Secondary metabolites in a soybean fermentation broth of Paecilomyces militaris

A methanolic extract from a soybean broth fermented by Paecilomyces militaris was analysed by HPLC–PDA–HRMS. It mainly contained mannitol, uracil, adenosine, cordycepin, daizein, genistein, and three new isoflavone glycosides. The isoflavonoids were isolated by preparative HPLC and identified with the aid of HPLC–PDA–HRMS, ¹H NMR, ¹³C, H–H COSY, HMBC, and HMQC NMR analysis as: 7,4′-dihydroxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside, 7,4′-dihydroxy-6-methoxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside, and 5,7,4′-trihydroxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside. The three novel compounds are transformation products of soybean isoflavones. The results of metabolites analysis revealed that the fermentation broth reserved the main functional molecules of soybean and P. militaris or Cordyceps militaris. This suggested that soybean fermented with P. militaris may be able to create a combined healthy food. However, isoflavones are also parts of anti-nutrients of soybean products, and cordycepin is a known antibiotic. This means that the fermentation broth may not suit to be used as normal food. At the same time the safety and bioactivity of the novel methylated glycosides need also further approval.     

Antioxidant capacity and hydrophilic phytochemicals in commercially grown native Australian fruits

Hydrophilic phytochemicals and antioxidant capacities of eight commercially grown native Australian fruits were determined. Kakadu plum (Terminalia ferdinandiana) contained a 6-fold higher level of total phenolic compounds and quandong (Santalum acuminatum) a 1.9-fold higher level of total phenolic compounds (TP, Folin–Ciocalteu assay) than blueberry (Vaccinum sp., cv. Biloxi). Both fruits displayed superior oxygen radical-scavenging capacity (ORAC-H assay) that was, respectively 4.1-fold and 6.5-fold of that of blueberry. The total reducing capacity (TRC; ferric reducing antioxidant power (FRAP) assay) of Kakadu plum and quandong exceeded the TRC of blueberry, respectively, 13.1- and 2.3-times. The primary sources of antioxidant capacities in the evaluated fruits were phenolic acids (benzoic and cinnamic) and flavonoids (flavonols, flavanones and anthocyanins) tentatively detected by liquid chromatography–mass spectrometry (LC–PDA–MS/MS). A high level of vitamin C was recorded for Kakadu plum and Australian citrus fruits. The major organic acids detected were citric and malic acid.     

HPLC–PDA–ESI–MS/MS profiling and chemopreventive potential of Eucalyptus gomphocephala DC

An HPLC–PDA–ESI/MS/MS method was developed to identify the phytoconstituents of the EtOAc fraction of Eucalyptus gomphocephala DC. The antioxidant effect of the EtOAc fraction together with its sub-fractions was determined in vitro. The cytotoxicity was evaluated on different cell lines. The EtOAc fraction exhibited strong antioxidant activity, reduced the viability of all cell lines and was more active on MCF-7 and HepG-2 cell lines. Subsequently, the cytotoxicity of the sub-fractions and the isolated compounds were tested on MCF-7, HepG-2. The EtOAc fraction possessed potential antitumour promoting properties. It inhibited the stimulated NO (20%), 5-LOX (48.0%) and COX-2 (49.7%) respectively (at concentration of 20 μg/ml). This study suggests that this fraction is a source of different antioxidant and cytotoxic compounds with potential chemopreventive properties that might prevent different stages of the carcinogenesis process.     

Direct enantiomeric analysis of Mentha essential oils

A rapid and fully automated screening of chiral compounds in essential oils, aimed to the selection of natural sources of pure enantiomers of limonene and carvone, is performed by using on-line coupled reversed phase liquid chromatography with gas chromatography and mass spectrometry (RPLC–GC–MS). Essential oils obtained from Mentha spicata and Mentha piperita were analysed by direct injection into RPLC. The reported procedure includes fractionation and clean-up in RPLC, selection of the fraction to be transferred from RPLC to GC, trapping and concentration of the target compounds in the interface, thermal desorption and, finally, enantiomeric resolution and identification of chiral compounds by GC–MS. The presence of (S)-limonene and (R)-carvone as the unique enantiomeric forms existing for both compounds could be unambiguously established by transferring different volume fractions from RPLC to GC. Data obtained demonstrate high separation efficiency and well tunable selectivity in the on-line coupled RPLC–GC–MS analysis of chiral compounds.     

Determination of glucosinolates and phenolic compounds in rocket salad by HPLC-DAD–MS: Evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic resources

The glucosinolate and phenolic profiles of 37 rocket salad accessions (32 Eruca sativa and 5 Diplotaxis tenuifolia) were obtained by liquid chromatography–mass spectrometry. Eleven desulpho-glucosinolates (DS-GLSs) were isolated and the glucosinolate profiles did not differ between the two species. Total DS-GLS content, expressed as sinigrin equivalents (SE) revealed a certain variability, ranging from 0.76 to 2.46 g kg⁻¹ d.w. but, again, the quantitative analysis did not discriminates Eruca from Diplotaxis. The polyphenol evaluation by HPLC-DAD–MS allowed the identification of two different classes of compounds in the two rocket salad species. Qualitative differences were observed between the polyphenol profiles at specific level: quercetin derivatives were the main phenolics of Diplotaxis, whereas kaempferol derivatives characterised Eruca samples. The contents of total flavonoids determined as rutin equivalents (RE) ranged from 4.68 to 31.39 g kg⁻¹ d.w. Kaempferol-3,4′-diglucoside (71.4–82.2%) and isorhamnetin-3,4′-di-glucoside (7.8–18.4%) were always isolated as first and second more abundant phenolic compounds in Eruca samples. No marker phenolic compounds were isolated in Diplotaxis samples.     

Identification and analysis of isothiocyanates and new acylated anthocyanins in the juice of Raphanus sativus cv. Sango sprouts

The freeze-dried sprouts’ juice of Raphanus sativus (L.) cv. Sango was prepared and analysed for the first time. HPLC analysis of total isothiocyanates, after protein displacement, resulted in 77.8 ± 3.0 μmol/g of dry juice while GC–MS analysis of hexane and acetone extracts showed E- and Z-raphasatin (8.9 and 0.11 μmol/g, respectively) and sulforaphene (11.7 μmol/g), summing up to 20.7 ± 1.7 μmol/g of free isothiocyanates. Sprouts’ juice contained an unprecedented wealth of anthocyanins and a new fractionation methodology allowed us to isolate 34 mg/g of acylated anthocyanins (28.3 ± 1.9 μmol/g), belonging selectively to the cyanidin family. Analysis was performed by HPLC–PDA–ESI–MSⁿ and extended to deacylated anthocyanins and aglycones, obtained, respectively, by alkaline and acid hydrolysis. This study identified 70 anthocyanins, 19 of which have never been described before and 32 of which are reported here in R. sativus for the first time. Sango radish sprouts are exceptional dietary sources of heath-promoting micronutrients.     

Structure–activity relationships of acetylated flavone glycosides from Galeopsis ladanum L. (Lamiaceae)

Antioxidant activity, neuroprotective effect and acetylcholinesterase inhibitor activity were studied in acetylated flavones from Galeopsis ladanum L. (Lamiaceae), previously isolated and identified by UV-spectra, UPLC–MS/MS and ¹H-NMR spectroscopy. Structure–activity relationships of flavonoids have been determined in many antioxidant assays, generally focused on hydroxyl groups. In this study we have detected new interesting structure–activity relationships for the isolated flavonoids due to the acetylation of sugar moiety of these flavonoids. Methylation at 4′-OH and monoacetylation is beneficial to inhibiting AChE and shows a neouroprotective effect.     

Chemical composition and antioxidant activities of Myrtus communis L. berries extracts

Myrtle (Myrtus communis L.) berries extracts were prepared with solvents at different polarity (water, ethanol, and ethyl acetate) and analysed using different in vitro tests in order to evaluate their antioxidant properties. Antiradical and total antioxidant activities were measured with DPPH and FRAP tests, respectively. Their ability to protect biological molecules was assessed using the cholesterol and LDL oxidation assays. In addition, phenolic compounds and unsaturated fatty acids composition was analysed by HPLC–DAD and HPLC–MS/MS. Ethanol and water extracts showed the highest amount of extracted compounds, but the highest antiradical and antioxidant activities were found in ethanol and ethyl acetate extracts. These extracts were also the ones with the highest content of phenolic compounds. In addition, our results showed a highly significant correlation between the amount of total phenols and antiradical (R² = 0.9993) or antioxidant activities (R² = 0.9985) in these extracts. HPLC–DAD and HPLC–MS analyses showed significant quantitative and qualitative differences among these three extracts. The ethyl acetate extract had the highest protective effect in assays of thermal (140 °C) cholesterol degradation and Cu²⁺-mediated LDL oxidation, inhibiting the reduction of polyunsaturated fatty acids and cholesterol, and the increase of their oxidative products. These results suggest that because of these properties, myrtle berries could be used in dietary supplements preparations or as food additives.     

Composition of peel essential oils from four selected Tunisian Citrus species: Evidence for the genotypic influence

The peel essential oils from four selected Tunisian Citrus species: sweet orange (Citrus sinensis Osbeck), mandarin (Citrus reticulata Blanco); sour orange (Citrus aurantium L.) and pummelo (Citrus grandis Osbeck), cultivated under the same pedoclimatic and cultural conditions have been analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The essential oils content ranged from 1.06% to 4.62% (w/w) in pummelo and mandarin, respectively. The qualitative and quantitative analysis led to the identification of 70 components in all oil samples. The analysed oils consist mainly in monoterpene hydrocarbons (97.59–99.3%), with limonene (92.52–97.3%) and β-pinene (1.37–1.82) being the major constituents. The remaining chemical classes were weakly represented (<1%). Both qualitative and quantitative differences between oil samples have been observed and numerous components have been proposed as marker compounds. Since the influence of different environmental factors has been eliminated, the observed chemical variability between the studied species and cultivars seems likely to results from the genetic variability.     

Studies on the antioxidant potential of flavones of Allium vineale isolated from its water-soluble fraction

The aim of this work was to examine the chemical constituents and antioxidant potential of water-soluble fractions from the commonly consumed vegetable, Allium vineale. The water-soluble fraction, containing phenolic compounds, was extracted with ethyl acetate to obtain flavonoids which were separated and purified by repeated column chromatography over Sephadex LH-20, RP C18 and silica gel. The isolated compounds were identified according to their physicochemical properties and spectral data (UV, HPLC–TOF/MS, ¹H NMR, ¹³C NMR and 2D NMR). Three flavonoids were isolated and identified as chrysoeriol-7-O-[2″-O-E-feruloyl]-β-d-glucoside (1), chrysoeriol (2), and isorhamnetin-3-β-d-glucoside (3). Antioxidant studies of the aqueous extract and three isolated compounds, 1, 2, 3, were undertaken and they were found to have significant antioxidant activity. Antioxidant activities were evaluated for total antioxidant activity by the ferric thiocyanate method, ferric ion (Fe³⁺) reducing antioxidant power assay (FRAP), ferrous ion (Fe²⁺) metal chelating activity, and DPPH free radical-scavenging activity. The water-soluble ethyl acetate and methanol extraction methods were also compared using HPLC–TOF/MS.     

A further amendment to the classical core structure of gum arabic (Acacia senegal)

Using the more recently available techniques such as methylation–GC–MS, 1D (¹H, ¹³C) and 2D (COSY, TOCSY, HMQC and HMBC) NMR spectral analysis, we have revisited the classical structure of gum arabic (Acacia senegal). Methylation and GC–MS analysis confirmed that gum arabic (A. senegal) is a highly branched polysaccharide with the backbone composed of 1,3-linked galactopyransyl (Galp) residues substituted at O-2, O-6 or O-4 positions. The terminal sugar residues are 59.5% of the total sugars. The residues of →2,3,6-β-d-Galp1→, →3,4-Galp1→, →3,4,6-Galp1→ and substitutions at O-2 and O-4 position were not identified in previous studies.     

Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat)

A standardised profiling method based on liquid chromatography with diode array and electrospray ionisation/mass spectrometry (LC–DAD–ESI/MS) was used to identify 46 flavonoids and 17 caffeic acid derivatives in the aqueous methanol extract of Flos chrysanthemum (Chrysanthemummorifolium Ramat) (Compositae), an important Chinese herb. Fifteen caffeoylquinic acids and 15 flavonoids were positively identified and the remaining compounds were provisionally identified. The detected phenolics were present at concentrations greater than 0.001% of the dry plant material. Many of these phenolics have been reported to have human health benefits. Twenty-five compounds, including C-glycosylated flavones and aliphatic acid-containing caffeoylquinic acids, are reported for the first time in chrysanthemum flower, and this comprehensive analysis of the phenolic components of herb Flos chrysanthemum will be helpful for the quality control of this herb and its products, and to understand the usage and function.     

Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry

A rapid, sensitive and accurate ¹H NMR method has been developed for the quantitation of curcumin isolated from Curcuma longa rhizome (turmeric) extract, the results of which were compared with a validated LC–MS/MS method. The relative standard deviations of the methods were found to be 2.49% and 3.48% for the ¹H NMR and LC–MS/MS methods, respectively. The correlation coefficients were 0.998 for ¹H NMR and 0.995 for LC–MS/MS assay in the calibration range. The recoveries at 5 mg mL⁻¹ and 50 μg mL⁻¹ concentrations averaged to 99–101% for both techniques, respectively. The uncertainty of the measurement of curcumin via ¹H NMR spectroscopy was determined to be 5.80% while in LC–ESI-MS/MS method was 7.38%.     

A new type of Brazilian propolis: Prenylated benzophenones in propolis from Amazon and effects against cariogenic bacteria

Ethanol extracts of four propolis samples (E1–E4) from Manaus (Brazilian Amazon) were analysed by HPLC/DAD/ESI–MS/MS and GC/EIMS. The major constituents of E2 and E4 were analysed by NMR (¹H and ¹³C) and ESI/MS/MS. The main constituents of E2 and E4 are polyprenylated benzophenones: 7-epi-nemorosone, 7-epi-clusianone (major E4 constituents), xanthochymol and gambogenone (major E2 constituents), making up a chemical profile so far unreported for Brazilian propolis. Aristhophenone, methyl insigninone, 18-ethyloxy-17-hydroxy-17,18-dihydroscrobiculatone B, and derivatives of dimethyl weddellianone A and B, propolones, and a scrobiculatone derivative, were detected as minor constituents. Triterpenoids (β-amyrins, β-amyrenone, lupeol and lupenone) were ubiquitous and predominant in E1 and E3. The extracts E2 and E4 were highly active against the cariogenic bacteria Streptococcusmitis, Streptococcusmutans and Streptococcussalivarius. E2 was more active than E4, probably due to a higher content of 2-epi-nemorosone, while the latter was richer in di-hydroxylated compounds.     

Effect of fermentation time on antioxidative activities of Ganoderma lucidum broth using leguminous plants as part of the liquid fermentation medium

Oxidative damage plays an important role in the pathology of human diseases. Ganoderma lucidum, a medicinal fungus, has been used for thousands of years in traditional Oriental medicine. It is reported to have antioxidant functions such as inhibition of lipid peroxidation. The objective of the present study was to investigate the effect of fermentation time on the antioxidative activities of G. lucidum broth filtrate using leguminous plants as part of the liquid fermentation medium. Inhibition of Cu²⁺-induced oxidation of human low-density lipoprotein (LDL), DPPH radical-scavenging activity, total phenolic compounds, isoflavones and protocatechuic acid were measured to evaluate the antioxidant activity of G. lucidum fermentation broth filtrate. Our results showed that black soybean and Astragalus membranaceus improved the antioxidant activity of the G. lucidum fermentation broth filtrate. Protocatechuic acid was identified by LC–MS as the antioxidant compounds whose relative potency of inhibiting LDL oxidation to Trolox is 1.55. Protocatechuic acid showed positive correlation with the antioxidant activity of the fermentation broth filtrate while isoflavones did not contribute to antioxidant activity.     

Rapid screening and identification of antioxidants in aqueous extracts of Houttuynia cordata using LC–ESI–MS coupled with DPPH assay

Houttuynia cordata Thunb. has been used traditionally as immune stimulant and anticancer agent. An aqueous extract of H. cordata tea showed high radical scavenging activity determined by off-line DPPH assays. Then, it was screened for its antioxidant components via an on-line DPPH radical scavenging technique coupled with a liquid chromatography–electrospray ionization mass spectrometer (LC–ESI–MS). Based on their mass spectra and fragmentation patterns; the antioxidant compounds were identified as quinic acid derivative, caffeic acid derivatives, procyanidin B, neo-chlorogenic acid, catechin, chlorogenic acid, crypto-chlorogenic acid and quercetin hexoside. LC–MS/MS in multiple reactions monitoring (MRM) mode was used to quantify these antioxidant compounds. Chlorogenic acid was found to be a major component in H. cordata tea.     

Determination of minor carbohydrates in carrot (Daucus carota L.) by GC–MS

Minor carbohydrates present in carrot (Daucus carota L.) have been studied by GC–MS analysis of their trimethylsilyl derivatives because of their remarkable role in a variety of biological functions. Scyllo-inositol and sedoheptulose (d-altro-2-heptulose), identified for the first time in this paper were present in all the carrots analysed in concentrations ranging 1.5–5.8 and 1.4–24.6 mg g⁻¹ dried weight, respectively. Other minor carbohydrates detected in carrot were myo- inositol (2.2–9.8 mg g⁻¹) and mannitol (traces-1.3 mg g⁻¹). Whereas small amounts (close to 2 mg g⁻¹) of scyllo-inositol were experimentally determined in other vegetables from the Apiaceae family (parsley, coriander and fennel), sedoheptulose was only detected at trace levels.     

Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm

Polyphenolic profiles in the berry samples of 344 European grape (Vitis vinifera) cultivars were evaluated for two consecutive years. These cultivars represent a diverse collection of V. vinifera germplasm maintained at the USDA-Agricultural Research Service Vitis Clonal Repository in Davis of California, USA. A total of 36 polyphenolic compounds, including 16 anthocyanins, 6 flavonols, 6 flavanols, 6 hydroxycinnamic acids and 2 hydroxybenzoic acids, were identified via HPLC–MS and quantified by HPLC–DAD. The mean contents for anthocyanins, flavanols, flavonols, hydroxycinnamic acids and hydroxybenzoic acids were 0.946 (coloured cultivars), 0.147, 0.043, 0.195 and 0.016 mg g⁻¹ FW, respectively. On average, wine grapes had higher concentrations than had table grapes for all of these compounds except hydroxycinnamic acids. Berry colours affected the total contents of anthocyanins, but not others. Positive correlations (0.151–0.535) were found among these groups of compounds. As expected, these groups of compounds were all negatively correlated with berry weight.     

Characterisation of phenolic antioxidants in aqueous extract of Orthosiphon grandiflorus tea by LC–ESI-MS/MS coupled to DPPH assay

Aqueous extract from Orthosiphon grandiflorus tea was on-line screened for its antioxidant components based on their capacity to scavenge free DPPH (1,1-diphenyl-2-picrylhydrazyl) radical after separation by a LC gradient. The structural elucidation of the active compounds was achieved by negative ionisation LC–ESI-MS/MS. Based on their mass spectra and fragmentation patterns related to antioxidant activity trace, seven compounds showing strong DPPH scavenging were identified to be danshensu, caffeic acid, caftaric acid, rosmarinic acid, caffeic acid derivative, sagerinic acid and salvianolic acid B. It is noted that danshensu, caftaric acid, sagerinic acid and salvianolic acid B have been reported in O. grandiflorus for the first time. In addition, the quantification of antioxidant compounds was performed using LC–MS/MS in multiple reaction monitoring (MRM) mode. Rosmarinic acid was found as a major component responsible for the antioxidant activity of this plant extract.     

Formation and characterisation of melanoidin-like polycondensation products from amino acids and lipid oxidation products

Various coloured water-soluble high molecular weight and water-nonsoluble reaction products were isolated from model reactions of an amino acid (glycine or lysine) and a lipid oxidation product (hexanal, (2E)-hexenal, (2E, 4E)-decadienal) with or without glucose. They were characterised by UV–visible absorbance measurements, elementary analysis, and thermal degradation followed by SPME–GC–MS analysis. The UV–visible absorbance spectra before and after dialysis indicated that the most important contributors to the formation of water-soluble coloured material were constituents of the low molecular fraction. Elementary analysis data indicated that a higher amount of nitrogen was incorporated in the high molecular weight fractions as compared to the water-nonsoluble fractions, except for the water-nonsoluble reaction products from amino acid/(2E, 4E)-decadienal interactions, which showed the lowest C/N ratio found. Volatile carbonyl compounds, furans, aliphatic compounds, pyridines, pyrroles and benzene derivatives were the main groups identified in the thermal degradation profile of each fraction tested. Especially pyridines seem typical indicators of amino acid–lipid oxidation product interactions.