Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by Fluorescence in situ hybridization (FISH)

As the bioavailability of flavonoids is influenced by intestinal metabolism, we have investigated the microbial deconjugation and degradation of several flavonols and flavonol glycosides using the pig cecum in vitro model system developed in our group. For this model system the microbiota was directly isolated from the cecal lumen of freshly slaughtered pigs. The characterization of the cecal microbiota by fluorescence in situ hybridization (FISH) with 16S rRNA-based oligonucleotide probes confirmed the suitability of the model system for studying intestinal metabolism by the human microbiota. We have investigated the microbial degradation of quercetin-3-O-beta-d-rutinoside 1, quercetin-3-O-beta-d-glucopyranoside 2, quercetin-4'-O-beta-d-glucopyranoside 3, quercetin-3-O-beta-d-galactopyranoside 4, quercetin-3- O-beta-d-rhamnopyranoside 5, quercetin-3- O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 6, kaempferol-3-O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 7, apigenin 8, apigenin-8- C-glucoside (vitexin) 9, and feruloyl-O-beta-d-glucopyranoside 10 (100 microM), representing flavonoids with different aglycones, sugar moieties, and types of glycosidic bonds. The degradation rate was monitored using HPLC-DAD. The flavonol O-glycosides under study were almost completely metabolized by the intestinal microbiota within 20 min and 4 h depending on the sugar moiety and the type of glycosidic bond. The degradation rates of the quercetin monoglycosides showed a clear dependency on the hydroxyl pattern of the sugar moiety. The degradation of 2 with all hydroxyl groups of the glucose in the equatorial position was the fastest. The intestinal metabolism of di- and trisaccharides was much slower compared to the monoglycosides. The structure of the aglycone has not much influence on the intestinal metabolism; however, the type of glycosidic bond ( C- or O-glycoside) has substantial influence on the degradation rate. The liberated aglycones were completely metabolized within 8 h. Phenolic compounds such as 3,4-dihydroxyphenylacetic acid 12, 4-hydroxyphenylacetic acid 13, and phloroglucinol 18 were detected by GC-MS as main degradation products.     

MALDI-TOF MS analysis of food flavonol glycosides

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that is having a great impact on food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify flavonol glycosides in food samples. 2',4',6'-Trihydroxyacetophenone was chosen as the best matrix because it worked for crude sample extracts and ionized flavonol glycosides in both positive and negative MALDI-TOF MS modes. In the positive mode, multiple ion forms were observed for flavonol glycosides, including [M + H](+), [M + Na](+), [M + K](+), and [M - H + Na + K](+), with further fragmentation through loss of glycosidic residues. The negative mode for all flavonol glycosides resulted in [M - H](-) ion formation without detectable fragmentation. The multiple ions in the positive mode gave more information on individual flavonol glycoside structures than the negative mode. Flavonol glycosides showed similar intensities or responses in the positive mode, while kaempferol glycosides exhibited much less response than quercetin glycosides in negative mode.     

Structural investigations of flavonol glycosides from sea buckthorn (Hippophaë rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS(n)

Four flavonol glycosides were isolated from an extract of sea buckthorn pomace (Hippopha? rhamnoides) by Sephadex LH-20 gel chromatography and semipreparative HPLC. Their structures were elucidated by hydrolysis studies, ESI-MS(n), UV, and (1)H and (13)C NMR spectroscopy. The occurrence of the major flavonol glycoside kaempferol 3-O-beta-sophoroside-7-O-alpha-rhamnoside in sea buckthorn is described here for the first time. A further 21 flavonol glycosides of Sephadex LH-20 fractions of sea buckthorn pomace were characterized by HPLC-DAD-ESI-MS. The characteristic MS-MS and MS(3) fragmentation pattern of flavonol glycosides previously identified in sea buckthorn juice and of flavonol glycosides identified by NMR spectroscopy gave valuable indications for their identification. The results demonstrate that loss of the sugar moiety from C-7 of the aglycon is more favored than fission of the glycosidic linkage at the C-3 position. Thus, most of the compounds identified were 7-rhamnosides of isorhamnetin, kaempferol, and quercetin, which exhibit different substitution patterns at the C-3 position, mainly glucosides, rutinosides, and sophorosides. In addition, numerous flavonol glycosides were detected lacking a sugar moiety at C-7. Finally, eight flavonol derivatives were identified that are acylated by hydroxybenzoic or hydoxycinnamic acids.     

The flavonoid glycosides and procyanidin composition of Deglet Noor dates (Phoenix dactylifera)

The fruits of the date palm (Phoenix dactylifera) are consumed throughout the world and are an important part of the diet in the Middle East. Dates at the rutab and tamar maturity and ripening stages contain a wide array of phenolic antioxidants, but little is known about the composition of phenolic compounds in dates at the khalal stage of ripening. In the current study, the flavonoid glycoside and procyanidin compositions of dates of the cultivar Deglet Noor harvested at the khalal stage of maturity were characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS/MS). Procyanidin oligomers through decamers were identified in extracts of these dates. Higher molecular weight polymers, undecamers through heptadecamers, were also apparent from mass spectra. Thirteen flavonoid glycosides of luteolin, quercetin, and apigenin, 19 when considering isomeric forms, were also identified. Mass spectra indicate that both methylated and sulfated forms of luteolin and quercetin are present as mono-, di-, and triglycosylated conjugates whereas apigenin is present as only the diglycoside. LC-ESI/MS/MS spectra indicate that quercetin and luteolin formed primarily O-glycosidic linkages whereas apigenin is present as the C-glycoside.     

Flavonoids in horse chestnut (Aesculus hippocastanum) seeds and powdered waste water byproducts

Horse chestnut extracts are widely used in pharmacy and cosmetic industries. The main active constituents are saponins of oleane type, but seeds of horse chestnut also contain flavonoids, being glycosides of quercetin and kaempferol. Their contribution to the overall activity of the extracts was not clear. In the present work, the main flavonoids from horse chestnut seeds were isolated and their structures established with spectral methods. Seven glycosides were isolated, out of which six ( 2, 3, 4, 7, 11, 13) were previously reported and one ( 9) was identified as a new tamarixetin 3- O- [beta- d-glucopyranosyl(1-->3)]- O-beta- d-xylopyranosyl-(1-->2)- O-beta- d-glucopyranoside. The structures of three additional compounds 1, 10, and 12, not previously reported, were deduced on the basis of their LC-ESI/MS/MS fragmentation characteristics. A new ultraperformance liquid chromatographic (UPLC) method has been developed for profiling and quantitation of horse chestnut flavonoids. The method allowed good separation over 4.5 min. Thirteen compounds could be identified in the profile, out of which di- and triglycoisdes of quercetin and kaempferol were the dominant forms and their acylated forms occurred in just trace amounts. The total concentration of flavonoids in the powdered horse chestnut seed was 0.88% of dry matter. The alcohol extract contained 3.46%, and after purification on C18 solid phase, this concentration increased to 9.40% of dry matter. The flavonoid profile and their content were also measured in the horse chestnut wastewater obtained as byproduct in industrial processing of horse chestnut seeds. The total flavonoid concentration in the powder obtained after evaporation of water was 2.58%, while after purification on solid phase, this increased to 11.23% dry matter. It was concluded that flavonoids are present in a horse chestnut extract in a relatively high amount and have the potential to contribute to the overall activity of these extracts. Industrial horse chestnut wastewater can be used to obtain quercetine and kaempferol glycosides for cosmetic, nutraceutical, and food supplement industries.     

Separation and characterization of phenolic compounds in fennel (Foeniculum vulgare) using liquid chromatography-negative electrospray ionization tandem mass spectrometry

Liquid chromatography (LC) diode array detection (DAD) coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for the rapid and sensitive identification of water-soluble phenolic compounds in fennel waste. The plant material was first extracted and then chromatographed on Sephadex LH-20 to afford seven fractions, each of them being subjected to LC-MS analysis. Identification of the compounds was carried out by interpretation of UV, MS, and MS/MS spectra. Forty-two phenolic substances were identified, 27 of which had not previously been reported in fennel, including hydroxycinnamic acid derivatives, flavonoid glycosides, and flavonoid aglycons.     

Structural identification of novel oligosaccharides produced by Lactobacillus bulgaricus and Lactobacillus plantarum

β-Galactosidases (β-Gal) of lactic acid bacteria produce oligosaccharides from lactose when suitable acceptor carbohydrates are present. This study aimed to elucidate the structure of oligosaccharides formed by galactosylation of N-acetylglucosamine (GlcNAc) and fucose. Crude cellular extract of Lactobacillus bulgaricus and LacLM of Lactobacillus plantarum were used as sources of β-Gal activity. Disaccharides obtained by galactosylation of GlcNAc were identified as Gal-β-(1→4)-GlcNAc or Gal-β-(1→6)-GlcNAc by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and comparison with external standards. Trisaccharides were identified as Gal-β-(1→6)-Gal-β-(1→[4 or 6])-GlcNAc by LC-MS, analysis of the MS/MS spectra of selected in-source fragment ions, and their relative retention times. LC-MS analysis revealed the presence of five galactosylated fucosides, but their linkage type could not be identified, partly due to the lack of reference compounds. β-Gal of lactic acid bacteria may serve as suitable tools for the chemoenzymatic synthesis of therapeutic oligosaccharides.     

UHPLC-PDA-ESI/HRMS/MS(n) analysis of anthocyanins, flavonol glycosides, and hydroxycinnamic acid derivatives in red mustard greens (Brassica juncea Coss variety)

An UHPLC-PDA-ESI/HRMS/MS(n) profiling method was used for a comprehensive study of the phenolic components of red mustard greens ( Brassica juncea Coss variety) and identified 67 anthocyanins, 102 flavonol glycosides, and 40 hydroxycinnamic acid derivatives. The glycosylation patterns of the flavonoids were assigned on the basis of direct comparison of the parent flavonoid glycosides with reference compounds. The putative identifications were obtained from tandem mass data analysis and confirmed by the retention time, elution order, and UV-vis and high-resolution mass spectra. Further identifications were made by comparing the UHPLC-PDA-ESI/HRMS/MS(n) data with those of reference compounds in the polyphenol database and in the literature. Twenty-seven acylated cyanidin 3-sophoroside-5-diglucosides, 24 acylated cyanidin 3-sophoroside-5-glucosides, 3 acylated cyanidin triglucoside-5-glucosides, 37 flavonol glycosides, and 10 hydroxycinnamic acid derivatives were detected for the first time in brassica vegetables. At least 50 of them are reported for the first time in any plant materials.     

Glycosidically bound flavor compounds of cape gooseberry (Physalis peruviana L.)

The bound volatile fraction of cape gooseberry (Physalis peruviana L.) fruit harvested in Colombia has been examined by HRGC and HRGC-MS after enzymatic hydrolysis using a nonselective pectinase (Rohapect D5L). Forty bound volatiles could be identified, with 21 of them being reported for the first time in cape gooseberry. After preparative isolation of the glycosidic precursors on XAD-2 resin, purification by multilayer coil countercurrent chromatography and HPLC of the peracetylated glycosides were carried out. Structure elucidation by NMR, ESI-MS/MS, and optical rotation enabled the identification of (1S,2S)-1-phenylpropane-1,2-diol 2-O-beta-D-glucopyranoside (1) and p-menth-4(8)-ene-1,2-diol 1-O-alpha-L-arabinopyranosyl-(1-6)-beta-D-glucopyranoside (2). Both glycosides have been identified for the first time in nature. They could be considered as immediate precursors of 1-phenylpropane-1,2-diol and p-menth-4(8)-ene-1,2-diol, typical volatiles found in the fruit of cape gooseberry.     

Characterization of cigarette tobacco by direct electrospray ionization-ion trap mass spectrometry (ESI-ITMS) analysis of the aqueous extract--a novel and simple approach

In support of the efforts to combat smuggling, as well as illegal sale and distribution of cigarettes, an analytical approach for the characterization of tobacco has been proposed and evaluated. It involves aqueous extraction of the filler tobaccos followed by direct analysis of the extracts by electrospray ionization-ion trap mass spectrometry (ESI-ITMS) in the negative mode. Typically, the deprotonated ions, [M - H](-), of organic acids (malic, citric, caffeic, quinic acid) and polyphenols (chlorogenic acid, rutin, scopoletin) were detected. MS/MS spectra of the ion at m/z 191, which is the [M - H](-) of quinic acid, citric acid, and scopoletin, and a fragment ion of chlorogenic acid were acquired. Significant differences in the MS and MS/MS spectra were observed between counterfeit samples and the corresponding authentic brand name cigarettes. Analysis of 25 commercial cigarettes showed that straight Virginia blends were readily distinguished from the blended products containing different tobacco types (Virginia, burley, and Oriental). The former exhibited consistently higher relative abundances of m/z 353 (chlorogenic acid) to m/z 133 (malic acid) in the MS spectra (0.9-1.2 vs 0.4-0.6) and higher intensity ratios of m/z 176 (scopoletin) to m/z 173 (0.4-0.8 vs 0.1-0.3) and of m/z 127 (quinic acid) to m/z 173 (0.7-1.0 vs 0.3-0.5) in the MS/MS spectra. Evidence is presented to demonstrate that the spectral differences were related not only to the tobacco type (Virginia, burley and Oriental) but also to the tobacco part (stem, lamina) used in the manufacture of the cigarettes.     

初级芳香胺的高分辨质谱裂解规律研究

为探究初级芳香胺类化合物的质谱裂解规律,本研究采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法,在电喷雾正离子模式下采集数据,根据一、二级质谱离子的精确质荷比推导14种初级芳香胺类化合物可能的裂解途径。结果表明,由于分子结构中存在氨基基团,因此初级芳香胺类化合物极易被质子化,更易形成[M+H]⁺。断裂过程主要发生NH₃中性碎片的丢失,产生[M+H-NH₃]⁺特征碎片离子,在此基础上,氯代苯胺类化合物发生碳正离子重排后,丢失氯原子形成[·M+H-NH₃-Cl]⁺碎片离子。甲氧基取代苯胺类化合物可丢失CH₃O基团产生碎片离子[M+H-NH₃-CH₃O]⁺,或发生碳正离子转移重排至苯甲基,进一步丢失CH₃和CH₃O基团。甲基或甲氧基取代的联苯胺类化合物还会发生CN键断裂形成[M+H-NH]⁺,继而丢失NH₃中性碎片形成[M+H-NH-NH₃]⁺。偶氮苯类化合物中高键能的偶氮键不易断裂,碎片离子主要通过两侧的CN键断裂形成。本研究提出的最佳电离方式和质谱裂解规律为初级芳香胺类化合物的快速鉴定提供了重要依据。     

Reactivity of epicatechin in aqueous glycine and glucose maillard reaction models: quenching of C2, C3, and C4 sugar fragments

Mechanisms of how epicatechin alters the pathways of the Maillard reaction were investigated. Carbon-13 and nitrogen-15 labeling studies were utilized to define the reactivity of epicatechin with glucose, glycine, and/or reaction products in an aqueous model (pH 7, 125 degrees C for 30 min) via GC, GC/MS and HPLC/MS analysis. Quantification of the volatile reaction product isotopomers by GC/MS from a 1:1 labeled to unlabeled glucose (carbohydrate module labeling technique) plus glycine model system indicated the formation of 2,3-butanedione and acetol were primarily formed via intact C4 and C3 sugar fragments, whereas pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and cyclotene were primarily formed via intact C2/C2, C2/C3, C3/C3, C3/C3, and C3/C3 sugar fragment pairs, respectively. The formation of these seven compounds was also reported by GC analysis to be dramatically inhibited when epicatechin was added to the glucose/glycine model system (observed 9-113-fold reduction). HPLC/MS analysis of both the glucose-labeled and glycine-labeled model systems with and without epicatechin indicated that epicatechin reacted directly with C2, C3, and C4 sugar fragments, while epicatechin did not report any direct reactivity with glycine. In conclusion, the quenching of sugar fragmentation products via epicatechin was correlated with the observed inhibition on volatile compound formation when epicatechin was added to a glucose/glycine aqueous reaction model system.     

Thermal degradation of onion quercetin glucosides under roasting conditions

Flavonoids are an important constituent of the human diet. In recent years, they have gained much attention due to their physiological properties, leading to an enormous increase in research on cancer prevention and reduction of cardiovascular diseases. Unfortunately, there is limited information about the fate of flavonoid glycosides during thermal treatment such as cooking, frying, roasting, etc. Such processing techniques may have an impact on the flavonoid structure, resulting in changes of the bioavailability and activity of the flavonoids. In this study, the stability of selected model and onion quercetin glycosides under roasting conditions (180 degrees C) was determined. The influence of the kind and position of the sugar moiety was investigated. As onions contain large amounts of quercetin glycosides and are often subject to thermal processes in food production, their major glycosides were isolated using counter current chromatography and roasted. The thermal treatment led to a degradation of the quercetin glycosides. The main product is the aglycone quercetin, which remained stable during further roasting. During the roasting process of the quercetin diglucoside isolated from onion, the formation of a monoglycoside as an intermediate product was observed. This underlined that the stability of the glycosides is dependent on the kind and position of the sugar moiety.     

Chemical ionization mass spectrometry of N-methylcarbamate insecticides, some of their metabolites, and related compounds.

The chemical ionization (CI) mass spectra with methane as the reagent gas are reported for 25 N-methylcarbamate insecticides or related compounds. The [M + 1]+ ions in the CI spectra are larger than the parent ions in the corresponding electron impact (EI) spectra of N-methyl- and N,N-dimethylcarbamates, but the contrary is true with N-hydroxymethylcarbamates. CI but not EI fragmentation of substituted-phenyl N-hydroxymethylcarbamates [ROC (O) NHCH2OH] appears to involve a 4-membered [ROC(O)NHCH2]+ intermediate which rearranges to give a [ROCH2]+ fragment. Other fragmentations unique to the CI spectra are also discussed.     

Crocetin esters, picrocrocin and its related compounds present in Crocus sativus stigmas and Gardenia jasminoides fruits. Tentative identification of seven new compounds by LC-ESI-MS

Crocetin esters present in saffron (Crocus sativus L.) stigmas and in Gardenia jasminoides Ellis fruit are the compounds responsible for their color. Of the fifteen crocetin esters identified in this study, five new compounds were tentatively identified: trans and cis isomers of crocetin (beta-D-triglucoside)-(beta-D-gentibiosyl) ester, trans and cis isomers of crocetin (beta-D-neapolitanose)-(beta-D-glucosyl) ester, and cis crocetin (beta-D-neapolitanose)-(beta-D-gentibiosyl) ester. The most relevant differences between both species were a low content of the trans crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) ester, the absence of trans crocetin di-(beta-D-glucosyl) ester in gardenia, and its higher content of trans crocetin (beta-D-gentibiosyl) ester and cis crocetin di-(beta-D-gentibiosyl) ester. With the same chromatographic method it was possible to identify, in a single run, ten glycosidic compounds in saffron extracts with a UV/vis pattern similar to that of picrocrocin; among them, 5-hydroxy-7,7-dimethyl-4,5,6,7-tetrahydro-3H-isobenzofuranone 5-O-beta-D-gentibioside and 4-hydroxymethyl-3,5,5-trimethyl-cyclohexen-2-one 4-O-beta-D-gentibioside were tentatively identified for the first time in saffron. Of these ten glycosides, only the O-beta-D-gentibiosyl ester of 2-methyl-6-oxo-2,4-hepta-2,4-dienoic acid was found in gardenia samples, but it was possible to identify the iridoid glycoside, geniposide.     

Determination of strophanthidin in ingesta and plant material by LC-MS/MS

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.     

Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse

Application of taste dilution analyses on freshly prepared black tea infusions revealed neither the high molecular weight thearubigen-like polyphenols nor the catechins and theaflavins, but a series of 14 flavon-3-ol glycosides as the main contributors to the astringent taste perceived upon black tea consumption. Among these glycosides, the apigenin-8-C-[alpha-l-rhamnopyranosyl-(1-->2)-O-beta-d-glucopyranoside] was identified for the first time in tea infusions. Depending on the structure, the flavon-3-ol glycosides were found to induce a velvety and mouth-coating sensation at very low threshold concentrations, which were far below those of catechins or theaflavins; for example, the threshold of 0.001 micromol/L found for quercetin-3-O-[alpha-l-rhamnopyranosyl-(1-->6)-O-beta-d-glucopyranoside] is 190000, or 16000 times below the threshold determined for epigallocatechin gallate or theaflavin, respectively. Moreover, structure/activity considerations revealed that, besides the type of flavon-3-ol aglycon, the type and the sequence of the individual monosaccharides in the glycosidic chain are key drivers for astringency perception of flavon-3-ol glycosides.     

Nectar Flavonol rhamnosides are floral markers of acacia (Robinia pseudacacia) honey

With the objective of finding floral markers for the determination of the botanical origin of acacia (robinia) honey, the phytochemicals present in nectar collected from Robinia pseudacacia flowers were analyzed by high-performance liquid chromatography-tandem mass spectrometry. Eight flavonoid glycosides were detected and characterized as kaempferol combinations with rhamnose and hexose. Acacia honey produced in the same location where the nectar was collected contained nectar-derived kaempferol rhamnosides. This is the first time that flavonoid glycosides have been found as honey constituents. Differences in the stability of nectar flavonoids during honey elaboration and ripening in the hive were shown to be due to hydrolytic enzymatic activity and to oxidation probably related to hydrogen peroxide (glucose-oxidase) activity. Acacia honeys contained propolis-derived flavonoid aglycones (468-4348 microg/100 g) and hydroxycinnamic acid derivatives (281-3249 microg/100 g). In addition, nectar-derived kaempferol glycosides were detected in all of the acacia honey samples analyzed (100-800 microg/100 g). These flavonoids were not detected in any of the different honey samples analyzed previously from different floral origins other than acacia. Finding flavonoid glycosides in honey related to floral origin is particularly relevant as it considerably enlarges the number of possible suitable markers to be used for the determination of the floral origin of honeys.     

Transglycosylation of naringin by Bacillus stearothermophilusMaltogenic amylase to give glycosylated naringin.

Naringin, a bitter compound in citrus fruits, was transglycosylated by Bacillus stearothermophilus maltogenic amylase reaction with maltotriose to give a series of mono-, di-, and triglycosylnaringins. Glycosylation products of naringin were observed by TLC and HPLC. The major glycosylation product was purified by using a Sephadex LH-20 column. The sturcture was determined by using MALDI-TOF MS, methylation analysis, and (1)H and (13)C NMR. The major transglycosylation product was maltosylnaringin, in which the maltose unit was attached by an alpha-1-->6 glycosidic linkage to the D-glucose moiety of naringin. This product was 250 times more soluble in water and 10 times less bitter than naringin.