Dietary‐flavonoid‐rich flowers of Rumex nervosus Vahl: Liquid chromatography with electrospray ionization tandem mass spectrometry profiling and in vitro anti‐inflammatory effects

Rumex nervosus is a plant species found widely in Eastern Africa and the Arabian Peninsula. In addition to its uses in traditional medicinal, the plant shows various biological activities, such as antiviral, antibacterial, and antioxidant activity. In this study, nine flavonols, six flavones, three flavanones, and one flavanol were characterized from the flowers of R. nervosus using liquid chromatography with electrospray ionization tandem mass spectrometry and literature data. Validation data indicated that the determination coefficients (R²) were ≥ 0.9914. The limits of detection and quantification were in the ranges of 0.15–1.24 and 0.50–4.13 mg/L, respectively. Recoveries at 10 and 50 mg/L were 71.1–110.2 and 65.4–115.1%, with relative standard deviations of 7.4–40.1 and 2.1–13.0%, respectively. Quercetin 3‐O‐rhamnoside ( 10 ) was the dominant component, contributing 30.8% of total flavonoids (1003.0 ± 26.2 mg/kg fresh flower sample), whereas luteolin 6‐C‐glucoside (3) was the lowest yielding compound (0.1%). The 19 flavonoids identified were characterized for the first time. In vitro anti‐inflammatory studies showed that this mixture can suppress the production of inflammatory mediators, including inducible nitric oxide synthase, cyclooxygenase‐2, kappa B inhibitor, and interleukin‐1β, by down‐regulating the nuclear factor‐kappa B and mitogen‐activated protein kinases pathways. The results of this study may provide information for processing R. nervosus as a potential source of functional food.     

Flavonoid Constituents from Spiraea brahuica

Sparins A–C ( 1 – 3 , resp.), three new flavonoids, were isolated from the CHCl₃ subfraction of the EtOH extract of the whole plant of Spiraea brahuica, along with 3′,7‐di‐O‐methylquercetin ( 4 ) and luteolin 7‐β‐D ‐glucopyranoside ( 5 ), reported for the first time from this species. The structures of the new compounds were elucidated by spectroscopic techniques including MS and 2D‐NMR spectroscopy.     

An accurate and reproducible method for simultaneous determination of four flavonoids in EtOAc extracts from Sophora flavescens Ait. in rat plasma based on UHPLC Q‐Exactive Mass spectrometry: Application to a pharmacokinetics study

In previous studies, it was revealed that ethyl acetate (EtOAc) extracts from Sophora flavescens Ait. improved glucose tolerance, reduced hyperglycemia, and restored insulin levels in diabetic patients. The aim of this study was to develop an accurate and sensitive UHPLC–MS method for simultaneous determination of flavonoids in EtOAc extracts of Kushen in rat plasma. Ethyl acetate–acetonitrile (2:1) was selected as the solvent to extract the four flavonoids from rat plasma. A BEH C₁₈ column (2.1 mm × 100 mm, 1.7 μm) with a C₁₈ guard cartridge was chosen as the separation plant using a gradient elution with acetonitrile (solvent A) and 0.1% formic acid (solvent B) in water. For all four analytes, the method showed good linearity (r² > 0.991) in 1–500 ng/mL. The inter‐ and intra‐day accuracy ranged from ?13.78 to 7.19%, and the precision (RSD) was <8.75%. Recoveries of all four flavonoids ranged from 85.9 to 101.3%. According to the results of multitarget pharmacokinetic studies, four active flavonoids in EtOAc extracts from Kushen have similar absorption kinetics but very different metabolic kinetics, and a double peak phenomenon was observed in the concentration–time curve of norkurarinone, which is different from the previous study. In conclusion, detection and multitarget pharmacokinetic studies successfully determined active flavonoids after oral administration of EtOAc extracts from Kushen by an efficient, sensitive and selective UHPLC–MS method, and the results may provide a foundation for future studies of Kushen.     

Atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite

Colloidal graphite is a promising matrix for atmospheric pressure laser desorption/ionization mass spectrometry. Intact [M+H]⁺ and [M–H]^? ions are readily produced from a wide range of small molecule plant metabolites, particularly anthocyanins, fatty acids, lipids, glycerides, and ceramides. Compared with a more traditional organic acid matrix, colloidal graphite provides more efficient ionization for small hydrophobic molecules and has a much cleaner background spectrum, especially in negative ion mode. Some important metabolites, e.g., fatty acids and glycosylated flavonoids, can be observed from Arabidopsis thaliana leaf and flower petal tissues in situ. Copyright © 2010 John Wiley & Sons, Ltd.     

Influence of Phytochemical Reductive Capacity on Ultraviolet–visible Spectroscopic Behavior of Silver Nanoparticles

ABSTRACT

A widely used method for obtaining silver nanoparticles uses plant extracts for reduction because of the presence of phytochemicals such as terpenoids, tannins, and flavonoids. Extracts of Flores sambuci, Hypericum perforatum, Lavandula angustifolia, Origanum vulgare, Rosmarinus officinalis, and Salvia officinalis were used for generating silver nanoparticles. The ultraviolet–visible spectra of silver nanoparticle solutions were correlated with variations of phytochemical characteristics to evaluate the plant extracts. These parameters were the antioxidant activity, total flavonoids, total tannins, total terpenoids, and total phenolics. Correlations between measurements of extracts’ phytoreductive characteristics were explained using Pearson coefficients. The results showed medium linear positive correlations for total tannins with the spectra of silver nanoparticle solutions. The antioxidant activity and total terpenoids presented medium linear negative correlations. Pearson coefficients between total phenolics and relative areas from ultraviolet–visible spectra from 350 to 600?nm were close to zero indicating no linear correlation.     

Flavonoids from the Flowers of Pueraria Lobata

A new isoflavonoid glycoside, gehuain, together with three flavonoids and eight isoflavonoids, was isolated from the flowers of Pueraria lobata. Three of them, apigenin, apigenin 4′‐O‐β‐D‐glucoside, and wistin, were reported the first time from this plant. Their structures were established on the basis of spectral evidence.     

Separation of Flavonoids from the Leaves of Oroxylum indicum by HSCCC

A high-speed counter-current chromatography system (HPCCC) capable of rapid processing has been employed to separate seven flavonoids from a methanolic extract of the leaves of Oroxylum indicum by a one-step isocratic elution using a chloroform–methanol–water (9.5:10:5) two-phase system. LC, MS and NMR have identified the components from the extract as chrysin, baicalein, baicalein-7-O-glucoside, baicalein-7-O-diglucoside, chrysin-7-O-glucuronide, baicalein-7-O-glucuronide, and a chrysin-diglucoside. Baicalein-7-O-glucuronide and chrysin-7-O-glucuronide have been separated from this plant by HSCCC for the first time. The present study also reports a new chrysin-diglucoside from the leaf extract. The results demonstrate that HSCCC is a powerful separation tool and can contribute to identifying and quantifying plant ingredients.

     

Determination of Flavonoids in Stellaria by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The genus Stellaria (Caryophyllaceae) presents widely distributed plants often used in traditional medicine. Flavonoids are highly active plant secondary metabolites that may be involved in some of the effects of Stellaria plants. In this study, two new high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS-MS) methods were developed for the determination of flavonoids and isoflavonoids in plant material. The separations were performed on a reverse-phase C₁₈ column with gradient elution using methanol and water with 0.5% acetic acid as the mobile phase. Multiple reaction monitoring was used for the tandem mass spectrometry detection and the two most intensive transitions were chosen for the identification of each analyte. The limits of detection and the limits of quantification were between 0.2 and 15.0 ng/mL and 0.6 and 50.0 ng/mL. The developed methods were successfully used for the analyses of four representatives of the genus Stellaria. All the studied herbs contained luteolin and its 7-O-glycosides, naringenin, kaempferol, quercetin, its glycoside rutin, apigenin, and its 7-O-glycoside. One coumarin, scopoletin, was also found. Isoflavones were primarily represented by genistein, genistin, and ononin. Some of the analytes were detected for the first time in Stellaria sp. The findings support that these methods are suitable for analyses of plant material.     

β‐Cyclodextrin‐modified three‐dimensional graphene oxide‐wrapped melamine foam for the solid‐phase extraction of flavonoids

A new three‐dimensional graphene oxide‐wrapped melamine foam was prepared and used as a solid‐phase extraction substrate. β‐Cyclodextrin was fabricated onto the surface of three‐dimensional graphene oxide‐wrapped melamine foam by a chemical covalent interaction. In view of a specific surface area and a large delocalized π electron system of graphene oxide, in combination with a hydrophobic interior cavity and a hydrophilic peripheral face of β‐cyclodextrin, the prepared extraction material was proposed for the determination of flavonoids. In order to demonstrate the extraction properties of the as‐prepared material, the adsorption energies were theoretically calculated based on periodic density functional theory. Static‐state and dynamic‐state binding experiments were also investigated, which revealed the monolayer coverage of flavonoids onto the β‐cyclodextrin/graphene oxide‐wrapped melamine foams through the chemical adsorption. ¹H NMR spectroscopy indicated the formation of flavonoids–β‐cyclodextrin inclusion complexes. Under the optimum conditions, the proposed method exhibited acceptable linear ranges (2–200 μg/L for rutin and quercetin‐3‐O‐rhamnoside; 5–200 μg/L for quercetin) with correlation coefficients ranging from 0.9979 to 0.9994. The batch‐to‐batch reproducibility (n = 5) was 3.5–6.8%. Finally, the as‐established method was satisfactorily applied for the determination of flavonoids in Lycium barbarum (Goji) samples with relative recoveries in the range of 77.9–102.6%.     

On-line screening and identification of polyphenolic antioxidant compounds of Convolvulus trabutianus

Abstract

Convolvulus trabutianus Schweinf. & Muschl. is an endemic plant from northern Sahara used in folk medicine. Herein we report, the isolation, characterization and evaluation of the radical scavenging properties of twenty three compounds from different extracts of this species by on-line HPLC-ABTS^?+ screening. These compounds include nine phenolic acids: 2, 6, 10–16, two phytosterols: 3–4, four coumarins: 5, 7–9, two quinic acids: 21 and 22 and six flavonoids: 1, 17–20 and 23 among which the most active were: 10, 16, 21 and 22. All the extracts showed a significant antioxidant activity on-line. These results were validated off-line by ORAC and TEAC assays. Four compounds: 1, 5, 18 and 19 were described for the first time from the Convolvulaceae family, whereas compounds 2, 6, 8, 10, 13 and 21 were new for the genus Convolvulus.     

Simultaneous determination of eight bioactive components of Cirsium setosum flavonoids in rat plasma using triple quadrupole LC/MS and its application to a pharmacokinetic study

Cirsium setosum (Willd.) MB. has been reported to exert significant anti‐hemorrhagic, anti‐inflammation, antimicrobial, sedative and detoxicating efficacy. It has been widely used to treat gastrointestinal bleeding, uterine bleeding, infectious hepatitis and cardiovascular disease in China. Recent studies have shown that flavonoids are the main active components in C. setosum. Nevertheless, to the best of our knowledge, there is no report concerning the simultaneous determinations and pharmacokinetics of constituents in C. setosum flavonoids in rat plasma. In this study, a rapid, sensitive and selective triple quadrupole liquid chromatography–mass spectrometry method was developed to determine eight analytes from the flavonoids of C. setosum in rat plasma. In addition, the pharmacokinetic study of the eight analytes in rats after oral administration of C. setosum flavonoids was successfully completed through this method. According to the pharmacokinetic parameters of the eight analytes, rutin, naringin, quercetin, acacetin, wogonin were the long‐acting components of the C. setosum flavonoids, with long elimination time and high bioavailability. Of note, the method developed in this study fills a blank in pharmacokinetic studies of C. setosum flavonoids. Our findings provide valuable views on the understanding of the absorption mechanism of C. setosum flavonoids and their clinical efficacy.     

Comprehensive metabolite profiling of Plantaginis Semen using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique

Plantaginis Semen is commonly used in traditional medicine to treat edema, hypertension, and diabetes. The commercially available Plantaginis Semen in China mainly comes from three species. To clarify the chemical composition and distinct different species of Plantaginis Semen, we established a metabolite profiling method based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique. A total of 108 compounds, including phenylethanoid glycosides, flavonoids, guanidine derivatives, terpenoids, organic acids, and fatty acids, were identified from Plantago asiatica L., P. depressa Willd., and P. major L. Results showed significant differences in chemical components among the three species, particularly flavonoids. This study is the first to provide a comprehensive chemical profile of Plantaginis Semen, which could be involved into the quality control, medication guide, and developing new drug of Plantago seeds.     

Liquid chromatography with mass spectrometry method based two‐step precursor ion scanning for the structural elucidation of flavonoids

Plant flavonoids are very important secondary metabolites for insect and virus control of their host plant and are potent nutrients for humans. To be able to understand the bioavailability and functions of plant flavonoids, it is necessary to reveal their exact chemical structures. Liquid chromatography with tandem mass spectrometry is a powerful approach for structural elucidation of metabolites. In this report, a two‐step precursor ion scanning based liquid chromatography with tandem mass spectrometry method was developed for the structural elucidation of plant flavonoids. The established method consists of the two‐step precursor ions scanning for possible flavonoids extraction, MS² fragment spectra acquisition and comparison with an online database, liquid chromatography retention rules correction, and commercial standards verification. The developed method was used for the structure elucidation of flavonoids in flowers and leaves of tobacco (Nicotiana tabacum), and 17 flavonoids were identified in the tobacco variety Yunyan 97. Nine of the 17 identified flavonoids were considered to be found in tobacco flowers or/and leaves for the first time based on the available references. This method was proved to be very effective and can be used for the identification of flavonoids in other plants.     

Liquid chromatography‐tandem mass spectrometry assay for the simultaneous determination of three major flavonoids and their glucuronidated metabolites in rats after oral administration of Artemisia annua L. extract at a therapeutic ultra‐low dose

The traditional antimalarial herb Artemisia annua L., from which artemisinin is isolated, is widely used in endemic regions. It has been suggested that artemisinin activity can be enhanced by flavonoids in A. annua; however, how fast and how long the flavonoids are present in the body remains unknown. In the present study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of three major flavonoids components, i.e. chrysosplenol D, chrysoplenetin, and artemetin and their glucuronidated metabolites in rats after oral administrations of A. annua extracts at a therapeutic ultra‐low dose. The concentration of the intact form was determined directly, and the concentration of the glucuronidated form was assayed in the form of flavonoids aglycones, after treatment with β‐glucuronidase/sulfatase. The method was linear in the range of 0.5–300.0 ng/mL for chrysoplenetin and artemetin, and 2–600 ng/mL for chrysosplenol D. All the validation data conformed to the acceptance requirements. The study revealed a significantly higher exposure of the flavonoid constituents in conjugated forms in rats, with only trace intact from. Multiple oral doses of A. annua extracts led to a decreased plasma concentration levels for three flavonoids.     

Enrichment of flavonoid-rich extract from Bidens bipinnata L. by macroporous resin using response surface methodology,UHPLC–Q-TOF MS/MS-assisted characterization and comprehensive evaluation of its bioactivities by analytical hierarchy process

The extract of Bidens bipinnata L. exhibited wide spectrum of bioactivities owing to the presence of flavonoids. In this study, a purification process was developed to enrich the flavonoid-rich extract from B. bipinnata L. (BBTF). AB-8 resin was selected for the purification of total flavonoids. Response surface methodology coupled with Box–Behnken design was employed to optimize the purification condition; it was optimized as pH 5.1, volume of ethanol 80 ml, flow rate of ethanol 1.8 bed volume per hour (BV/h) and concentration of ethanol 76.0%. The total flavonoid content of BBTF was 56.48% under the optimal conditions. The identification of flavonoids in BBTF was conducted using UHPLC–ESI-Q-TOF MS. A total of 14 compounds, including 12 flavonoids, were identified in BBTF. Finally, the in vitro antioxidant activities, α-glucosidase and α-amylase inhibitory activities of BBTF were comprehensively analyzed by an analytical hierarchy process. The results indicated that it exhibited higher bioactivities than the crude extract. These findings suggested that the optimized process could significantly enhance the purity of flavonoids and their bioactivities. This study showed a comprehensive analysis of a total flavonoid extract of B. bipinnata L. for the first time, which could provide a useful approach for its purification process and quality control as well as bioactivities.     

An overview of techniques and strategies for isolation of flavonoids from the genus Erythrina

Plants in the genus Erythrina is a potential source of chemical constituents, one of which is flavonoids, which have diverse bioactivities. To date, literature on the flavonoids from the genus Erythrina has only highlighted the phytochemical aspects, so this review article will discuss isolation techniques and strategies for the first time. More than 420 flavonoids have been reported in the Erythrina genus, which are grouped into 17 categories. These flavonoid compounds were obtained through isolation techniques and strategies using polar, semi-polar, and non-polar solvents. Various chromatographic techniques have been developed to isolate flavonoids using column flash chromatography, quick column chromatography, centrifugally accelerated thin-layer chromatography, radial chromatography, medium-pressure column chromatography, semi-preparative high-performance liquid chromatography, and preparative high-performance liquid chromatography. Chromatographic processes for isolating flavonoids can be optimized using multivariate statistical applications such as response surface methodology with central composite design, Box–Behnken design, Doehlert design, and mixture design.     

Evaluation of the antioxidant and anti-inflammatory activities of solvent extracts of Tricholepis chaetolepis (Boiss) Rech. f. whole plant

Abstract

The whole plant, Tricholepis chaetolepis, powder was investigated using proximate and fluorescence analysis along with determining the extractive values. Total phenolics, flavonoids and total protein contents of n-hexane, chloroform and methanolic extracts of the whole plant were also determined. The anti-diabetic activity of all the three extracts of the plant was determined by in vitro alpha-amylase inhibition assay. The anti-oxidant potential was evaluated using Phosophomolybdenum and DPPH methods. The anti-inflammatory potential of all extracts were determined by carrageenan-induced rat paw oedema model. The evaluation of the plant extracts exhibited the anti-diabetic, anti-oxidant, and anti-inflammatory activities in dose dependent fashion. The research concludes that Tricholepis chaetolepis extracts contain phenol, flavonoids, and tannins that show observable anti-oxidant and anti-diabetic potential. It is also concluded that the methanol extract of the plant showed the maximum effect against inflammation induced by carrageenan in rat paw oedema as compared with n-hexane and chloroform extracts.     

Quality evaluation of six bioactive constituents in goji berry based on capillary electrophoresis field amplified sample stacking

Goji berry, fruits of the plant Lycium barbarum L., has long been used as traditional medicine and functional food in China. In this work, a simple and easy‐operation on‐line concentration capillary electrophoresis (CE) for detection flavonoids in goji berry was developed by coupling of field amplified sample stacking (FASS) with an electroosmotic (EOF) pump driving water removal process. Due to the EOF pump and electrokinetic injection showing different influence on the concentration, the analytes injection condition should be systemically studied. Thereafter, the verification of the analytes injection conditions was achieved using response surface experimental design. Under the optimum conditions, 86–271 folds sensitivity enhancement upon normal capillary zone electrophoresis (CZE, 50 mbar × 5 s) were achieved for six flavonoids, and the detection limits ranged from 0.35 to 1.82 ng/mL; the LOQ ranged from 1.20 to 6.01 ng/mL. Eventually, the proposed method was applied to detect flavonoids in 30 goji berry samples from different habitats of China; and the results indicated that the flavonoids were rich in the eluent of 30–60% methanol, which provided a reference for extraction of goji berry flavonoids.     

UVB Radiation Induced Increase in Quercetin: Kaempferol Ratio in Wild-Type and Transgenic Lines of Petunia

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild-type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho-cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla-vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra-violet-B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul-traviolet-B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.