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Anthocyanins from the fruit Lonicera caerulea L. var. kamtschatica (blueberry honeysuckle, Caprifoliaceae) were studied via (semi)preparative chromatographic fractionation followed by MS and μLC/MS analysis. The extraction procedure was optimized with respect to analytical purposes as well as its potential use for the preparation of nutraceuticals. The highest yield of anthocyanins was obtained using acidified methanol as the extraction medium. A comparable total anthocyanin content was obtained using a mixture of methanol and acetone. However, when Lonicera anthocyanins were in contact with acetone, a condensation reaction occurred to a large extent and related 5-methylpyranoanthocyanins were found. The effect of other extraction media, including ethanol as a "green" solvent, is also discussed. The potential of two fractionation procedures for extract purification differing in their chromatographic selectivity and scale was studied (i.e. using a Sephadex LH-20 gel column and a reversed phase). Fractions obtained by both procedures were used for a detailed analysis. MS and μLC/MS(2) methods were used for monitoring anthocyanin and 5-methylpyranoderivatives content as well as identifying less common and more complex dyes (dimer of cyanidin-3-hexoside, cyanidin-ethyl-catechin-hexosides, etc.). These more complex dyes are most likely formed during fruit treatment.

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called alpha- and beta-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MS(n) data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H](+) and [M-H](-) ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.

Capillary zone electrophoresis was applied to the investigation of the interaction of sanguinarine and chelerythrine with mercapto compounds including albumins at pH 7.4. Mercaptoethanol and L-cysteine were chosen for the identification of the type of interaction and for the identification of the interacting chemical form of these alkaloids. It was evidenced that sanguinarine and chelerythrine do not react chemically with these mercapto compounds at pH 7.4 and that non-covalent products form in this interaction. Their interaction is a fast and reversible complexation and is based on non-bonding intermolecular interactions. Conditional binding constants measured at pH 7.4 and 5.0 indicate that only uncharged forms of sanguinarine and chelerythrine (pseudobases) participate in complexation. A negatively charged group, either bound to the mercapto ligand or supplied by the solution, enters in the complexation. The simple 1 : 1 interaction scheme holds, therefore, only for mercapto compounds bearing an anionic group. Constants corrected for the abundance of the uncharged alkaloid form are of the order of magnitude of 104 l mol−1 and depend on the chemical composition of buffer. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins is qualitatively identical with interaction of these alkaloids with simple mercapto compounds. Constants for the binding of uncharged form of sanguinarine with human and bovine serum albumins in sodium phosphate buffer at pH 7.4, corrected for abundance of the interacting uncharged form, are 332 000±38 400 and 141 000±14 400 l mol−1, respectively. The former agrees well with the value K=385 000 (or log K=5.59) reported from static experiments. For the uncharged form of chelerythrine, the constants are 2 970 000±360 000 and 1 380 000±22 600 l mol−1 for human and bovine serum albumins, respectively. Copyright © 2003 John Wiley & Sons, Ltd.

Capillary electrophoresis was employed to determine the principal quaternary benzo[ c ]phenanthridine alkaloids, sanguinarine and chelerythrine, in two plant extracts and one oral hygiene product. Phosphate–Tris buffer of pH 2.5 was used as a background electrolyte, limits of detection were 3 μmol l −1 (sanguinarine) and 2.4 μmol l −1 (chelerythrine) using UV detection at 270 nm. The method, which correlated well with HPLC, is suitable for serial determination of sanguinarine and chelerythrine in plant products and pharmaceuticals.

The isoquinoline alkaloids adlumiceine, adlumidiceine, coptisine, corytuberine, cryptopine, fumaricine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, palmatine, parfumine, protopine, sinactine, stylopine, and N-methylstylopine were determined by reversed phase high performance liquid chromatography in Fumaria agraria, F. capreolata, F. densiflora, F. muralis, F. officinalis, F. parviflora, and F. vaillantii. Organic acids, namely citric, coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic, protocatechuic acid and caffeic acid (and its methylester) were identified by gas chromatography—mass spectrometry. The total content of phenolic constituents was quantified by a colorimetric method using a phosphomolybdic–phosphotungstic reagent. The chemotaxonomic significance of the results is discussed. Copyright © 1999 John Wiley & Sons, Ltd.

To study the ability of silymarin, a standardized mixture of antioxidant flavonolignans from the medicinal plant Silybum marianum, and of silybin, the main flavonolignan of silymarin, to inhibit the development of diet-induced hypercholesterolemia the rats were fed high cholesterol diet (HCD). Silymarin or silybin were given as dietary supplements, and their influences on serum cholesterol levels were compared to those of probucol, an antioxidant hypocholesterolemic drug. Anticholesterolemic effect of silymarin was parallel to that of probucol, and dose-dependent at dietary drug concentrations of 0.1-0.5-1.0% (w/w). However, in contradistinction to probucol, silymarin caused an increase in high density lipoprotein (HDL)-cholesterol and a decrease in liver cholesterol content, changes considered to be of benefit. In addition to its anticholesterolemic effect silymarin partially prevented the HCD-induced decrease in liver reduced glutathione, an endogenous antioxidant. Silybin was not so effective as silymarin suggesting that either other constituent(s) of silymarin may be responsible for its anticholesterolemic effect or the bioavailability of silybin alone might be lower than that of silybin as a compound of silymarin.

A high-performance liquid chromatographic method for the determination of colchicinoid alkaloids in plant material is described. The determination is performed separately in neutral and basic alkaloid fractions using a C18-bonded silica column. Nine alkaloids, i.e., 3-demethylcolchicine, 2-demethylcolchicine, colchifoline, N-deacetyl-N-formylcolchicine, colchicine, cornigerine, 2-demethyldemecolcine, 3-demethyldemecolcine and demecolcine, in seven Colchicum plants were assayed. For identification of phenolic compounds, a method using gas chromatography-mass spectrometry was elaborated and twenty phenolic compounds were identified in extracts from five Colchicum species. As the presence of luteolin appeared to be of value for chemotaxonomic purposes, its simple densitometric determination was developed.

Transformation of ten colchicinoids by isolated rat liver microsomes resulted in the mixture of C-2, C-3, and C-10 O-demethylated metabolites. Colchicinoids administered i.p. to rats (2.5 mumol/kg) increased serum and liver activities of alkaline phosphatase and decreased liver microsomal demethylase activity as well as cytochrome P-450 content. The changes of acid phosphatase level were less pronounced. The aspartate and alanine aminotransferase activities were significantly increased only in colchicine treated rats. No relations between enzyme activity changes and colchicinoid hydrophobicities quantified by partition coefficients (log P) were found. However, the enzyme activity changes were related to the type of substitution at C-3, C-7, and C-10 of colchicinoids. Particularly, O-demethylation at C-3 resulted into the fall of alkaline phosphatase response. On the other hand, the microsomal demethylation and cytochrome P-450 content were related to the modification of the nitrogen substituent at C-7.

The isoquinoline alkaloids, protopine and allocryptopine are components of numerous phytopreparations. The wide spectrum of biological activities reported for these alkaloids include multiple actions on the cardiovascular system, anti-thrombotic, anti-inflammatory, anti-spasmodic, neuroprotective, anti-bacterial, anti-viral, anti-fungal and anti-parasitic activities. This review aims to summarize recent knowledge on the basic chemistry, analysis, above biological activities, and application of both alkaloids published within the period 1995-2010.

Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline alkaloids which are used in phytopreparations with anti-inflammatory and antimicrobial activities. In this study, the alkaloids sanguinarine, chelerythrine, their dihydro derivatives, protopine and allocryptopine and phenolics, gallic, protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in extracts prepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV detection and/or LC/MS with electrospray ionization. The highest content of sanguinarine and chelerythrine was found in capsules. Protopine and allocryptopine were major alkaloids in leaves including footstalks. The seed oil contained dihydrosanguinarine, dihydrochelerythrine and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In addition, sanguinarine reductase, a key enzyme in sanguinarine/dihydrosanguinarine equilibrium in plants, was found for the first time, in the soluble proteins of leaves. Finally, extracts were tested for antimicrobial activity using the microdilution method on standard reference bacterial strains.

The Cu(II)-complexes of colchiceine (1) and N-deacetylcolchiceine (2) were prepared. They have been shown to be potent inhibitors of the yeast alcoholdehydrogenase. The results indicate the formation of a ternary enzyme-Cu(II)-alkaloid complex.

N-Deacetylcolchiceine (DAC) administered i.p. to rats decreased the level of serum cholesterol and apoB. It was accompanied by the fall of HDL-C due to the decrease of both HDL subfractions HDLa and HDLb, and by a rather weaker decrease of LDL-C. The levels of serum and lipoprotein triacylglycerols were not significantly affected. The "clearing reaction" to heparin in DAC rats differed from controls with regard to plasma cholesterol resulting in its accumulation in HDLa and LDL simultaneously with an increased activity of post-heparin lipoprotein lipase. These results suggest that the DAC accelerates the lipoprotein cholesterol metabolism.

The fruit of Lonicera caerulea L. (blue honeysuckle; Caprifoliaceae) and its phenolic fraction were analyzed for nutrients and micronutrients. The phenolic fraction was prepared from berries percolated with 0.1% H3PO4 and SPE using Sepabeads SP207. The sugar and lipid content was analyzed by HPLC and GC-MS. The total content of anthocyanins was determined using the pH differential absorbance method and aliphatic acids by capillary electrophoresis. MicroLC-MS/MS was used for determination of cyanidin-3-glucoside (the predominant anthocyanin), 3,5-diglucoside, and 3-rutinoside, paeonidin-3-glucoside, 3,5-diglucoside, and 3-rutinoside, delphinidin-3-glucoside and 3-rutinoside, pelargonidin-3-glucoside, 3,5-diglucoside, and 3-rutinoside, quercetin, its 3-glucoside, and 3-rutinoside, epicatechin, protocatechuic, gentisic, ellagic, ferulic, caffeic, chlorogenic, and coumaric acids. The phenolic fraction displayed Folin-Ciocalteu reagent reducing (335 +/- 15 microg of gallic acid equivalent/mg) and DPPH and superoxide scavenging activity (IC50 12.1 +/- 0.1 and 115.5 +/- 6.4 microg/mL) and inhibited rat liver microsome peroxidation (IC50 160 +/- 20 microg/mL). The freeze-dried fruit and its phenolic fraction reduced the biofilm formation and adhesion to the artificial surface of Candida parapsilosis, Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, and Streptococcus mutans.

Periodontitis is a chronic disease associated with inflammation of the tooth-supporting tissues. The inflammation is initiated by a group of gram-negative anaerobic bacteria. These express a number of irritating factors including a lipopolysaccharide (LPS), which plays a key role in periodontal disease development. Plant extracts with anti-inflammatory and anti-microbial properties have been shown to inhibit bacterial plaque formation and thus prevent chronic gingivitis. In this study we tested effects of Prunella vulgaris L. extract (PVE; 5, 10, 25microg/ml) and its component rosmarinic acid (RA; 1microg/ml) on LPS-induced oxidative damage and inflammation in human gingival fibroblasts. PVE and RA reduced reactive oxygen species (ROS) production, intracellular glutathione (GSH) depletion as well as lipid peroxidation in LPS-treated cells. Treatment with PVE and RA also inhibited LPS-induced up-regulation of interleukin 1beta (IL-1beta), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and suppressed expression of inducible nitric oxide synthase (iNOS). The results indicate that PVE and RA are able to suppress LPS-induced biological changes in gingival fibroblasts. The effects of PVE and RA are presumably linked to their anti-inflammatory activities and thus use of PVE and RA may be relevant in modulating the inflammation process, including periodontal disease.

The cyclization of 3-bromo-6-methoxy-2-(naphtho[2,3-d[1,3]dioxol-5-ylaminomethyl)phenol (2) using tributyltinhydride under radical-mediated conditions was accompanied by spontaneous oxidation and afforded directly isodecarine (2-methoxy[1,3]benzodioxolo[5,6-c]phenanthridin-1-ol, (3). 2-Methoxy-6-(naphtho[2,3-d [ 1,3]dioxol-5-ylaminomethyl)-phenol (4) was isolated as a side product. Isodecarine was also prepared by the catalytic debenzylation of 7-benzyloxy-8-methoxy-2,3-methylendioxybenzo-[c]phenanthridine (5). This compound was efficiently converted to 7-benzyloxy-8-methoxy-5 -methyl-2,3-methylenedioxybenzo[c]phenanthridinium trifluoromethanesulfonate (6) in high yield and with a short reaction time.

A new bisbenzylisoquinoline alkaloid (−)-(1S,1'R)-O,O'-dimethylgrisabine was isolated from the leaves of Phaeanthus vietnamensis Ban. Its structure was determined on the basis of the extensive 2-D and 1-D nmr long-range heteronuclear correlations. Its antibacterial activity is also described

Fluorescence data for the interaction of acetylcholinesterase (EC 3.1.1.7, ACHE) from Electrophorus electricus with berberine, 13-ethylberberine, coralyne, and chelerythrine have been presented. By a comparison with known inhibitors of acetylcholinesterase, tetramethylammonium and tacrine, the electrostatic interaction of quaternary alkaloids with the γ-anionic site of enzyme could be confirmed. During the interaction, two molecules of the ligand are bound to the molecule of the enzyme.

The isotachophoretic separation of some isoquinoline alkaloids in acid-base partially purified extracts from Bulgarian FUMARIA PARVIFLORA was studied. The electrolyte system for model mixtures of alkaloids (-)-stylopine, (-)-canadine, coptisine, berberine, protopine, cryptopine, chelidonine, bulbocapnine, papaverine, parfumine and for the plant extracts contained the leading ion K + (0.01 M), counter ion CH 3 COO - , pH L 5.5, and the terminating ion H + (acetic acid 0.01 M). In the plant extracts, protopine and parfumine were quantitatively determined during F. PARVIFLORA growth and development in the term May-June 1984.

It was found that the so far described variations in the content of colchicine alkaloids in leaves and flowers of Colchicum autumnale L. and C. byzantinum Ten. are due to enzymatic O-demethylation and degradation of colchicine (I), N-deacetyl-N-formylcolchicine (XI), and demecolcine (XVII) during drying and storing of the plant material. A simplified method was elaborated to separate the neutral, phenolic, basic, and basic-phenolic fractions of tropolone alkaloids.

The fraction of quaternary benzophen-anthridine alkaloids from roots of CHELIDONIUM MAJUS L., containing chelerythrme and sanguinarme, has been tested for its antiinflammatory activity. On the basis of its low toxicity, high antiinflammatory activity and antimicrobial action, it is recommended for medical use in the treatment of oral inflammatory processes.

A total of 29 associate components were identified in technical 9,10-anthraquinone. The product contained all the prominent impurities present in the starting anthracene except for 9,10-dihydroanthracene and fluorene. 9-(9'-Anthryl)-carbazole and Diels-Alder type adducts of anthracene with maleic anhydride and with maleinimide were also detected. The isolated and identified by-products of oxidation of anthracene were 1,4-anthraquinone, 4,4'-dioxo-1,1'-bianthrylidene, 4,10'-dioxo-1,9'-bianthrylidene, 1-(4'-oxy-1'-naphthylidene)-4-oxyanthracene, 2,3-naphthalenedicarboxylic acid and anhydride, and 5,7,12,14-tetrahydro-5,14;7,12-di(o-benzeno)pentacene-6,13-dione. Of the compounds formed by oxidation of the anthracene impurities, isolated and identified were dibenzo[b,d]pyrone, xanthone, 1,8-naphthalenedicarboxylic acid anhydride,9-fluorenone, and naphthol[2,3-b]thiophene-4,9-dione. As products of a deeper oxidation of the starting compounds, phthalic anhydride, maleic anhydride, maleinimide, phthalic acid, and maleic acid were found. Four additional components whose structure could not be determined were also isolated. The pathway of the anthracene oxidation is suggested.

The tropolone alkaloids colchicine, colchiceine, demecolcine, demecolceine, isocolchicine, isocolchiceine, N-deacetylcolchicine, N-deacetylcolchiceine, N-deacetyl-N-formylcolchicine, N-deacetyl-N-formylcolchiceine, were separated by thin-layer chromatography on silica gel in a new solvent system. Conditions for quantitative fluorodensitometric determination of tropolone alkaloids were investigated.

FUMARIA DENSIFLORA DC., collected near Varna (Bulgaria) was investigated for its alkaloidal content. Protopine, cryptopine, coptisine, palmatine, adlumidiceine, (+/-)-sinactine, and a new spirobenzylisoquinoline alkaloid densiflorine were isolated. Benzophenanthridine alkaloids were not detected.

A rapid isotachophoretic separation of benzo[ C]phenanthridine alkaloids from CHELIDONIUM MAJUS L. is described. The method allows the determination of sanguinarine and chelerythrine in the presence of other isoquinoline alkaloids in crude alkaloids extracts.

The distributions of the alkaloids colchicine, 2-demethylcolchicine, 3-demethylcolchicine, demecolcine, 2-demethyldemecolcine, and 3-demethyldemecolcine in the corms, stems, leaves, flowers, and seeds of COLCHICUM CILICICUM were analysed by HPLC. In addition, the alkaloids cornigerine, N-formyl- N-deacetylcolchicine, N-methyl-demecolcine, beta-lumicolchicine, gamma-lumicolchicine, beta-lumidemecolcine, gamma-lumidemecolcine, and N-ethoxycarbonyldemecolcine were identified, the last named being reported for the first time from a COLCHICUM species. The flavones luteolin and apigenin were isolated and benzoic, 2-hydroxy-6-methoxybenzoic, and vanillic acids were also detected.

From FUMARIA PARVIFLORA L AM. were isolated the major alkaloids protopine and adlumidiceine and the minor alkaloids parfumine, fumariline, dihydrofumariline, cryptopine, (-)-stylopine, 8-oxocoptisine, sanguinarine, and oxysanguinarine. The quaternary protoberberine fraction gave coptisine. Adlumidiceine, dihydrofumariline, 8-oxocoptisine, sanguinarine, and oxysanguinarine were found in F. PARVIFLORA for the first time. F. KRALIKII J ORDAN gave, besides the major alkaloids protopine, fumarophycine, and O-methylfumarophycine, the alkaloids adlumidiceine, berberine, coptisine, cryptopine, (-)-stylopine, and (-)-canadine. Only protopine and cryptopine have been isolated from F. KRALIKII earlier.

The inhibition of butyrylcholinesterase activity in human serum by protoberberine, benzophenanthridine and aporphine alkaloids as well as four model compounds has been studied. The mechanism of the inhibition is discussed on the basis of the different types of interaction of these compounds with butyrylcholinesterase and acetylcholinesterase.