Chronic inflammation on gingiva-derived mesenchymal stem cells.

The impact of inflammatory response on the biological characteristics of GMSCs has been the subject of studies, with conflicting findings. In order to more fully understand the effects of the localized inflammatory environment, the current study assessed the intensity and differentiating capacity of GMSCs derived from healthy periodontal tissues (H-GMSC) and GMSC derived from inflamed periodontal tissues (I-GMSC) tissues. Cells from every well were taken out and counted using a hemocytometer every three days for a total of 12 days. The findings of the cell proliferation assay, which involved quantifying the cells with the help of a hemocytometer at 0th day, 3rd day, 6th day, and 9th day, are shown. On day nine of culture, there was a considerable (P = 0.02) variation in the rate of multiplication between GMSCs from healthy gingival tissues and GMSCs from gingival tissues having inflammation. Additionally, I-GMSCs had a higher cell concentration on day twelve than that of H-GMSCs. However, there was no significant variance in PDT values comparing GMSCs from healthy gingival tissues and GMSCs from gingival tissues having inflammation (P > 0.05). The mean PDT findings of 66.7 h and 53.4 h have been documented for Healthy-GMSCs and Inflamed-GMSCs, respectively. In addition, compared to GMSCs from healthy gingival tissues, GMSCs from inflammatory tissues had decreased osteogenesis and increased adipogenic potential. To evaluate the efficacy of GMSCs derived from patients suffering periodontitis utilising human models for cell-based treatments, additional study is necessary.

Energy resolved mass spectrometry of chlorogenic acids and its application to isomer quantification by direct infusion tandem mass spectrometry.

INTRODUCTION: With the advent of high-perfomance liquid chromatography (HPLC)-tandem mass spectrometry (MS) using ion trap mass analysers it is possible to acquire unambigious structural information in particular with respect to aspects of regiochemistry and stereochemistry of organic compounds present in complex mixtures such as coffee extracts. However, HPLC-MS methods are resource extensive, laborious and lacking user friendliness. OBJECTIVE: To introduce a simple parameter - the energy threshhold for fragmentation - determined using energy resolved MS and demonstrate its value for the complete structural characterisation and even relative quantification of individual isomeric chlrogenic acids in direct infusion experiments. METHODOLOGY: Monocaffeoyl and dicaffeoyl quinic acids were investigated by direct infusion energy resolved mass spectrometry (ER-MS) in negative in mode, using a quadrupole ion trap and quadrupole time-of-flight (Q-TOF) mass spectrometer. Methanolic coffee extracts were quantitatively investigated by HPLC-MS and direct infusion ER-MS. RESULTS: Fragmentation occurs with retention of regiochemistry and regiochemistry of fragment ions can be determined using ER-MS. Analysis of breakdown graphs allows extraction of a single numerical parameter that allows assignment of regiochemistry. Analysis of monocaffeoyl and dicaffeoyl quinic acids revealed that regiosiomers could be distinguished and assigned based on their dissociation energies in collisional induced activation. Furthermore relative quantification of regioisomers by direct infusion ER-MS is possible within an error range of ±10% if compared with a conventional quantitative LC-MS method. CONCLUSION: ER-MS can be exploited in determining relative isomers quantities of chlorogenic acids (CGAs) in crude plant extracts by direct infusion tandem MS omitting time and resource intensive chromatographic separation.

Profiling and Quantification of Regioisomeric Caffeoyl Glucoses in Berry Fruits.

On the basis of a recently developed tandem mass spectrometry-based hierarchical scheme for the identification of regioisomeric caffeoyl glucoses, selected berry fruits were profiled for their caffeoyl glucose ester content. Fresh edible berries profiled, including strawberries, raspberries, blueberries, blackberries, red currant, black currant, lingonberries, gooseberries, and juices of elderberries, goji berries, chokeberries, cranberries, açai berries, sea buckthorn berries, Montmorency sour cherries, and pomegranates, were investigated. 1-Caffeoyl glucose was found to be the predominant isomer in the majority of samples, with further profiling revealing the presence of additional hydroxycinnamoyl glucose esters and O-glycosides with p-coumaroyl, feruloyl, and sinapoyl substituents. A quantitative liquid chromatography-mass spectrometry-based method was developed and validated, and all caffeoyl glucose isomers were quantified for the first time in edible berries.

Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables.

Based on the recently developed tandem MS based hierarchical scheme for the identification of regioisomeric caffeoyl glucoses, selected vegetables were profiled with respect to their caffeoyl glucose content. The dietary plants profiled were tomato, pepper, chilli and aubergine, all members of the Solanaceae family. 6-O-caffeoyl glucose was found to be the predominant isomer. In processed food such as tomato puree and ketchup a larger number of caffeoyl-glucose isomers formed through acyl migration reactions were observed. A LC-MS based quantitative method was developed, validated and caffeoyl glucose regioisomers quantified for the first time in dietary plants with quantitative data obtained from representative 30 food samples.

Synthesis, Structure, and Tandem Mass Spectrometric Characterization of the Diastereomers of Quinic Acid.

(-)-Quinic acid possess eight possible stereoisomers, which occur both naturally and as products of thermal food processing. In this contribution, we have selectively synthesized four isomers, namely, epi-quinic acid, muco-quinic acid, cis-quinic acid, and scyllo-quinic acid, to develop a tandem LC-MS method identifying all stereoisomeric quinic acids. Four derivatives have been unambiguously characterized by single-crystal X-ray crystallography. The missing diastereomers of quinic acid were obtained by nonselective isomerization of (-)-quinic acid using acetic acid/concentrated H2SO4 allowing chromatographic separation and assignment of all diastereomers of quinic acid. We report for the first time that a full set of stereoisomers are reliably distinguishable on the basis of their tandem mass spectrometric fragment spectra as well as their elution order. A rationale for characteristic fragmentation mechanisms is proposed. In this study, we also observed that muco-quinic acid, scyllo-quinic acid, and epi-quinic acid are present in hydrolyzed Guatemalan roasted coffee sample as possible products of roasting.

Neuraminidase inhibition of Dietary chlorogenic acids and derivatives - potential antivirals from dietary sources.

Plants rich in chlorogenic acids (CGAs), caffeic acids and their derivatives have been found to exert antiviral effects against influenza virus neuroaminidase. In this study several dietary naturally occurring chlorogenic acids, phenolic acids and derivatives were screened for their inhibitory activity against neuroaminidases (NAs) from C. perfringens, H5N1 and recombinant H5N1 (N-His)-Tag using a fluorometric assay. There was no significant difference in inhibition between the different NA enzymes. The enzyme inhibition results indicated that chlorogenic acids and selected derivatives, exhibited high activities against NAs. It seems that the catechol group from caffeic acid was important for the activity. Dietary CGA therefore show promise as potential antiviral agents. However, caffeoyl quinic acids show low bioavailibility and are intensly metabolized by the gut micro flora, only low nM concentrations are observed in plasma and urine, therefore a systemic antiviral effect of these compounds is unlikely. Nevertheless, gut floral metabolites with a catechol moiety or structurally related dietary phenolics with a catechol moiety might serve as interesting compounds for future investigations.

LC-MSn study of the chemical transformations of hydroxycinnamates during yerba maté (Ilex paraguariensis) tea brewing.

Yerba maté is one of the most popular beverages in South American countries and its consumption is associated with a wide array of health effects. In this study, we used advanced HPLC-ESI-MSn and HPLC-ESI-HRMS methods for the identification and characterization of hydroxycinnamates and their derivatives formed during the brewing process of yerba maté. We report on the hydroxylation of the hydroxycinnamates cinnamoyl substituent by conjugate addition of water to form 3-hydroxy-dihydrocinnamic acid derivatives using a series of model compounds, including caffeoylglucoses, dicaffeoylquinic acids, methyl caffeoylquinate and mono caffeoylquinic acids. The regiochemistry of conjugate addition was determined by targeted tandem MS experiments performed on authentic standards. It was interesting to note that hydroxylation of hydroxycinnamates produced cis and acyl-migration isomers, which is in line with previously reported data.

Differentiation of prototropic ions in regioisomeric caffeoyl quinic acids by electrospray ion mobility mass spectrometry.

RATIONALE: A series of dietary important regioisomeric chlorogenic acids were investigated by ion mobility mass spectrometry (IM-MS). The existence of prototropic isomers separated in the drift dimension was observed and investigated further using tandem mass spectrometry (MS/MS) and compared with suitable synthetic analogues. METHODS: Using a quadrupole ion mobility time-of-flight mass spectrometer, the IM-MS and IM-MS/MS spectra of selected chlorogenic acids were recorded in the negative ion mode and compared with synthetic analogues. RESULTS: Regioisomeric di- and monocaffeoyl quinic acids can be readily separated and investigated using IM-MS. Comparison of drift times allows assignment of the regiochemistry of precursor ions as well as for fragment ions. For 5-caffeoyl quinic acid the existence of prototropic ions was suggested and probed using synthetic analogues, unable to show this type of isomerism. These investigations suggest the presence of prototropic isomers with carboxylate and phenolate sites, respectively. CONCLUSIONS: We report on IM-MS measurements on regioisomeric mono- and dicaffeoyl quinic acids, which are important dietary natural products. Both classes of compounds can be readily separated by IM-MS in the drift time dimension and, following MS(2) experiments, fragment ion regiochemistry unambiguously determined. 5-Caffeoyl quinic acid shows two IM-MS signals, which we assign to prototropic isomers after comparison with suitable synthetic analogues, with a negative charge located at the carboxalate or phenolate functionality, respectively.

Investigation of the photochemical changes of chlorogenic acids induced by ultraviolet light in model systems and in agricultural practice with Stevia rebaudiana cultivation as an example.

Mono- and diacyl chlorogenic acids undergo photochemical trans-cis isomerization under ultraviolet (UV) irradiation. The photochemical equilibrium composition was established for eight selected derivatives. In contrast to all other dicaffeoylquinic acid derivatives, cynarin (1,3-dicaffeoylquinic acid) undergoes a [2 + 2] photochemical cycloaddition reaction, constituting a first example of Schmidt's law in a natural product family. The relevance of photochemical isomerization in agricultural practice was investigated using 120 samples of Stevia rebaudiana leave samples grown under defined cultivation conditions. Ratios of cis to trans chlorogenic acids were determined in leaf samples and correlated with climatic and harvesting conditions. The data indicate a clear correlation between the formation of cis-caffeoyl derivatives and sunshine hours prior to harvesting and illustrate the relevance of UV exposure to plant material affecting its phytochemical composition.

Hierarchical key for the LC-MSn identification of all ten regio- and stereoisomers of caffeoylglucose.

A chromatographic method was developed to separate all 10 regio- and stereoisomers of caffeoylglucose. Following chromatographic separation on reversed phase, the fragmentation behavior of all 10 regio- and stereoisomers of caffeoylglucose has been investigated using LC-MS(n). It is possible to discriminate between each of the isomers based on their characteristic fragment spectra and order of elution, including those for which commercial standards are not available. On the basis of the synthesis of authentic standards for 6-caffeoylglucose and 3-caffeoylglucose and nonselective further synthesis of suitable mixtures of isomers, it was possible to fully assign regiochemistry of all 10 isomeric compounds and stereochemistry of eight isomeric compounds. Their fragmentation pattern was rationalized based on assuming different hydrogen-bonding arrays of gas-phase ions opening distinct fragmentation pathways. An analysis of yerba maté extract showed all 10 regio- and stereoisomers of caffeoylglucose to be present in this dietary material, which could all be assigned to regioisomeric level and eight to stereoisomeric level.

Identification and characterisation of phenolics from Ixora coccinea L. (Rubiaceae) by liquid chromatography multi-stage mass spectrometry.

INTRODUCTION: Ixora coccinea L. leaves and stem are used in traditional Sudanese and Ayurvedic medicinal systems for the treatment of diarrhoea, fever, headache, skin diseases, eye trouble, wounds, sores and ulcers. Recent studies show that I. coccinea has anti-oxidant, anti-bacterial, anti-cancer, anti-inflammatory, analgaesic, anti-diarrhoeal, hepatoprotective, cardioprotective, anti-mutagenic, wound healing and anti-tumour activities. Ixora coccinea is a rich source of polyphenols such as proanthocyanidins, flavonoids, flavonoids glycosides and tannins. OBJECTIVES: To develop a LC-MS(n) method for the identification and characterisation of phenolic compounds of I. coccinea L. leaves and stem. METHODS: Aqueous methanolic (70% methanol) extracts of I. coccinea leaves and stem were used for LC-MS(n) to ensure efficient extraction of phenolics. A C18 amide reverse-phase HPLC column allowed separation of the phenolic compounds, including different isomers. For the LC-MS measurements, negative ion mode was used in order to obtain better tandem mass spectra and high-resolution mass spectra. RESULTS: The phenolics were identified by their typical UV absorptions at 254, 280 and 320 nm. All the flavonol glycosides showed a neutral loss of the glycan part; hydroxycinnamates showed loss of the cinnamoyl/cinnamic acid part; while proanthocyanidins showed a Diels-Alder fragment in negative ion mode mass spectra. CONCLUSION: It was possible to identify C-3 and C-7 flavonol glycosides by their order of elution and it was also possible to predict the glycosylation position in flavonol diglycosides from their tandem mass spectra.

Identification and characterization of chlorogenic acids, chlorogenic acid glycosides and flavonoids from Lonicera henryi L. (Caprifoliaceae) leaves by LC-MSn.

The chlorogenic acids, chlorogenic acid glycosides and flavonoids of the leaves of Lonicera henryi L. (Caprifoliaceae) were investigated qualitatively by liquid chromatography tandem mass spectrometry. Thirty-one chlorogenic acids and their glycosides were detected and characterized to their regioisomeric level on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. All of them were extracted for the first time from this source and thirteen of them were not reported previously in nature. For the positive identification of chlorogenic acid glycosides by LC-MS(n), multiple reaction monitoring and targeted MS(n) experiments were performed. We have developed an LC-MS(n) method for the systematic identification of chlorogenic acid glycosides and were also able to discriminate between chlorogenic acids and their isobaric glycosides. It was also possible to discriminate between 5-O-(3'-O-caffeoyl glucosyl)quinic acid and 5-O-(4'-O-caffeoyl glucosyl)quinic acid by LC-MS(n). This method can be applied for the rapid and positive identification of chlorogenic acids and their glycosides in plant materials, food and beverages.

Identification and characterisation of the phenolics of Ilex glabra L. Gray (Aquifoliaceae) leaves by liquid chromatography tandem mass spectrometry.

The phenolics of the leaves of Ilex glabra L. Gray (Aquifoliaceae) were investigated qualitatively by LC-MS(n). Thirty-two phenolics were detected and characterised on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. All of them were extracted for the first time from this source and fifteen of them were not reported previously in nature. For the positive identification of phenolic glucosides by LC-MS(n) a series of authentic standards and experiments were carried out. This is the first report of a full characterisation of 3,4-dihydroxybenzoyl glucosides, 3,4-dihydroxybenzyl glucosides, 4-hydroxybenzoyl glucosides, chlorogenic acid glucosides and vanillic acid glucosides by LC-MS(2-4).

Investigation of acyl migration in mono- and dicaffeoylquinic acids under aqueous basic, aqueous acidic, and dry roasting conditions.

Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties from one quinic acid alcohol group to another, thus interconverting chlorogenic acid regioisomers. It therefore constitutes a special case of transesterification reaction. Acyl migration constitutes an important reaction pathway in both coffee roasting and brewing, altering the structure of chlorogenic acid initially present in the green coffee bean. In this contribution we describe detailed and comprehensive mechanistic studies comparing inter- and intramolecular acyl migration involving the seven most common chlorogenic acids in coffee. We employe aqueous acidic and basic conditions mimicking the brewing of coffee along with dry roasting conditions. We show that under aqueous basic conditions intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl migration. 3-Caffeoylquinic acid was shown to be most labile to basic hydrolysis. We additionally show that the acyl migration process is strongly pH dependent with increased transesterification taking place at basic pH. Under dry roasting conditions acyl migration competes with dehydration to form lactones. We argue that acyl migration precedes lactonization, with 3-caffeoylquinic acid lactone being the predominant product.

Identification and characterization of the phenolic glycosides of Lagenaria siceraria Stand. (bottle gourd) fruit by liquid chromatography-tandem mass spectrometry.

Bottle gourd, Lagenaria siceraria Stand. (Cucurbitaceae), fruit is used in folk medicines and for culinary purposes in Asia. The phenolics of bottle gourd fruit were investigated qualitatively by LC-MS(n). Twenty-two phenolic glycosides were detected and characterized on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. Twenty of them were extracted for the first time from this source, and twelve of them have not been reported previously in nature. It was also possible to distinguish between the individual classes of isobaric phenolic glycosides by tandem and high-resolution mass spectrometry. In this study we also discuss the mass spectrometric fragmentation mechanism of 6-(hydroxycinnamoyl)glucoses. This is the first report of the full characterization of phenolic glycosides of bottle gourd fruit by LC-MS²â»4.

Identification of phenolic compounds in plum fruits (Prunus salicina L. and Prunus domestica L.) by high-performance liquid chromatography/tandem mass spectrometry and characterization of varieties by quantitative phenolic fingerprints.

Plums (Prunus domestica L. and Prunus salicina L.) are edible fruits mostly consumed in America, China, and Europe. We have used LC-MS(n) to detect and characterize the phenolic compounds in plum varieties. Forty-one phenolics were detected comprising caffeoylquinic acids, feruloylquinic acid, p-coumaroylquinic acids, methyl caffeoylquinates, methyl p-coumaroylquinate, caffeoylshikimic acids, catechin, epicatechin, rutin, esculin, quercetin, quercetin-3-O-hexosides, dimeric proanthocyanidins, trimeric proanthocyanidins, caffeoyl-glucoside, feruloyl-glucoside, p-coumaroyl-glucoside, vanillic acid-glucosides, 3,4-dihydroxybenzoyl-glucoside, quercetin-3-O-pentosides, quercetin-3-O-rhamnoside, quercetin-pentoside-rhamnosides, and 3-p-methoxycinnamoylquinic acid. This is the first time when 3-p-methoxycinnamoylquinic acid is reported in nature. Chlorogenic acids and proanthocyanidins were the major phenolics present in plums. Furthermore, HPLC with DAD and chemical reaction detection was used to generate quantitative phenolic fingerprints from the fruit flesh of 33 plum varieties. The predominant compound was 3-caffeoylquinic acid in nearly all varieties studied; generally, however, the qualitative and quantitative profiles showed high diversity even among closely related progenies.

What is under the hump? Mass spectrometry based analysis of complex mixtures in processed food--lessons from the characterisation of black tea thearubigins, coffee melanoidines and caramel.

In this contribution we review our work on the characterisation of processed food. We review novel methods and analysis strategies developed to account for the composition of extraordinarily complex materials such as black tea thearubigins, coffee melanoidines and thermally treated carbohydrates. Our methods are mainly based on modern mass spectrometry and are introduced and critically discussed. A series of novel previously unpublished data interpretation strategies are presented as well. Finally an evaluation of the insight obtained in the composition of selected processed foods is given discussing potential consequences for assessing beneficial and adverse health effects of processed food.

Phenolic promiscuity in the cell nucleus--epigallocatechingallate (EGCG) and theaflavin-3,3'-digallate from green and black tea bind to model cell nuclear structures including histone proteins, double stranded DNA and telomeric quadruplex DNA.

Flavanols from tea have been reported to accumulate in the cell nucleus in considerable concentrations. The nature of this phenomenon, which could provide novel approaches in understanding the well-known beneficial health effects of tea phenols, is investigated in this contribution. The interaction between epigallocatechin gallate (EGCG) from green tea and a selection of theaflavins from black tea with selected cell nuclear structures such as model histone proteins, double stranded DNA and quadruplex DNA was investigated using mass spectrometry, Circular Dichroism spectroscopy and fluorescent assays. The selected polyphenols were shown to display affinity to all of the selected cell nuclear structures, thereby demonstrating a degree of unexpected molecular promiscuity. Most interestingly theaflavin-digallate was shown to display the highest affinity to quadruplex DNA reported for any naturally occurring molecule reported so far. This finding has immediate implications in rationalising the chemopreventive effect of the tea beverage against cancer and possibly the role of tea phenolics as "life span essentials".

Investigating the chemical changes of chlorogenic acids during coffee brewing: conjugate addition of water to the olefinic moiety of chlorogenic acids and their quinides.

Coffee is one of the most popular and consumed beverages in the world and is associated with a series of benefits for human health. In this study we focus on the reactivity of chlorogenic acids, the most abundant secondary metabolites in coffee, during the coffee brewing process. We report on the hydroxylation of the chlorogenic acid cinnamoyl substituent by conjugate addition of water to form 3-hydroxydihydrocaffeic acid derivatives using a series of model compounds including monocaffeoyl and dicaffeoylquinic acids and quinic acid lactones. The regiochemistry of conjugate addition was established based on targeted tandem MS experiments. Following conjugate addition of water a reversible water elimination yielding cis-cinnamoyl derivatives accompanied by acyl migration products was observed in model systems. We also report the formation of all of these derivatives during the coffee brewing process.

Understanding the fate of chlorogenic acids in coffee roasting using mass spectrometry based targeted and non-targeted analytical strategies.

Coffee is one of mankind's most popular beverages obtained from green coffee beans by roasting. Much effort has been expended towards the chemical characterisation of the components of the roasted coffee bean, frequently termed melanoidines, which are dominated byproducts formed from its most relevant secondary metabolites - chlorogenic acids. However, impeded by a lack of suitable authentic reference standards and analytical techniques sufficiently powerful for providing insight into an extraordinarily complex enigmatic material, unsurprisingly little structural and mechanistic information about the products of coffee roasting is available. Here we report on the characterisation of low molecular weight melanoidine fractions of roasted coffee using a conceptually novel combination of targeted and non-targeted mass spectrometrical techniques. We provide an unprecedented account of the chemical composition of roasted coffee beans. Using a targeted analytical approach we show for the first time, by comparison to authentic reference standards obtained by chemical synthesis, that chlorogenic acids follow four distinct reaction pathways including epimerization, acyl migration, lactonisation and dehydration. The analytical strategy employed in a non-targeted approach uses high resolution mass spectrometry to identify the most abundant molecular formulas present in roasted coffee samples and model roasts followed by van Krevelen and homologous series analysis. We identified the molecular formulas formed from reactions of chlorogenic acids, carbohydrates and proteins, both between classes of compounds and within same classes of compounds. Furthermore, we identified two new classes of compounds formed from chlorogenic acids during roasting, chlorogenic acid acetates and O-phenolic quinoyl and shikimoyl esters of chlorogenic acids.