Publications by authors named "Vassiliki Exarchou"

30 Publications

  • Page 1 of 1

Synthesis of 2,4,6-Trinitrotoluene (TNT) Using Flow Chemistry.

Molecules 2020 Aug 6;25(16). Epub 2020 Aug 6.

Joint Research Centre, European Commission, Retieseweg 111, 2440 Geel, Belgium.

This paper describes the nitration of 2,4-dinitrotoluene (DNT) and its conversion to 2,4,6-trinitrotoluene (TNT) at a gram scale with the use of a fully automated flow chemistry system. The conversion of DNT to TNT traditionally requires the use of highly hazardous reagents like fuming sulfuric acid (oleum), fuming nitric acid (90-100%), and elevated temperatures. Flow chemistry offers advantages compared to conventional syntheses including a high degree of safety and simpler multistep automation. The configuration and development of this automated process based on a commercially available flow chemistry system is described. A high conversion rate (>99%) was achieved. Unlike established synthetic methods, ordinary nitrating mixture (65% HNO/98% HSO) and shorter reaction times (10-30 min) were applied. The viability of flow nitration as a means of safe and continuous synthesis of TNT was investigated. The method was optimized using an experimental design approach, and the resulting process is safer, faster, and more efficient than previously reported TNT synthesis procedures. We compared the flow chemistry and batch approaches, including a provisional cost calculation for laboratory-scale production (a thorough economic analysis is, however, beyond the scope of this article). The method is considered fit for purpose for the safe production of high-purity explosives standards at a gram scale, which are used to verify that the performance of explosive trace detection equipment complies with EU regulatory requirements.
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http://dx.doi.org/10.3390/molecules25163586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465666PMC
August 2020

In vitro and in vivo antiplasmodial activity of extracts and isolated constituents of Alstonia congensis root bark.

J Ethnopharmacol 2019 Oct 11;242:111736. Epub 2019 Feb 11.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.

Ethnopharmacological Relevance: An aqueous decoction of root bark of Alstonia congensis Engl. (Apocynaceae) is used in several African countries to treat various ailments including malaria.

Materials And Methods: Extracts of different polarity and isolated constituents were tested in vitro for their antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and the chloroquine-sensitive strain P. falciparum NF54A19A, as well as for their cytotoxic effects againt MRC-5 cells (human lung fibroblasts). Extracts and fractions were evaluated in vivo against the chloroquine-resistant strain P. yoelii N67 and the chloroquine-sensitive strain P. berghei berghei ANKA.

Results: The aqueous extract, the 80% methanol extract and the alkaloid-enriched extract exhibited strong antiplasmodial activity against P. falciparum K1 with IC values < 10 µg/ml and against P. falciparum NF54 A19A with IC values < 0.02 µg/ml. In vivo against P. yoelii N67, at the highest oral dose of 400 mg/kg body weight, all extracts produced 70-73% chemosuppression, while against P. berghei berghei, more than 75% chemosuppression was observed. With regard to the isolated constituents, boonein was inactive in vitro against P. falciparum K-1 (IC > 64 µM), while echitamine, 6,7-seco-angustilobine B and β-amyrin exhibited moderate activity (IC < 30 µM). Against P. falciparum NF54 A19A, boonein was inactive (IC > 64 µM), while echitamine, 6,7-secoangustilobine and β-amyrin showed moderate IC values of 11.07, 21.26 and 40.70 µM, respectively.

Conclusion: These results demonstrated that all extracts from A. congensis root bark possessed antiplasmodial activity in vitro and in vivo. They can be used as raw materials for the preparation of ameliorated remedies for the treatment of uncomplicated malaria. The observed antiplasmodial activity may be due in part to the presence of indole alkaloids.
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http://dx.doi.org/10.1016/j.jep.2019.02.019DOI Listing
October 2019

and Antidiabetic and Antimicrobial Evaluation of Constituents from ().

Front Pharmacol 2017 1;8:232. Epub 2017 May 1.

Laboratory of Natural Products and Food Research and Analysis, Department of Pharmaceutical Sciences, University of AntwerpAntwerp, Belgium.

(Wall.) Janch (or (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in assays related to the traditional use. Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated and in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of advanced glycation endproducts) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Phytochemical analysis of the extracts and fractions led to isolation of compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The assays for the α-glucosidase and 15-LOX assays confirmed the results of respective assays. Pectolinarigenin showed moderate antimicrobial activity against , K1, and , but it was not cytotoxic on a human MRC-5 cell line. Our findings may in part contribute to explain the traditional use of .
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http://dx.doi.org/10.3389/fphar.2017.00232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410595PMC
May 2017

Isolation and Structure Elucidation by LC-DAD-MS and LC-DAD-SPE-NMR of Cyclopeptide Alkaloids from the Roots of Ziziphus oxyphylla and Evaluation of Their Antiplasmodial Activity.

J Nat Prod 2016 11 10;79(11):2865-2872. Epub 2016 Nov 10.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, and ‡Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp , Universiteitsplein 1, 2610 Antwerp, Belgium.

Nine cyclopeptide alkaloids (1-9), of which five (compounds 2, 3, 5, 8, and 9) are described herein for the first time, were isolated from roots of Ziziphus oxyphylla by means of conventional separation methods as well as semipreparative HPLC with DAD and ESIMS detection and LC-DAD-SPE-NMR. Structure elucidation was done by spectroscopic means. Nummularine-R (1), a previously known constituent from this species, was isolated along with its new derivatives O-desmethylnummularine-R (2) and O-desmethylnummularine-R N-oxide (3). In addition, the known compounds hemsine-A (4) and ramosine-A (6), as well as hemsine-A N-oxide (5), were isolated. Moreover, oxyphylline-C (7), a known constituent of Z. oxyphylla stems, was obtained, and two new compounds were identified, oxyphyllines-E (8) and -F (9). Just like oxyphylline-C, oxyphyllines-E and -F belong to the relatively rare class of neutral cyclopeptide alkaloids. The antiplasmodial activity and cytotoxicity of compounds 1, 2, 4, 6, and 9 were evaluated, and the most promising activity was found for O-desmethylnummularine-R (2), which exhibited an IC value of 3.2 ± 2.6 μM against Plasmodium falciparum K1, whereas an IC value of >64.0 μM was evident for its cytotoxicity against MRC-5 cells.
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http://dx.doi.org/10.1021/acs.jnatprod.6b00633DOI Listing
November 2016

Saponins and Flavonoids from an Infusion of Herniaria hirsuta.

Planta Med 2016 Dec 24;82(18):1576-1583. Epub 2016 Oct 24.

Natural Products and Food - Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.

Stone diseases present a major health problem in the Western society, since both urinary and biliary stones occur with a relatively high prevalence of 10-12 % and 10-20 %, respectively, and demonstrate a high recurrence rate. At the moment treatment is mainly based on interventional procedures, or prophylactic and dissolution therapy. However, many of the current drugs cause severe side effects, and therefore, there is an increasing interest in natural medicines. At the moment no registered herbal medicinal products are available for treatment of gallstones. Since an infusion of L. has a proven efficacy against urolithiasis and cholelithiasis, its phytochemical composition has been investigated. Two previously undescribed triterpene saponins, 28--{[-D-xylopyranosyl-(1 → 4)--L-rhamnopyranosyl-(1 → 2)]-[-D-glucopyranosyl-(1-6)]--D-glucopyranosyl}-medicagenic acid and 3--[-L-rhamnopyranosyl-(1 → 3)--D-glucuronopyranosyl]-28--{[-D-glucopyranosyl-(1 → 3)--D-xylopyranosyl-(1 → 4)]-[-D-apiofuranosyl-(1 → 3)]--L-rhamnopyranosyl-(1 → 2)--D-fucopyranosyl}-medicagenic acid and three known flavonoids, quercetin-3--(2″---L-rhamnopyranosyl)--D-glucuronopyranoside, rutin, and narcissin (isorhamnetin-3--rutinoside), were isolated using flash chromatography and successive semi-preparative HPLC and were well characterized by MS and 1D and 2D NMR spectroscopic techniques. These findings could contribute to the development of a standardized extract that can be used in prophylaxis and treatment of gall and kidney stones.
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http://dx.doi.org/10.1055/s-0042-118710DOI Listing
December 2016

Antiprotozoal and Antiglycation Activities of Sesquiterpene Coumarins from Ferula narthex Exudate.

Molecules 2016 Sep 26;21(10). Epub 2016 Sep 26.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.

The exudate of Boiss. (Apiaceae) is widely used in the Indian subcontinent as a spice and because of its health effects. Six sesquiterpene coumarins have been isolated from this exudate: feselol, ligupersin A, asacoumarin A, 8'--acetyl-asacoumarin A, 10'-karatavacinol and 10'-acetyl-karatavacinol. Based on its use in infectious and diabetic conditions, the isolated constituents were evaluated for antimicrobial and antiglycation activities. Some compounds showed activity against protozoal parasites, asacoumarin A being the most active one against K1 (IC 1.3 μM). With regard to antiglycation activity, in the BSA-glucose test, ligupersin A displayed the highest activity (IC 0.41 mM), being more active than the positive control aminiguanidine (IC 1.75 mM). In the BSA-MGO assay, the highest activity was shown by 8'--acetyl-asacoumarin A (IC 1.03 mM), being less active than aminoguanidine (IC 0.15 mM). Hence, the antiglycation activity of the isolated constituents was due to both oxidative and non-oxidative modes of inhibition.
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http://dx.doi.org/10.3390/molecules21101287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274357PMC
September 2016

Treatment with Rhus tripartita extract curtails isoproterenol-elicited cardiotoxicity and oxidative stress in rats.

BMC Complement Altern Med 2016 Sep 8;16:351. Epub 2016 Sep 8.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610, Antwerp, Belgium.

Background: Consumption of plant-derived nutraceuticals and crude drugs in traditional medicine is widely believed to confer beneficial effects in thwarting the progression of cardiovascular diseases. Rhus tripartita (family Anacardiaceae) has been traditionally used to treat a wide range of ailments.

Methods: In the present study we investigated the protective effects of an alcoholic extract of the stem part of Rhus tripartita male genotype (RTSM) on experimentally induced myocardial injury in rats. To this end, cardiac injury was induced by administration of isoproterenol (ISO) and serum enzyme markers, lipid profiles and cardiac tissue redox status were determined following RTSM treatment (250 and 500 mg/kg).

Results: As a result, RTSM treatment significantly mitigated ISO-triggered upregulation of cardiac-specific markers of injury creatine kinase and lactate dehydrogenase. RTSM treatment significantly attenuated ISO-induced increase in serum cholesterol and triglycerides as well alterations in serum lipoproteins. Determination of oxidative balance showed that RTSM treatment significantly blunted ISO-induced increase in malondialdehyde and decrease in nonprotein sulfhydryl in cardiac tissue. Six compounds were isolated and identified as gallocatechin 1, taxifolin 2, myricetin-3-O-β-glucoside 3, catechin 4, epicatechin 5, and 3',8-binaringenin 6. Compound 6 was isolated for the first time from the stem part of Rhus tripartita. Furthermore, RTSM treatment enhanced the survival fraction of cardiac cells exposed to oxidative stress in vitro.

Conclusion: We conclude that the antioxidant properties of RTSM treatment underpin its cardioprotective pharmacological effects, thus, providing biological evidence for the treatment of cardiovascular diseases using Rhus tripartita in indigenous medicine.
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http://dx.doi.org/10.1186/s12906-016-1318-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017009PMC
September 2016

In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues.

Molecules 2016 Jul 29;21(8). Epub 2016 Jul 29.

Centro de Estudios Investigaciones y Evaluaciones Biológicas (CEIEB), Instituto de Farmacia y Alimentos (IFAL), Universidad de la Habana, Calle 222 #2317/23 y 31 La Coronela, La Lisa, CP 13600 La Habana, Cuba.

Two spirosteroid analogues were synthesized and evaluated for their in vitro neuroprotective activities in PC12 cells, against glutamate-induced excitotoxicity and mitochondrial damage in glucose deprivation conditions, as well as their anti-inflammatory potential in LPS/IFNγ-stimulated microglia primary cultures. We also evaluated the in vitro anti-excitotoxic and anti-inflammatory activities of natural and endogenous steroids. Our results show that the plant-derived steroid solasodine decreased PC12 glutamate-induced excitotoxicity, but not the cell death induced by mitochondrial damage and glucose deprivation. Among the two synthetic spirosteroid analogues, only the (25R)-5α-spirostan-3,6-one (S15) protected PC12 against ischemia-related in vitro models and inhibited NO production, as well as the release of IL-1β by stimulated primary microglia. These findings provide further insights into the role of specific modifications of the A and B rings of sapogenins for their neuroprotective potential.
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http://dx.doi.org/10.3390/molecules21080992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274191PMC
July 2016

Cyclopeptide Alkaloids from Hymenocardia acida.

J Nat Prod 2016 07 28;79(7):1746-51. Epub 2016 Jun 28.

Research and Valorization Center on Medicinal Plants , Dubréka, Guinea-Conakry.

Four cyclopeptide alkaloids (1-4) were isolated from the root bark of Hymenocardia acida by means of semipreparative HPLC with DAD and ESIMS detection and conventional separation methods. Structure elucidation was performed by spectroscopic means. In addition to the known compound hymenocardine (1), three other alkaloids were isolated for the first time from a natural source. These included a hymenocardine derivative with a hydroxy group instead of a carbonyl group that was named hymenocardinol (2), as well as hymenocardine N-oxide (3) and a new cyclopeptide alkaloid containing an unusual histidine moiety named hymenocardine-H (4). The isolated cyclopeptide alkaloids were tested for their antiplasmodial activity and cytotoxicity. All four compounds showed moderate antiplasmodial activity, with IC50 values ranging from 12.2 to 27.9 μM, the most active one being hymenocardine N-oxide (3), with an IC50 value of 12.2 ± 6.6 μM. Compounds 2-4 were found not to be cytotoxic against MRC-5 cells (IC50 > 64.0 μM), but hymenocardine (1) showed some cytotoxicity, with an IC50 value of 51.1 ± 17.2 μM.
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http://dx.doi.org/10.1021/acs.jnatprod.6b00131DOI Listing
July 2016

Phytochemical and Pharmacological Investigations on Nymphoides indica Leaf Extracts.

Phytother Res 2016 Oct 10;30(10):1624-1633. Epub 2016 Jun 10.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610, Antwerp, Belgium.

Nymphoides indica (L.) Kuntze (Menyanthaceae) is traditionally used in the Indian subcontinent. However, scientific data reporting its constituents are poor. This study aimed at evaluating its phytochemical constituents and various biological activities. Phytochemical investigations of the extracts and fractions resulted in the isolation of 5 lipophilic compounds, i.e. azelaic (nonanedioic) acid (1) and 4-methyl-heptanedioic acid (3), hexadecanoic (2) and stearic acid (5) and the fatty alcohol hexadecanol (4); 3 seco-iridoids, i.e. 7-epiexaltoside (6), 6″,7″-dihydro-7-epiexaltoside (7) and menthiafolin (8); 3 flavonoids, i.e. 3,7-di-O-methylquercetin-4'-O-β-glucoside (9), 3-O-methylquercetin-7-O-β-glucoside (10) and 3,7-di-O-methylquercetin (11); scopoletin (12) and ferulic acid (13); and the monoterpenoids foliamenthoic acid (14) and 6,7-dihydrofoliamenthoic acid methyl ester (15). Compounds 1-5 showed moderate antimicrobial activities, whereas compound 9 presented mild antiprotozoal activities against Trypanosoma brucei (IC 8 μM), Leishmania infantum (IC 32 μM) and Trypanosoma cruzi (IC 30 μM). Antiglycation activity was shown by compounds 7 (IC 0.36 mM), 10 (IC 0.42 mM) and 15 (IC 0.61 mM). Finally α-glucosidase inhibition was shown by compounds 7, 9, 11 and 13-15. It could be concluded that N. indica leaf extracts possess mild to moderate antimicrobial, antiprotozoal, antioxidant and antidiabetic activities. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/ptr.5663DOI Listing
October 2016

Edible coating composed of chitosan and Salvia fruticosa Mill. extract for the control of grey mould of table grapes.

J Sci Food Agric 2017 Jan 17;97(2):452-460. Epub 2016 May 17.

Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus.

Background: Consumer concerns regarding high-quality produce, free of pesticide residues, direct research towards disease management strategies that minimise or even exclude the use of synthetic chemistries in crop production. The efficacy of a chitosan-based edible coating combined with the acetonic extract of Salvia fruticosa Mill. (ASF) was assessed against the grey mould of table grapes.

Results: HPLC-SPE-NMR and q-NMR analyses defined major constituents of ASF to be the flavonoids hispidulin, salvigenin and cirsimaritin and the diterpenes carnosic acid, carnosol and the 12-methoxycarnosic acid. The extract was found to be efficacious in reducing spore germination and mycelial growth of Botrytis cinerea in vitro at 10 and 25 °C. However, the combination of the ASF with chitosan 1% (w/v; CHIT) significantly improved fungal inhibition. Similarly, in fruit inoculation trials at 10 °C, the efficacy of the combined application of the ASF at 500 mg L with CHIT against grey mould was statistically equal to the synthetic fungicide thiabendazole, ranging from 98.4% to 92.7% at 12 and 21 days post-inoculation, respectively. Furthermore, chitosan coating alone and in combination with ASF decreased the rate of fruit weight loss during cold storage, while preserved soluble solids content and titratable acidity. Chitosan-based coatings did not affect quality attributes and the bioactive compounds in table grapes.

Conclusion: The combined application of the ASF in the form of an edible coating with chitosan could effectively control B. cinerea without deteriorating quality and physico-chemical properties of grapes. © 2016 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.7745DOI Listing
January 2017

Antioxidant and Antiglycating Constituents from Leaves of Ziziphus oxyphylla and Cedrela serrata.

Antioxidants (Basel) 2016 Mar 17;5(1). Epub 2016 Mar 17.

Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.

Ziziphus oxyphylla and Cedrela Serrata plants have a folkloric use in Pakistan for treatments of different ailments, i.e., Jaundice, Hepatitis, Diabetes, and antimicrobial. Until now, none of the research studies have reported any phytochemical work on leaves of these two plants. This study aimed to isolate and perform phytochemical analysis in order to search for the constituent having the active role in treatment of the aforementioned ailments. A bioassay-guided fractionation and isolation procedure was used to isolate the concerned phytochemicals present in leaf extracts of Z. oxyphylla and C. serrata. The process involved the hyphenated techniques, i.e., Flash Chromatography, Semi-Preparative HPLC/DAD, UPLC/MS, and NMR in order to isolate and elucidate the structure of the phytochemicals. Seven compounds (1-7) were isolated and identified as flavonoids, more in particular glycosides of quercetin and kaempferol. They showed DPPH scavenging activity, compound 3 (isoquercitrin) being the most active one with an IC50 of 10.8 µg/mL (positive control quercetin; IC50 3.6 µg/mL). The superoxide-radical scavenging and total antioxidant (ABTS) assays indicated IC50 values ranging from 200 to 910 µg/mL and 170 to 320 µg/mL, respectively (positive control quercetin: 374 and 180 µg/mL, respectively). Furthermore, these compounds had low IC50 values for inhibition of protein glycation (AGEs inhibition), ranging from 530 to 818 µg/mL, comparable to aminoguanidine (510 µg/mL) used as a positive control. This study resulted in the identification of seven flavonoid glycosides for the first time from the leaves of Z. oxyphylla and C. serrata with antioxidative and antiglycating activities.
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http://dx.doi.org/10.3390/antiox5010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808758PMC
March 2016

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum.

Toxicol Appl Pharmacol 2015 Apr 11;284(2):246-53. Epub 2015 Feb 11.

Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium. Electronic address:

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography-tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis-Menten model. Apparent Km values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148μM, respectively. Apparent Vmax values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment.
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http://dx.doi.org/10.1016/j.taap.2015.01.021DOI Listing
April 2015

HPLC-SPE-NMR characterization of major metabolites in Salvia fruticosa Mill. extract with antifungal potential: relevance of carnosic acid, carnosol, and hispidulin.

J Agric Food Chem 2015 Jan 12;63(2):457-63. Epub 2015 Jan 12.

Laboratory of Natural Products and Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp , Universiteitsplein 1, 2610 Antwerp, Belgium.

Plant pathogenic fungi are considered of significant economic importance for adversely affecting both quantitatively and qualitatively fresh and processed produce. Extracts of Salvia fruticosa were initially screened for their antifungal activity, and the ethyl acetate fraction, being the most active, was further analyzed using HPLC-SPE-NMR hyphenation. The methoxylated flavones hispidulin, salvigenin, and cirsimaritin and the diterpenes carnosic acid, carnosol, and 12-methoxycarnosic acid were identified as the major components of the extract. In addition, the concentration levels of all identified components were determined using q-NMR. The antifungal activity of the crude extract and selected phytochemicals was estimated against the fungal species Aspergillus tubingensis, Botrytis cinerea, and Penicillium digitatum. The estimated MIC and MFC values of the ethyl acetate extract of S. fruticosa, as well as three of its major constituents, carnosic acid, carnosol, and hispidulin, support their antifungal activity, especially against B. cinerea and P. digitatum, suggesting their potential use in food and agricultural systems.
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http://dx.doi.org/10.1021/jf5050734DOI Listing
January 2015

Automated analytical standard production with supercritical fluid chromatography for the quantification of bioactive C17-polyacetylenes: a case study on food processing waste.

Food Chem 2014 Dec 29;165:371-8. Epub 2014 May 29.

Flemish Institute for Technological Research (VITO), Business Unit Separation and Conversion Technology (SCT), Boeretang 200, 2400 Mol, Belgium. Electronic address:

Food processing enterprises produce enormous amounts of organic waste that contains valuable phytochemicals (e.g. C17-polyacetylenes). Knowledge on the phytochemicals content is a first step towards valorisation. Quantification of C17-polyacetylenes is however often hampered by the lack of commercially available standards or by tedious multistep in-house standard production procedures. In the current study, a new and straightforward supercritical fluid chromatography purification procedure is described for the simultaneous production of 2 analytical C17-polyacetylene standards. Respectively, 5 and 6 mg of falcarinol and falcarindiol were purified in 17 h on analytical scale. After confirming the identity and quality (97% purity) by Nuclear Magnetic Resonance, accurate mass-Mass Spectrometry (am-MS) and Photo Diode Array (PDA) detection the C17-polyacetylene standards were used for the analysis of industrial vegetable waste with Liquid Chromatography coupled to PDA and am-MS detection. Measurements showed varying concentrations of C17-polyacetylenes in the organic waste depending on its nature and origin.
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http://dx.doi.org/10.1016/j.foodchem.2014.05.093DOI Listing
December 2014

Hydrogen bonding probes of phenol -OH groups.

Org Biomol Chem 2013 Feb 7;11(6):1013-25. Epub 2013 Jan 7.

Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, Ioannina GR-45110, Greece.

Correlations between hydrogen bonds and solvent effects on phenol -OH proton shieldings, temperature coefficients (Δδ/ΔT) and effects on OH diffusion coefficients for numerous phenolic acids, flavonols, flavones, and oleuropein derivatives of biological interest were investigated in several organic solvents and were shown to serve as reliable indicators of hydrogen bonding and solvation state of -OH groups. The temperature coefficients span a range of -0.5 to -12.3 ppb K(-1). Shielding differences of 2.0 to 2.9 ppm at 298 K were observed for solvent exposed OH groups between DMSO-d(6) and CD(3)CN which should be compared with a shielding range of ~7 ppm. This demonstrates that the solvation state of hydroxyl protons is a key factor in determining the value of the chemical shift. For -OH protons showing temperature gradients more positive than -2.5 ppb K(-1), shielding changes between DMSO-d(6) and CD(3)CN below 0.6 ppm, and diffusion coefficients significantly different from those of traces of H(2)O, there is an intramolecular hydrogen bond predictivity value of 100%. The C-3 OH protons of flavonols show very significant negative temperature coefficients and shielding changes between DMSO-d(6) and CD(3)CN of ~2.3 ppm, which indicate the absence of persistent intramolecular hydrogen bonds, contrary to numerous X-ray structures.
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http://dx.doi.org/10.1039/c2ob27117fDOI Listing
February 2013

Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts.

J Agric Food Chem 2012 May 30;60(18):4508-13. Epub 2012 Apr 30.

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina GR-45110, Greece.

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H₂O₂ at ∼10.30 ppm in DMSO-d₆ and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H₂O₂ below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 μmol L⁻¹. Application of an array of NMR experiments, including 2D ¹H-¹³C HMBC, spiking of the samples with H₂O₂, and variable temperature experiments, resulted in the unequivocal assignment of H₂O₂ precluding any confusion with interferences from intrinsic phenolics in the extract.
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http://dx.doi.org/10.1021/jf205003eDOI Listing
May 2012

Unprecedented ultra-high-resolution hydroxy group (1)H NMR spectroscopic analysis of plant extracts.

J Nat Prod 2011 Nov 20;74(11):2462-6. Epub 2011 Oct 20.

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, GR-45110, Greece.

A general method is demonstrated for obtaining ultra-high resolution in the phenolic hydroxy group 1H NMR spectroscopic region, in DMSO-d6 solution, with the addition of picric acid. Line-width reduction by a factor of over 100 was observed, which resulted in line-widths ranging from 1.6 to 0.6 Hz. This unprecedented resolution, in combination with the shielding sensitivity of the hydroxy group absorptions to substituent effects at least up to 11 bonds distant and the application of 2D 1H-13C HMBC techniques, allows the unequivocal structure analysis of natural products with phenolic hydroxy groups in complex plant extracts.
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http://dx.doi.org/10.1021/np200329aDOI Listing
November 2011

Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against gram-positive pathogenic bacteria.

PLoS One 2011 Apr 4;6(4):e18127. Epub 2011 Apr 4.

Laboratory of Analytical Chemistry, University of Ioannina, Ioannina, Greece.

Background: Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria.

Methodology/principal Findings: In this study we report the identification and characterization of 4',5'-O-dicaffeoylquinic acid (4',5'-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3',5'-ODCQA, 4',5'-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4',5'-ODCQA with pump inhibitory activity whereas 3',5'-ODCQA was ineffective. These initial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology.

Conclusions/significance: These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4',5'-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018127PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070693PMC
April 2011

A new method for the determination of free L-carnitine in serum samples based on high field single quantum coherence filtering 1H-NMR spectroscopy.

Anal Bioanal Chem 2011 Feb 11;399(6):2285-94. Epub 2011 Jan 11.

NMR Center, University of Ioannina, 45110 Ioannina, Greece.

The rapid and accurate determination of specific metabolites present in biofluids is a very demanding task which is essential in both medicine and chemistry. L-carnitine (3-hydroxy-4-N-trimethylammonium butyrate) is an important metabolite which participates in a series of biological paths and therefore its determination is of diagnostic importance. A single quantum coherence filtering (1)H NMR methodology was used for the accurate and rapid determination of L-carnitine in human serum samples. The methodology is based on spectral simplification, and specifically on the distinction of the N-methyl proton signal of L-carnitine that is greatly overlapped in the (1)H-NMR spectrum of serum. The quantitative results provided by the proposed method are in excellent agreement with those obtained by the enzymatic method, which is widely used. The proposed method is rapid (~20 min of experimental time), selective, sensitive, and has good analytical characteristics (accuracy, reproducibility). Selected protein precipitation methods were also investigated and sample pretreatment with EtOH is suggested.
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http://dx.doi.org/10.1007/s00216-010-4600-0DOI Listing
February 2011

Tris-(hydroxyamino)triazines: high-affinity chelating tridentate O,N,O-hydroxylamine ligand for the cis-V(V)O2(+) cation.

Dalton Trans 2010 Oct 24;39(38):9032-8. Epub 2010 Aug 24.

Department of Chemistry, Section of Inorganic and Analytical Chemistry, University of Ioannina, 45110, Greece.

The treatment of the trichloro-1,3,5-triazine with N-methylhydroxylamine hydrochloride results in the replacement of the three chlorine atoms of the triazine ring with the function -N(OH)CH(3) yielding the symmetrical tris-(hydroxyamino)triazine ligand H(3)trihyat. Reaction of the ligand H(3)trihyat with NaV(V)O(3) in aqueous solution followed by addition of Ph(4)PCl gave the mononuclear vanadium(V) compound Ph(4)P[V(V)O(2)(Htrihyat)] (1). The structure of compound 1 was determined by X-ray crystallography and indicates that this compound has a distorted square-pyramidal arrangement around vanadium. The ligand Htrihyat(2-) is bonded to vanadium atom in a tridentate fashion at the triazine ring nitrogen atom and the two deprotonated hydroxylamido oxygen atoms. The high electron density of the triazine ring nitrogen atoms, which results from the resonative contribution of electrons of exocyclic nitrogen atoms, leads to a very strong V-N bond. The cis-[V(V)O(2)(Htrihyat)](-) species exhibits high hydrolytic stability in aqueous solution over a wide pH range, 2.5-11.5, as was evidenced by potentiometry.
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http://dx.doi.org/10.1039/c0dt00574fDOI Listing
October 2010

Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts.

J Agric Food Chem 2010 Mar;58(6):3303-8

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece.

The contribution of flavonoids to the overall radical scavenging activity of olive leaf polar extracts, known to be good sources of oleuropein related compounds, was examined. Off line and on line HPLC-DPPH(*) assays were employed, whereas flavonoid content was estimated colorimetrically. Individual flavonoid composition was first assessed by RP-HPLC coupled with diode array and fluorescence detectors and verified by LC-MS detection system. Olive leaf was found a robust source of flavonoids regardless sampling parameters (olive cultivar, leaf age or sampling date). Total flavonoids accounted for the 13-27% of the total radical scavenging activity assessed using the on line protocol. Luteolin 7-O-glucoside was one of the dominant scavengers (8-25%). Taking into consideration frequency of appearance the contribution of luteolin (3-13%) was considered important, too. Our findings support that olive leaf, except for oleuropein and related compounds, is also a stable source of bioactive flavonoids.
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http://dx.doi.org/10.1021/jf903823xDOI Listing
March 2010

Unexpected synthesis of an unsymmetrical mu-oxido divanadium(V) compound through a reductive cleavage of a N-O bond and cleavage-hydrolysis of a C-N bond of an N,N-disubstituted bis-(hydroxylamino) ligand.

Inorg Chem 2010 Jan;49(1):52-61

Department of Chemistry, Section of Inorganic and Analytical Chemistry, University of Ioannina, 45110, Greece.

Reaction of the N,N-disubstituted bis-(hydroxylamino) ligand 2,6-bis[hydroxy(methyl)amino]-4-morpholino-1,3,5-triazine, H(2)bihyat, with V(IV)OSO(4).5H(2)O in water for 6 h followed by the addition of methyl alcohol resulted in the isolation of the unsymmetrical mu-oxido divanadium(V) compound [V(V)(2)O(2)(mu(2)-O)(bihyat)(hyta)(hyto)].3H(2)O (1.3H(2)O) and of the methylhydroxylamido derivative [V(V)O(bihyat)(CH(3)NHO)].H(2)O (2.H(2)O). The N,N-disubstituted mono(hydroxylamino) ligands Hhyta, Hhyto, and CH(3)NHOH were formed by the decomposition of the ligand H(2)bihyat in the presence of vanadium. The structures of compounds 1.3H(2)O and 2.H(2)O were determined by X-ray crystallography. The structure of 1.3H(2)O consists of one five-coordinate vanadium(V) atom and one six-coordinate vanadium(V) atom bridged by an oxido group and ligated to a tridentate bihyat(2-) and two bidentate hyta(-) and hyto(-) ligands, respectively. The two terminal oxido groups in 1.3H(2)O are syn-directed, lying on the same side of the V-O-V plane. The coordination environment of the vanadium atom in 2.H(2)O approximates to a highly distorted pentagonal pyramid with the oxido ligand occupying the apical position. Compounds 1.3H(2)O and 2.H(2)O were studied by multinuclear NMR ((1)H, (13)C, and (51)V) to elucidate their solution structures. The (51)V NMR of 1.3H(2)O in anhydrous CD(2)Cl(2) gave two signals at -199 and -508 ppm, which were assigned to the five- and six-coordinate vanadium(V) atoms, respectively. The resonance of the five-coordinate vanadium nucleus, in a field much lower than that expected from Rehder's [Inorg. Chem., 1988, 27, 584-587] referencing scale, was attributed to the low-energy ligand-to-metal charge transfer transition at 605 nm [epsilon(M) = 5050 M(-1) cm(-1)] of 1.3H(2)O according to Pecoraro et al. [J. Am. Chem. Soc., 1992, 114, 9925-9933]. Electrospray ionization-mass spectrometry studies were used to follow the decomposition products of H(2)bihyat in the presence of vanadium.
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http://dx.doi.org/10.1021/ic901809fDOI Listing
January 2010

Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy-Comparison with HPLC methods.

Anal Chim Acta 2009 Mar 18;635(2):188-95. Epub 2009 Jan 18.

Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, Ioannina GR-45110, Greece.

A novel strategy for NMR analysis of mixtures of oleanolic and ursolic acids that occur in natural products is described. These important phytochemicals have similar structure and their discrimination and quantification is rather difficult. We report herein the combined use of proton-carbon heteronuclear single-quantum coherence ((1)H-(13)C HSQC) and proton-carbon heteronuclear multiple-bond correlation ((1)H-(13)C HMBC) NMR spectroscopy, in the identification and quantitation of oleanolic acid (OA) and ursolic acid (UA)in plant extracts of the Lamiaceae and Oleaceae family. The combination of (1)H-(13)C HSQC and (1)H-(13)C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of oleanolic and ursolic acid without resorting to physicochemical separation of the components. The quantitative results provided by 2D (1)H-(13)C HSQC NMR data were obtained within a short period of time ( approximately 14min) and are in excellent agreement with those obtained by HPLC, which support the efficiency of the suggested methodology.
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http://dx.doi.org/10.1016/j.aca.2009.01.021DOI Listing
March 2009

Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells.

Mol Nutr Food Res 2009 May;53(5):600-8

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece.

Olive oil compounds is a dynamic research area because Mediterranean diet has been shown to protect against cardiovascular disease and cancer. Olive leaves, an easily available natural material of low cost, share possibly a similar wealth of health benefiting bioactive phytochemicals. In this work, we investigated the antioxidant potency and antiproliferative activity against cancer and endothelial cells of water and methanol olive leaves extracts and analyzed their content in phytochemicals using LC-MS and LC-UV-SPE-NMR hyphenated techniques. Olive-leaf crude extracts were found to inhibit cell proliferation of human breast adenocarcinoma (MCF-7), human urinary bladder carcinoma (T-24) and bovine brain capillary endothelial (BBCE). The dominant compound of the extracts was oleuropein; phenols and flavonoids were also identified. These phytochemicals demonstrated strong antioxidant potency and inhibited cancer and endothelial cell proliferation at low micromolar concentrations, which is significant considering their high abundance in fruits and vegetables. The antiproliferative activity of crude extracts and phytochemicals against the cell lines used in this study is demonstrated for the first time.
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http://dx.doi.org/10.1002/mnfr.200800204DOI Listing
May 2009

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups.

Anal Chim Acta 2008 Jan 3;607(2):219-26. Epub 2007 Dec 3.

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, GR-45110, Greece.

The (1)H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD(3)OH solutions indicate significantly deshielded signals in the region of 14-15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional (1)H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14-15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV-vis and LC/MS measurements.
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http://dx.doi.org/10.1016/j.aca.2007.11.040DOI Listing
January 2008

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS.

Phytochemistry 2007 Feb 29;68(3):383-93. Epub 2006 Dec 29.

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, University Campus, Ioannina, Epirus GR-45110, Greece.

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.
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http://dx.doi.org/10.1016/j.phytochem.2006.11.026DOI Listing
February 2007

Hyphenated chromatographic techniques for the rapid screening and identification of antioxidants in methanolic extracts of pharmaceutically used plants.

J Chromatogr A 2006 Apr 15;1112(1-2):293-302. Epub 2005 Dec 15.

NMR Center, University of Ioannina, Ioannina GR-45110, Greece.

Phytochemical analysis is an important scientific research area, which normally relies on a number of rather laborious and time-consuming techniques for compound identification. Isolation of the ingredients of plant extracts in adequate quantities for spectral and biological analysis was the basis of this research. In this paper the possibility of on-line rapid screening of antioxidant components in methanolic plant extracts and their subsequent identification is reported. Based exclusively on hyphenated chromatographic techniques the methanolic extracts of Tilia europea, Urtica dioica, Lonicera periclymenum and Hypericum perforatum are initially screened for their antioxidant components via an on-line DPPH and ABTS radical scavenging technique. Structural elucidation of the active analytes is achieved by means of LC-MS and LC-UV-SPE-NMR. After the determination of the appropriate LC gradient, a minimal number of chromatographic runs with these hyphenated techniques are adequate for the acquisition of the necessary data, leading to the identification of the targeted compounds. Based on their UV, NMR and MS spectra, the antioxidant compounds identified in the extracts under study were found to be either flavonoid glycosides or mono- and dicaffeoylquinic acids. Although the aim of the study was to show the great potential of the LC-UV-NMR-DPPH/ABTS approach for the rapid screening and identification of plant constituents, the results produced in the course of this study also have some merit by themselves. Some of the compounds detected are reported for the first time in the specific plant extracts.
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http://dx.doi.org/10.1016/j.chroma.2005.11.077DOI Listing
April 2006

LC-NMR coupling technology: recent advancements and applications in natural products analysis.

Magn Reson Chem 2005 Sep;43(9):681-7

NMR Center, Department of Chemistry, University of Ioannina, Greece.

An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.
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http://dx.doi.org/10.1002/mrc.1632DOI Listing
September 2005

LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano.

Anal Chem 2003 Nov;75(22):6288-94

Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.
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http://dx.doi.org/10.1021/ac0347819DOI Listing
November 2003
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