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.     

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.     

Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase

To determine the compounds responsible for its anti-influenza activities, we isolated the three flavonoids, 6-hydroxyluteolin 7-O-β-d-glucoside (1), nepitrin (2), homoplantaginin (3) from the MeOH extract of Salvia plebeia R.Br. and identified them by comparing the spectroscopic data with that reported in the literature. The contents of the three flavonoids in the whole extract were 108.74 ± 0.95, 46.26 ± 2.19, and 69.35 ± 1.22 mg/g for 6-hydroxyluteolin 7-O-β-d-glucoside, nepitrin, and homoplantaginin, respectively, which demonstrates that they are the major constituents of this plant. The three flavonoids were evaluated for their inhibitory activities against influenza virus H1N1 A/PR/9/34 neuraminidase and H1N1-induced cytopathic effect (CPE) on Madin-Darby canine kidney (MDCK) cells. Our results demonstrated the following arrangement for their anti-influenza activities: nepitrin (2) > 6-hydroxyluteolin 7-O-β-d-glucoside (1) > homoplantaginin (3). The potent inhibitory activities of these flavonoids against influenza suggested their potential to be developed as novel anti-influenza drugs in the future.     

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%.     

Application and recovery of ionic liquids in the preparative separation of four flavonoids from Rhodiola rosea by on‐line three‐dimensional liquid chromatography

A novel on‐line three‐dimensional liquid chromatography method was developed to separate four main flavonoids from Rhodiola rosea. Ethyl acetate/0.5 mol/L ionic liquid 1‐butyl‐3‐methylimidazolium chloride aqueous solution was selected as the solvent system. In the first‐dimension separation, the target flavonoids were entrapped and subsequently desorbed into the second‐dimension high‐speed countercurrent chromatographic column for separation. In the third‐dimension chromatography, the residual ionic liquid in the four separated flavonoids was removed and the used ionic liquid was recovered. As a result, 35.1 mg of compound 1 , 20.4 mg of compound 2 , 8.5 mg of compound 3, and 10.6 mg of compound 4 were obtained from 1.53 g R. rosea extract. They were identified as rhodiosin, rhodionin, herbacetin, and kaempferol, respectively. The recovery of ionic liquid reached 99.1% of the initial amount. The results showed that this method is a powerful technology for the separation of R. rosea flavonoids and that the ionic‐liquid‐based solvent system has advantages over traditional solvent systems in renewable and environmentally friendly properties.     

Antimicrobial flavonoids isolated from Indian medicinal plant Scutellaria oblonga inhibit biofilms formed by common food pathogens

Scutellaria oblonga Benth., a hitherto phytochemically unexplored Indian medicinal folklore plant was extracted with acetone and subjected to chromatography to yield nine flavonoids, for the first time from this plant. Antimicrobial assays were performed against 11 foodborne pathogens, and three molecules (Techtochrysin, Negletein and Quercitin-3-glucoside) depicted significant activity. These molecules were assessed for their rate of antibacterial action using time–kill curves which depicted complete inhibition of most of the bacteria within 12–16 h. The significant biofilm-reducing capability exhibited by these three molecules formed a significant finding of the current study. In most of the experiments, a 90–95% reduction in biofilms was observed. Thus, flavonoids as natural molecules from S. oblonga could be further researched to be used as potent antimicrobial and antibiofilm agents.     

Towards Adsorptive Enrichment of Flavonoids from Honey Using h-BN Monolayer

Density functional theory (DFT) was used to explore the possibility of h-BN monolayer acting as an adsorbent for the flavonoids. Four flavonoids named apigenin, kaempferol, myricetin, and quercetin as well as glucose (Glu) were selected as representatives of honey. DFT and ab initio molecular dynamics simulation results show that the four flavonoids interact with the h-BN monolayer much stronger than the Glu does in both vacuum and solutions, indicating a good adsorptive selectivity of the flavonoids over Glu. The interaction of the flavonoids and the Glu with water as well as the solvation energy of the flavonoids in water, methanol and ethanol was obtained using both the PBE-D and B3LYP-D functionals. It is shown that the h-BN monolayer can provide high selective adsorption of the flavonoids from bee honey and ethanol can be used as an elution solvent to recover the adsorbed flavonoids.     

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.     

Separation and Determination of Low Abundant Flavonoids in Scutellaria Baicalensis Georgi by Micellar Electrokinetic Capillary Electrophoresis

Abstract

Flavonoids are secondary metabolites in many plants with diverse biological activities. A simple and rapid micellar electrokinetic capillary electrophoresis method was developed for simultaneous determination of three low abundant flavonoids (chrysin, 5,7,4′-trihydroxy-8-methoxyflavone, and skullcapflavone II) in Scutellaria baicalensis Georgi within 12 min. The calibration curves revealed a good linear relationship between the peak areas of analytes and their concentrations (11.38 to 436.67 mg/L). The standard deviations of the migration time of the three flavonoids were determined to be 0.64%, 0.83%, and 1.17%, and peak areas were 6.14%, 7.57%, and 8.33%. Recoveries ranged between 90.37 and 109.4%.     

Chromatographic investigations of flavonoid compounds in the leaves and flowers of some species of the genusAlthaea

Summary The flavonoids present in the leaves and flowers ofAlthaea armeniaca Ten., A. cannabina L., A. narbonesis Pourr. andA. broussonetiifolia Iljin were investigated and compared to the flavonoids present in the leaves and flowers ofA. officinalis L. The inliquid chromatography and two-dimensional paper chromatography. The same flavonoids were found in the flowers of all the investigated species while differences could be noted in the flavonoid composition of the leaves. Both the qualitative and quantitative difference was the greatest in the flavonoids of the leaves ofA. cannabina L.     

Determination of Flavonoids from Paulownia tomentosa (Thunb) Steud by Micellar Electrokinetic Capillary Electrophoresis

A micellar electrokinetic capillary electrophoresis (MEKC) method was developed for the determination of three flavonoids: diplacone (DI), mimulone (MI) and apigenin (AP) in the flowers of Paulownia tomentosa (Thunb) Steud. A buffer solution (pH 10.00) of 20 mM sodium borate, 10 mM sodium dodecyl sulfate (SDS) and 5% methanol was found to be the most suitable electrolyte for this separation. Regression equations revealed linear relationships (correlation coefficients: 0.9997 and 0.9998) between the peak area of each compound (DI, MI and AP) and its concentration. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of pH, surfactant (SDS) concentration and organic modifier (methanol) on migration were also studied. The contents of three flavonoids in the extracts from the flowers of P. tomentosa applying different solvents were successfully determined within 12 min.     

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.     

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.     

Novel Flavonoids in Dragon's Blood of Daemonorops draco

The three novel methylene bis[flavonoids] 1 – 3 , the novel 2‐flavene 4 , the new naturally occurring flavan 5 , and the new retro‐dihydrochalcone 6 were isolated from dragon's blood of Daemonorops draco, together with seven known compounds. The structures were elucidated by extensive 1D‐ and 2D‐NMR spectroscopic analysis.     

Integrated extraction and purification of total bioactive flavonoids from Toona sinensis leaves

An integrated procedure was developed to extract and purify total flavonoids from Toona sinensis leaves for the first time, in which pressurized liquid extraction was performed in tandem with HPD100 macroporous resin column. Consequently, the total flavonoids can be extracted using 10% EtOH, and the recovery and purity of total flavonoids was 71.05% and 66.60%. Moreover, products of high quality were obtained in an environmentally friendly process with lower consumption of time and solvent. The results demonstrated that the integrated extraction-adsorption procedure was an efficient process for the preparation of total bioactive flavonoids from Toona sinensis leaves.     

The Chemical Constituents from the Aerial Part of Rosa laevigata

Eleven chemical constituents were isolated from the ethyl acetate soluble fraction of the aerial part of Rosa laevigata Michx. These compounds include the Henze's ketol (16), diethyl malate (17), three γ-lactones (18-20), loliolide (21), p-coumaric acid (22), 6,7-dimethoxycoumarin (23) and three flavonoids (24-26). The new compounds 19 and 20 were determined to be the cis- and trans-isomers of ethyl 2-benzyl-3-hydroxy-5-oxo-3-furancarboxylate.     

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.     

Constituents of Leaves of Tetradium Glabrifolium

Twenty-nine compounds including three alkaloids, live triterpenoids, five coumarins, seven benzenoids, two tannins, five flavonoids, one sugar and one nucleoside are isolated from leaves of Tetradium glabrifolium. Their structures are characterized on comparison of spectral data with literature values.     

Extraction technology,component analysis,and in vitro antioxidant and antibacterial activities of total flavonoids and fatty acids from Tribulus terrestris L. fruits

The current study focused on the extraction technology, components analysis, in vitro antioxidant and antibacterial activities of total flavonoids and fatty acids from Tribulus terrestris L. fruits. The extraction process of total flavonoids and fatty acids was optimized by the response surface method, and the compositions were identified from the two extracts by HPLC–DAD–ESI–MS^? and GC–MS, respectively. In addition, the antioxidant and antibacterial activities were evaluated by assay of ABTS, DPPH radical scavenging activity, ferric reducing antioxidant power and minimal inhibitory concentration. The yields of total flavonoids and fatty acids were 0.46 and 9.76% under the optimized conditions. Moreover, nine and eight compositions were identified from the two extracts based on the related references, respectively. In addition, total flavonoids and fatty acids extracts both exhibited certain antioxidant and antibacterial activities. The present findings suggest that total flavonoids extracted from T. terrestris L. fruits comprised a more interesting candidate than fatty acids for the research and development of natural and healthy antioxidants and antibacterial agents for the pharmaceutical and food industries.     

A New Megastigmane Glucoside and Three New Flavonoid Glycosides from Spiraea prunifolia var. simpliciflora

A new megastigmane glucoside, simplicifloranoside ( 1 ), and three new flavonol glycosides, prunifolianosides A–C ( 2 – 4 , resp.), were isolated from the aerial parts of Spiraea prunifolia var. simpliciflora. In addition, fifteen known compounds, including five phenolic acids, three lignans, four flavonoids, one eugenol glycoside, and two alkyl‐primeverosides, were also identified. Their structures were elucidated on the basis of detailed spectroscopic analyses and acid hydrolysis.