Spruce Balm-Based Semisolid Vehicles for Wound Healing: Effect of Excipients on Rheological Properties and Ex Vivo Skin Permeation.

The treatment of chronic wounds, an important issue with the growing elderly population, is increasingly hindered by antibiotic resistance. Alternative wound care approaches involve the use of traditional plant-derived remedies, such as purified spruce balm (PSB), with antimicrobial effects and the promotion of cell proliferation. However, spruce balm is difficult to formulate due to its stickiness and high viscosity; dermal products with satisfying technological properties and the scientific literature on this topic are scarce. Thus, the aim of the present work was to develop and rheologically characterize a range of PSB-based dermal formulations with different hydrophilic/lipophilic compositions. Mono- and biphasic semisolid formulations based on different compounds (petrolatum, paraffin oil, wool wax, castor oil, and water) were developed and characterized by their organoleptic and rheological measurements. A chromatographic method of analysis was established, and skin permeation data were collected for pivotal compounds. The results showed that the dynamic viscosity ranged from 10 to 70 Pas at 10/s for the different shear-thinning systems. The best formulation properties were observed for water-free wool wax/castor oil systems with 20% w/w PSB followed by different water-in-oil cream systems. Skin permeation through porcine skin was observed for different PSB compounds (e.g., pinoresinol, dehydroabietic acid, and 15-hydroxy-dehydroabietic acid) using Franz-type diffusion cells. The permeation potential of wool wax/castor oil- and lard-based formulations was shown for all the analyzed substance classes. The varying content of pivotal compounds in different PSB batches collected at different timepoints from different spruce individuals might have contributed to observed differences in vehicle performance.

Ultra High-Performance Supercritical Fluid Chromatography for the Quantitation of Diterpene Resin Acids in Norway Spruce Samples.

Picea abies (L.) H. Karst. (Pinaceae) is native to Northern, Central and Eastern Europe. The fast-growing tree reaches up to 50 m in height, has modest nutritional requirements and depends on sufficient water supply. The conifer, commonly called Norway spruce, produces exudates which are traditionally used to treat skin wounds in Northern European countries. Major bioactive constituents of the conifer oleoresin are diterpene resin acids (DRAs) of the abietane and the pimarane type. To assure consistent pharmaceutical quality of Norway spruce balm and commercial products thereof, an analytical method for the quantitation of DRAs is the prerequisite. However, high structural similarity among DRAs and their poor UV absorption makes chromatographic separation and detection challenging: Conventional liquid chromatography systems often fail to achieve sufficient separation, moreover, they are not sustainable. Gas chromatography on the other hand requires time-consuming derivatization prior to unacceptably long analyses (>60 min). These drawbacks prompted the development of the first validated supercritical fluid-based protocol for the separation and quantitation of eight DRAs, i.e., pimaric acid (1), sandaracopimaric acid (2), palustric acid (3), isopimaric acid (4), levopimaric acid (5), abietic acid (6), dehydroabietic acid (7), and neoabietic acid (8). By using an ultra high-performance supercritical fluid chromatography (UHPSFC) device hyphenated to a quadrupole mass detector, the DRAs were separated in less than 20 min on a Torus 2-Picolylamin (2-PIC) column (3.0 mm × 100 mm; 1.7 µm particle size) applying supercritical CO2 and ethanol as mobile phase. Regarding selectivity, accuracy (recovery rates: 87-108%), intermediate precision (between 6.6 and 11.1%), and linearity (R2 ≥ 0.99; linear between 0.75 µg/ml and 2.5 mg/ml), results were obtained in line with ICH guidelines. The lowest limit of detection (LOD) was at 0.75 µg/ml (7) and the lowest limit of quantitation (LOQ) at 2 µg/ml (8). As application examples, 22 Norway spruce balm samples and five commercial products were analyzed. The here presented protocol not only simplifies and shortens the analytical workflow, but also reduces the amount of organic solvent waste by about two thirds compared to conventional liquid chromatographic set-ups. These advantages qualify this fast and efficient method as an ideal tool for an environmentally friendly quality control of traditionally used wound-healing Norway spruce balm products.

Exudates of Picea abies, Pinus nigra, and Larix decidua: Chromatographic Comparison and Pro-Migratory Effects on Keratinocytes In Vitro.

Balms and resins of Picea abies, Larix decidua, and Pinus nigra are traditionally used to treat wounds. Three chromatographic techniques differing in separation capacity and technical demands were employed to distinguish among these plant exudates. A TLC method was established for fingerprint comparison, providing a quick overview of a large number of samples at low cost. HPLC-DAD (RP18) and UHPSFC-DAD (Torus 2-Picolylamin), hyphenated to ESI-MS, represented orthogonal chromatographic systems with high separation performance. The developed methods allow for the separation and detection of major and minor constituents belonging to different compound classes (phenyl carboxylic acids, lignans, diterpene resin acids). The qualitative compositions of the diterpene resin acids, the main compounds in the exudates, were comparable in all three genera. Differences were detected in the distribution of hydroxylated diterpene resin acids, pinoresinol, and hydroxycinnamic acids. The three tested chromatographic systems with varying demands on lab equipment offer appropriate tools for the quality assessment of Picea abies, Larix decidua, and Pinus nigra. The extracts were furthermore tested at three different concentrations (10 µg/mL, 3 µg/mL, and 1 µg/mL) for boosted re-epithelialization, a crucial step in the wound-healing process, in an in vitro HaCaT keratinocyte-based scratch assay. Lysophosphatidic acid (LPA, 10 µM) and extracts of several medicinal plants well known for their wound-healing properties (birch, marigold, St. John's wort, manuka honey) were used as positive controls. Picea abies and Pinus nigra showed concentration dependency; significant activity was measured for Larix decidua at 3 µg/mL.

Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro.

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids ( 1: - 3: ), the lignan pinoresinol ( 4: ), four hydroxylated derivatives of dehydroabietic acid (DHAA) ( 5:  -  8: ), and dehydroabietic acid ( 9: ) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids-pimaric acid ( 10: ) and isopimaric acid ( 11: )-and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

Microscopic and Phytochemical Comparison of the Three Leonurus Species L. cardiaca, L. japonicus, and L. sibiricus.

At least three Leonurus species, Leonurus cardiaca, Leonurus japonicus, and Leonurus sibiricus, are used in various traditional medicinal systems for different therapeutic purposes. The plant names "L. sibiricus" and "L. japonicus" are often used as synonyms, which causes confusion in literature and implies that several studies may be based on improperly identified plant material. To avoid further confusion, the current status of the identification of these three species is illustrated. Characteristics for their unequivocal identification are presented using stereo and light microscopy as well as HPTLC.The establishment of the species-specific anatomical, morphological, and phytochemical characters was based on reference specimens in comparison with wild collected or commercially obtained material. Morphologically, the species differed in shape and length ratio of the upper and lower lip of the corolla. Differentiating anatomical characters involve the presence, density, and dimension of trichomes on different organs. Detailed anatomical descriptions of the leaves and corollae of L. japonicus and L. sibiricus are given and compared to L. cardiaca. These three sets of characters facilitate fast and reliable identification. Complementary HPTLC fingerprints show type-specific patterns that allow the differentiation of L. japonicus from the other two species. For the distinction of L. cardiaca and L. sibiricus, flower morphology has to be considered.The presented data contribute to the quality control of the three Leonurus species and additionally meet a definite and timely need regarding the introduction of L. japonicus to the European Pharmacopoeia in addition to the already existing monograph of L. cardiaca.

HPTLC Bioautography Guided Isolation of α-Glucosidase Inhibiting Compounds from Justicia secunda Vahl (Acanthaceae).

INTRODUCTION: α-Glucosidase inhibitors form an essential basis for the development of novel drugs in diabetes type 2 treatment. Searching for α-glucosidase inhibitors in plants, TLC bioautographic assays have been established and improved within the last years. In traditional medicine, extracts from the leaves of Justicia secunda Vahl are used to treat diabetes mellitus symptoms. OBJECTIVE: To screen for α-glucosidase inhibitors in J. secunda via HPTLC bioautography. Methodology - Extracts from the leaves of J. secunda and fractions thereof were evaluated in terms of their α-glucosidase inhibiting potential by subjecting them to HPTLC bioautography. The aqueous (AQ) fraction deriving from the methanol extract was further fractionated via column chromatography on polystyrene Diaion® HP-20. Two AQ subfractions revealed active compounds, which were isolated via preparative HPTLC and semipreparative HPLC. Their identification and structure elucidation was achieved employing HPLC-ESI-MSn , HRESI-MS, and NMR analyses. RESULTS: α-Glucosidase inhibitors were visualised as white zones on violet background on the TLC plate. The crude water extract, the methanol extract, and the methanol extract derived AQ fraction showed α-glucosidase inhibiting effects. In the latter, two diastereomeric mixtures responsible for the α-glucosidase inhibition were enriched. They were identified as the novel 2-caffeoyloxy-4-hydroxy-glutaric acid and the diastereomers secundarellone B and C. CONCLUSION: The current study presents the α-glucosidase inhibiting potential of J. secunda supporting its traditional medicinal use in diabetes mellitus treatment. HPTLC bioautography screening for α-glucosidase inhibitors provides a simple and effective method for the investigation of complex samples, such as plant extracts. Copyright © 2016 John Wiley & Sons, Ltd.

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells.

BACKGROUND/AIMS: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. METHODS: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. RESULTS: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. CONCLUSION: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae).

INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.

Walnut leaf extract inhibits PTP1B and enhances glucose-uptake in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut, Juglans regia L. (Juglandaceae), is one of the medicinal plants used to treat diabetic symptoms in Austrian folk medicine. The air-dried green leaves are either used as aqueous decoctions or liquor preparations and are consumed on a daily basis. We investigated the hypoglycemic effect of a methanolic Juglans regia leaf extract on glucose uptake, protein tyrosine phosphatase 1B (PTP1B) inhibition and peroxisome proliferator-activated receptor gamma (PPARγ) activation. MATERIAL AND METHODS: Hypoglycemic activity was assessed by glucose-uptake in C2C12 myocytes, inhibition of PTP1B and activation of PPARγ. Phytochemical characterization of the extract was carried out by LC-MS and GC-MS. RESULTS: Methanolic Juglans regia leaf extract enhanced the glucose uptake rate in C2C12 myocytes at concentrations of 25µg/mL compared to untreated cells. This activity may partly be explained by the inhibition of PTP1B but not PPARγ agonism. LC-MS analyses revealed chlorogenic acid (1), 3-p-coumaroylquinic acid (2), a trihydroxynaphthalene-hexoside (3), as well as eight flavonoids (4-11) as main phenolic constituents in the active extract. CONCLUSIONS: The finding that Juglans regia leaf extract enhances glucose uptake and inhibits PTP1B provides an in vitro-based rationale for the traditional use of walnut leaf preparations against elevated blood-glucose levels.

HPLC determination of flavonoid glycosides in Mongolian Dianthus versicolor Fisch. (Caryophyllaceae) compared with quantification by UV spectrophotometry.

INTRODUCTION: Dianthus versicolor is used in traditional Mongolian medicine against liver impairment. Fractions enriched in flavone-di- and triglycosides were shown to enhance bile secretion. Therefore, reliable and accurate analytical methods are needed for the determination of these flavonoids in the crude drug and extracts thereof. OBJECTIVE: To provide a validated HPLC-DAD (diode array detector) method especially developed for the separation of polar flavonoids and to compare the data obtained with those evaluated by UV spectrophotometry. METHODOLOGY: Separations were carried out on an Aquasil® C18-column (4.6 mm × 250.0 mm, 5 µm) with a linear gradient of acetonitrile and water (adjusted to pH 2.8 with trifluoroacetic acid) as mobile phase. Rutoside was employed as internal standard with linear behavior in a concentration range of 0.007-3.5 mg/mL. Accuracy was determined by spiking the crude drug with saponarin resulting in recoveries between 92% and 102%. RESULTS: The method allows the quantification of highly polar flavonoid glycosides and the determination of their total content. For saponarin a linear response was evaluated within the range 0.007-3.5 mg/mL (R² > 0.9999). It was proven that threefold sonication represents a time-saving, effective and cheap method for the extraction of the polar flavonoid glycosides. The contents determined by HPLC were shown to be in agreement with those obtained employing UV spectrophotometry. CONCLUSION: The study has indicated that the newly developed HPLC method represents a powerful technique for the quality control of D. versicolor. Ultraviolet spectrophotometry may be used alternatively provided that the less polar flavonoids are removed by purification.

Flavonoid C- and O-glycosides from the Mongolian medicinal plant Dianthus versicolor Fisch.

Eighteen flavonoids were identified from an aqueous extract of the aerial parts of Dianthus versicolor, a plant used in traditional Mongolian medicine against liver diseases. The flavonoid C- and O-glycosides isoorientin-7-O-rutinoside, isoorientin-7-O-rhamnosyl-galactoside, isovitexin-7-O-rutinoside, isovitexin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-rutinoside, isoscoparin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-galactoside, and isoorientin-7-O-galactoside were isolated and structurally elucidated. Their structures were established on the basis of extensive spectroscopic techniques including LC-UV-DAD, LC-MS(n), LC-HRMS, 1D and 2D NMR spectroscopy, and by GC-MS analysis after hydrolysis. Flavonoids with such a high glycosylation pattern are rare within the genus Dianthus. Furthermore, isovitexin-7-O-glucoside (saponarin), isovitexin-2″-O-rhamnoside, apigenin-6-glucoside (isovitexin), luteolin-7-O-glucoside, apigenin-7-O-glucoside, as well as the aglycons luteolin, apigenin, chrysoeriol, diosmetin, and acacetin were identified by TLC and LC-DAD-MS(n) in comparison to reference substances or literature data. The NMR data of seven structures have not been reported in the literature to date.

Secondary metabolites of Centaurea calolepis and evaluation of cnicin for anti-inflammatory, antioxidant, and cytotoxic activities.

CONTEXT: Centaurea L. (Astreaceae) species are used as herbal remedies in Turkey. Centaurea calolepis Boiss. is an endemic species of Anatolia that has not been subjected to phytochemical studies except essential oil analysis. OBJECTIVE: Secondary metabolite determination, isolation and structure elucidation of pure compounds were performed on C. calolepis. Cnicin, which is the main component of several Centaurea species, was tested for its in vitro anti-inflammatory, antioxidant and cytotoxic activities. MATERIALS AND METHODS: Chloroform and methanol extracts of the aerial parts of C. calolepis were subjected to isolation process using column chromatography. The structures of the compounds were characterized by 1D- and 2D-NMR experiments. Thin-layer chromatography and high performance liquid chromatography were used in determination of phenolics. Cnicin was subjected to a panel of cellular assays to test for inhibition of nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS), reactive oxygen species and cytotoxicity. RESULTS: Cnicin, lucenin-2, schaftoside and 3-O-feruloylquinic acid were isolated from C. calolepis extracts. Vicenin-2, vitexin, isovitexin, homoorientin, rutin, orientin, luteolin-7-O-glycoside and chlorogenic acid were determined in fractions. Cnicin showed inhibition of NF-κB and inhibition of iNOS activity with IC50 Values of 1.8 and 6.5 µM, respectively. Cytotoxic activity of cnicin was observed toward pig kidney epithelial (LLC-PK11), human malignant melanoma (SK-MEL) and human ductal carcinoma (BT-549) cells with IC50 values of 23.3, 14.0 and 18.3 µM, respectively. DISCUSSION AND CONCLUSION: This is the first detailed report of secondary metabolites of C. calolepis. Evaluation of biological activity of cnicin establishes the potential of this compound as an anti-inflammatory and cytotoxic agent.

Quantification of flavonoid glycosides in an aqueous extract from the traditional Mongolian medicinal plant Dianthus versicolor FISCH.

An HPLC-diode array detection (DAD) method was established in order to investigate dried aerial parts of Dianthus versicolor FISCH. (Caryophyllaceae), a plant used in traditional Mongolian medicine against liver impairment. Aqueous extracts were separated on an Aquasil(®) C(18) column with a linear gradient of acetonitrile (ACN) and water (adjusted to pH 2.8 with formic acid) as the mobile phase. LC-IT-MS facilitated the assignment of 26 flavonoids, among them a series of rare C-glycosylated as well as O-glycosylated derivatives, which are assumed to be the active principles. Quantification was performed and validated using isovitexin-7-O-glucoside (saponarin) as the external standard. The method showed good linear behaviour (r(2) ≥0.9999) over the investigated concentration range (0.007-3.5 mg/mL). The good precision of the method allowed the successful qualitative and quantitative analysis of flavonoid-glycosides in the aqueous extracts prepared from five different D. versicolor samples. Depending on the origin of the samples, the total flavonoid content was found to vary considerably from 0.41 to 3.30% in the aqueous extracts and from 0.07 to 0.57% in the crude drug. In addition, the relative composition of the various flavonoids was found to differ strongly. These results highlight the need for proper quality control for this herbal drug.

Extracts from the Mongolian traditional medicinal plants Dianthus versicolorFisch. and Lilium pumilum Delile stimulate bile flow in an isolated perfused rat liver model.

AIM OF THE STUDY: Dianthus versicolor (Caryophyllaceae) and Lilium pumilum (Liliaceae) are two medicinal plants used in traditional Mongolian medicine to treat hepatic and gastrointestinal disorders. In this study aqueous (AE) and methanolic (ME) extracts of Dianthus versicolor and Lilium pumilum were investigated for their influence on the bile flow. The aqueous extracts of both plants were tested in absence and presence of 10 µM taurocholic acid at three different concentrations (100, 250, and 500 mg/L). The aqueous extract of Dianthus versicolor was further purified in order to locate the active principles. Two resulting fractions, one enriched in flavonoids and the other in sugars, were investigated for their influence on the bile flow in absence of taurocholic acid at 10, 20, and 40 mg/L. The aqueous extracts of both plants were analysed qualitatively by LC-MS(n) and quantitatively by UV-spectrophotometry. MATERIALS AND METHODS: The bile flow experiments were performed in the isolated perfused rat liver. The compounds were identified by LC-DAD-MS(n) and TLC using references. The UV-spectrophotometric analysis was based on the monograph "Passiflorae herba" of the European Pharmacopoeia, and the total flavonoid contents were calculated and expressed as vitexin. RESULTS: AE and ME of both plants increased the bile flow dose-dependently (between 9% and 30%), and no hepatotoxic effect was seen even during longer perfusions. Stimulation of bile secretion was comparable in the presence and in the absence of taurocholic acid. The flavonoid fraction of Dianthus versicolor increased the bile flow by 18% (p<0.05) at 40 mg/L, which was comparable to the positive control cynarin. The phytochemical investigations of the Dianthus versicolor AE (total flavonoid content 1.78%) revealed the presence of the isovitexin derivative saponarin. In the AE of Lilium pumilum (total flavonoid content 1.04%) the flavonoids rutoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were detected. CONCLUSIONS: The results show that choleresis under extract application is due to a stimulation of the bile-salt-independent bile flow which might be caused by the osmotic power of the extracts (hydrocholeresis). The flavonoids seem to contribute to the bile-flow-stimulating effect of Dianthus versicolor. Both plants exhibit a considerable choleretic effect that contributes to their use in traditional Mongolian medicine against gastrointestinal disorders.

Choleretic effects of the Mongolian medicinal plant Saussurea amara in the isolated perfused rat liver.

Saussurea amara is used in traditional Mongolian medicine for the treatment of hepato-biliary disorders. To determine the plant's effect on the bile-salt independent bile flow (hydrocholeresis) as a measure of liver exocrine functions, different extracts were investigated in the isolated rat liver perfusion system. The methanolic extract (3) exerted a dose-dependent increase in bile flow (16%, 37%, 53%, 61%) in concentrations of 50 mg/L, 100 mg/L, 250 mg/L and 500 mg/L. The aqueous crude extract (1) and the ethyl acetate extract (2) also showed a dose-dependent increase, whereas at the highest concentrations (1000 mg/L and 100 mg/L, respectively) a continuous decrease in bile flow could be observed. Cynaropicrin also provoked a dose-dependent increase in bile flow, but caused liver damage at the highest dose tested (20 mg/L). Apigenin 7- O-glucoside, present in extracts 2 and 3, induced a dose-dependent increase of 20%, 30% and 40% (5 mg/L, 10 mg/L, 20 mg/L) and showed a significantly higher effect than the reference substance cynarin. The total flavonoid content was determined by spectrophotometry. To quantify the absolute amount of cynaropicrin in the crude drug and in the tested extracts, an HLPC system was established with santonin as internal standard.

Infrared spectroscopy as a tool for chemotaxonomic investigations within the Achillea millefolium group.

An FT-IR-based method for the rapid characterization and identification of Achillea species was developed. With the IR spectra of the CH2Cl2 extracts of A. aspleniifolia, A. collina, A. millefolium, A. millefolium ssp. sudetica, A. pannonica, A. pratensis, A. roseoalba, A. setacea, and A. styriaca, a spectrum library was created. This database allowed the identification of the respective taxa by comparison of IR spectra. Moreover, information concerning dominant structural elements of sesquiterpene constituents was obtained, providing hints about the composition of a plant sample of unknown origin.

Phenolic and terpenoid compounds from Chione venosa (sw.) urban var. venosa (Bois Bandé).

The Caribbean island of Grenada furnishes the popular aphrodisiac drug Bois Bandé, which consists of the stem bark and the roots of Chione venosa (sw.) URBAN var. venosa (Rubiaceae), a native tree growing in the islands' rain forest. The phytochemical investigation of dichloromethane and methanolic-aqueous extracts of the bark and the roots yielded three acetophenone derivatives described for the first time in plants - ortho-hydroxy-acetophenone-azine (1), acetophenone-2-O-[beta-D-apiofuranosyl-(1''-->6')-O-beta-D-glucopyranoside] (2) and acetophenone-2-O-beta-D-glucopyranoside (3) - along with five known compounds, alpha-morroniside (4), sweroside (5), diderroside (6), daucosterol (7) and beta-sitosterol (8). Their structures were elucidated by 1D and 2D NMR analysis, UV-Vis and ESI-MS.

Morphological, chemical and functional analysis of catuaba preparations.

Fourteen commercial samples of the popular Brazilian aphrodisiac Catuaba specified as bark drugs of Anemopaegma, Erythroxylum and Trichilia species were examined for identity and purity. Only a minority of the examined Catuaba samples contained the crude drugs claimed on the labels. More than half of the products were adulterated with different crude drugs. The majority of the samples contained a bark originating from Trichilia catigua. The TLC fingerprints confirmed the heterogeneity, in 50% of the samples tropane alkaloids of various concentrations were detected. TLC and HPLC methods for separation and identification of the tropane alkaloids were developed and their analytical data (RF values, retention times, ESI-MS) given. The structure elucidation of the two main alkaloids, catuabine D and its hydroxymethyl derivative, is presented. The 1H- and 13C-NMR assignments of these alkaloids are discussed with regard to literature data. Neither aqueous nor methanolic extracts of the Trichilia catigua reference material nor alkaloid-enriched fractions of commercial samples showed any effect on the rabbit corpus cavernosum in an in vitro test.

Sesquiterpenes from Achillea pannonica Scheele.

From dichloromethane extracts of flowerheads of Achillea pannonica Scheele three sesquiterpenes were isolated and identified: 11,13-dehydrodesacetylmatricarin, (6E)-5-tigloxy-9-hydroxynerolidol and alpha-longipin-2-en-1-one. The structures were determined by MS, IR and NMR spectroscopic analyses, (6E)-5-Tigloxy-9-hydroxynerolidol is reported here for the first time. Additionally spathulenol, a compound of the essential oil was identified using GC-MS and GC-FTIR.