diamonsil C18 column (4.6 mm x 200 mm, 5 microm) with water with 50 mM ammonium acetate and 2% acetic acid (A) and acetonitrile (B) as the mobile phase. According to the characteristic UV spectra, the information of molecular weight and structure provided by ESI-MS/MS, 13 coumarin and 7 chromone components were detected and identified. This method is rapid and reliable for identification of the constituents in the complex herbal system, and the fragmentation patterns proposed could be extended to the similar compounds.     

Anti-allergic prenylated flavonoids from the roots of Sophora flavescens

The bioassay-guided fractionation of the MeOH extract from the root of Sophora flavescens led to the isolation of eight known prenylated flavonoids ( 1 - 8) responsible for the IN VITRO anti-allergic activity. Among them, kushenol N ( 3), sophoraflavanone G ( 6), and leachianone A ( 7) demonstrated significant inhibition of the release of beta-hexosaminidase from cultured RBL-2H3 cells with IC (50) values ranging from 15 to 30 muM.     

Authentication of Senecio scandens and S. vulgaris based on the comprehensive secondary metabolic patterns gained by UPLC-DAD/ESI-MS

A secondary metabolic pattern using ultra-performance liquid chromatography (UPLC)-DAD/ESI-MS was constructed to gain chemical information for authentication of Senecio scandens (SS) and Senecio vulgaris (SV), the two representative species containing hepatotoxic pyrrolizidine alkaloids (HPAs). The metabolic pattern showed three groups of bioactive constituents: phenolic/aromatic acids, flavonoid glycosides and the HPAs. 47 peaks were identified including 19 phenolic/aromatic acids, 10 flavonoid glycosides and 18 PAs by direct comparison with the available reference compounds or deduced from the UV absorption and their ESI-MS fragmentation patterns.The two species could be authenticated diagnostically by their metabolic profiling of the three chromatographic fingerprints. Although both SS and SV contain PAs as the characteristic constituents, only 2 PAs, adonifoline and adonifoline N-oxide were detected in SS, while other 16 PAs were detected in SV, including the highly toxic senecionine, retrorsine, seneciphylline and their corresponding N-oxides. The concentration of PAs in SV is also higher than that in SS. The number and concentration of the phenolic compounds in SS were higher than in SV. Jacaranone derivatives were only detected in SS and jacaranone ethyl ester was detected as the predominant constituent.In the fingerprint of the n-butanol extracts, 10 quercetin and kaempferol glycosides derivatives were detected. 9 were found in SS and only 2 in SV. PAs, jacaranone derivatives and flavonoid glycosides can serve as the metabolic markers to distinguish the Senecio plants from each other, and provide evidence for their clinical application in the consideration of safety and efficacy.     

Identification and characterization of antioxidants from Sophora flavescens

The objectives of this study were to investigate the radical-scavenging activity and protective potential of Sophora flavescens from oxidative damage by the radical generator 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) in renal epithelial LLC-PK(1) cells and to identify the active components using the bioassay-linked fractionation method. The MeOH extract and fractions of CH(2)Cl(2), BuOH, and H(2)O from S. flavescens showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging effects in a dose-dependent manner (p<0.01),whereas only the BuOH and CH(2)Cl(2) fractions showed protective effects against LLC-PK(1) cellular damage induced by AAPH in a dose-dependent manner (p<0.01). In particular, the BuOH fraction had the most effective (p<0.05) antioxidative capacity. Employing a bioassay-linked HPLC/MS method, the active constituents from the BuOH fraction of S. flavescens were isolated and characterized as sophoraflavanone G and kurarinone with potent antioxidant effects against the DPPH radical, with IC(50) values of 5.26 and 7.73 microg/ml, respectively. Moreover, the compounds dose dependently recovered cell viability decreased by AAPH treatment (p<0.01), suggesting their protective roles against cellular oxidative damage. The results of this study suggest that S. flavescens has excellent antioxidative and kidney-protective potential and that flavonoids from S. flavescens, i.e., sophoraflavanone G and kurarinone, are the active constituents.