Novel Piperidine and 3,4-dihydro-2H-pyrrole Alkaloids from Tilia platyphyllos and Tilia cordata Flowers.

Lime flowers, traditionally used for medical purposes for the treatment of symptoms of the common cold and mental stress, consist of the dried inflorescences including the floral bracts of Tilia cordata, Tilia platyphyllos, Tilia × vulgaris, or mixtures thereof. During phytochemical investigations, 6 different alkaloids - not described until now - were detected in T. cordata and T. platyphyllos flowers. They have been isolated and characterized as alkaloids with a dihydro-pyrrole and a piperidine substructure, respectively. Compounds 1A: and 1B: (tiliines A and B) are characterized as 2 diastereomers containing a 2-methyl-3,4-dihydro-2H-pyrrol-3-ol, connected via a C-10 alkyl chain to a O-glucosylated hydroquinone moiety. Compounds 2A: and 2B: (tiliamines A and B) are diastereomers of a 2-methyl-substituted piperidin-3-ol, coupled via a C-9 alkyl chain again to an O-glucosylated hydroquinone moiety. Compounds 3A: and 3B: (tilacetines A and B) are 3-O-acetylated derivatives of tiliamines. Quantification of the 6 alkaloids by HPLC-ESI-qTOF analysis indicated the presence of all alkaloids in T. cordata flowers and T. platyphyllos flowers, bracts, and leaves, with tiliines A and B and tilacetines A and B being the major compounds. Acetone/water turned out be the best extraction solvent for the alkaloids, but ethanol and ethanol/water mixtures also can be used for effective extraction. Furthermore, the alkaloids are found in hot water extracts, which are typically used in the traditional medicine.

Multistep Analysis of Diol-LC-ESI-HRMS Data Reveals Proanthocyanidin Composition of Complex Plant Extracts (PAComics).

Proanthocyanidins (PACs) are complex oligomeric or polymeric phenolic biopolymers composed of flavan-3-ol building blocks. PACs exert manifold functional bioactivities and are assessed as bioactive ingredients in a variety of food products, beverages, medicinal plants, and phytopharmaceuticals. Although analytical methods for PACs with low degree of polymerization (DP) are well established, a lack of methods for the detailed analysis of higher oligomers and polymers from complex plant extracts is obvious. For this, the present study investigated PAC-enriched extracts from four different plants, traditionally used for medical purpose (Lime flower, Hawthorn leaf and flower, Japanese Wisteria fruits without seeds, and Common Sorrel herb). PACs were separated on diol stationary-phase high-performance liquid chromatography according to the respective DP and detected by fluorescence and quadrupole time-of-flight mass spectrometry (qTOF-MS). The qTOF-MS contour plots [tR → m/z] provided a sufficient overview on the respective PAC distribution. Subsequently, high-resolution mass spectrometry data were used for Kendrick mass defect (KMD) analysis, with (epi)catechin, the main flavan-3-ol unit in PACs, as the reference unit. The resulting KMD plots enabled an elucidation of the general polymer chain composition with regard to DP, building blocks, and potential secondary modifications (e.g., galloylation). Subsequently, analysis of MS2 fragmentation patterns of PAC oligomers confirmed the structural features obtained from the KMD plots. While Lime flower contained oligomeric A- and B-type PACs, composed of (epi)catechin and (epi)afzelechin, Japanese Wisteria fruit contained PACs consisting of three different hydroxylated flavan-3-ols. Cinchonains, A-type PACs, and B-type PACs were detected in the Hawthorn plant material. Galloylated oligo- and polymeric PACs were detected in Common Sorrel herb. This multistep analysis reveals collective insights into the PAC composition of the extracts. The protocol offers a fast and reliable methodology to be used in a standard laboratory. On the other hand, this methodology reaches its limits for higher oligomeric PACs, and further optimization is necessary for a better detection of the polymers, as the optimal DP cluster detection depends on the resolution of diol stationary-phase chromatography and is therefore limited.

Isoflavonoids with inhibiting effects on human hyaluronidase-1 and norneolignan clitorienolactone B from Ononis spinosa L. root extract.

Human hyaluronidase-1 (Hyal-1) is one of the main enzymes in the homeostasis of hyaluronic acid (HA), the main polysaccharide of extracellular matrix. Development of specific Hyal-1 inhibitors might be a promising target for improved wound healing, tissue regeneration, and looking at renal function for diuresis. By using surface-displayed Hyal-1 on Escherichia coli F470 cells, HA as substrate and stains-all method for quantification of undegraded HA, the respective enzyme activity can be determined easily. Based on the traditional use of extracts from the roots from Ononis spinosa L. (Restharrow root) as a weak diuretic to achieve flushing of the urinary tract and as an adjuvant in minor urinary complaints the herbal material was selected for bioactivity guided fractionation for compounds with Hyal-1 inhibition activity. Hot water and hydroalcoholic extracts showed moderate inhibiting effects (IC50 1.36 resp. 0.73 mg/mL) while dichloromethane extract exerted an IC50 of 190 µg/mL. Bioassay guided fractionation of the dichloromethane extract yielded four isoflavonoids with anti Hyal-1 activity: onogenin 1, sativanone 2, medicarpin 3 and calycosin-D 4 with inhibition rates of 25.4, 61.2, 22.4 and 23.0%, respectively at test concentration level of 250 µM. The norneolignan clitorienolactone B 5, the first time described for the genus Ononis, was inactive. The IC50 of sativanone, the most active compound was determined with 1501 µM, which was better than that of the positive control glycyrrhizinic acid (177 µM). Thus, a possible explanation for diuretic properties of Ononis spinosa L. root extract may be postulated from the results so far obtained.

Flavan-3-ols and proanthocyanidins from Limonium brasiliense inhibit the adhesion of Porphyromonas gingivalis to epithelial host cells by interaction with gingipains.

Porphyromonas gingivalis is a pathogen strongly involved in chronic and aggressive forms of periodontitis. Natural products, mainly polyphenols, have been described for advanced treatment of periodontitis by inhibition of the bacterial adhesion of P. gingivalis to the epithelial host cells. An acetone:water extract (LBE) from the rhizomes of Limonium brasiliense (Boiss.) Kuntze was tested under in vitro conditions for potential antiadhesive effects against P. gingivalis to human KB cells and for inhibition of the proteolytic activity of gingipains, the main virulence factor of P. gingivalis. LBE≤100µg/mL had no cytotoxicity against the bacteria and did not influence the cell physiology of human epithelial KB cells. At 100µg/mL LBE reduced the adhesion of P. gingivalis to KB cells significantly by about 80%. LBE at 20µg/mL reduced the proteolytic activity of the arginin-specific Rgp gingipain by about 75%. Chemical profiling of LBE indicated the presence of gallic acid, epigallocatechin-3-O-gallate and samarangenins A and B as lead compounds. UHPLC by using MS and UV detection displays a suitable method for quality control of the extract for identification and quantification of the lead compounds.

Qualitative and quantitative phytochemical characterization of Myrothamnus flabellifolia Welw.

The upper aerial parts and leaf tips of Myrothamnus flabellifolia Welw. (Myrothamnaceae), a resurrection plant indigenous to southern Africa, are used in African traditional medicine for infections of the respiratory and urinary system as well as for inflammation of mucosa and skin. Within a phytochemical investigation of the herbal material from M. flabellifolia the flavonoid fraction was shown to contain quercetin 10 as well as the respective 3-O-ß-d-galactosides, glucosides, -glucuronides and 3-O-α-l-rhamnosides of quercetin (6, 7, 8, 26) and kaempferol (1, 2, 3, 9). Additionally mono-galloylated 12, 13, di-galloylated 15 and tri-galloylated 16 flavonol glycosides were identified. 3,4,5-Tri-O-galloyl- 14 and 1,3,4,5-tetra-O-galloyl quinic acid 28 were isolated and characterized, beside arbutin 27 and 2″,3″-di-O-galloyl-arbutin 11. Furthermore, the depside 2-O-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxyphenylacetic acid 5, 1,2,3,4,6-penta-O-galloyl-ß-d-glucose 22 and seven ellagitannins were found: 1,2-di-O-galloyl-(4,6-(S)-hexahydroxydiphenoyl)-ß-d-glucose 20, 1,3-di-O-galloyl-(4,6-(S)-hexahydroxydiphenoyl)-ß-d-glucose 21, 2,3-di-O-galloyl-(4,6-(S)-hexahydroxydiphenoyl)-ß-d-glucose (tellimagrandin-I) 19, 1,2,3-tri-O-galloyl-(4,6-O-hexahydroxydiphenoyl)-ß-d-glucose (tellimagrandin-II) 23, and two so far not described dimers of tellimagrandin-I and -II (myrodigamin-I and -II, 24, 25). The presence of trehalose (3.3%), raffinose (0.2%) and stachiose (0.2%) beside a fructan (2.1%) was determined. Two ICH2-validated UHPLC methods have been developed and used within batch analysis for unambiguous identification of the herbal material and quantification of the major compounds myrodigamin-I (0.9 to 1.7%), tellimagrandin-II (0.3 to, 0.9%) and 3,4,5-tri-O-galloyl quinic acid (0.1 to 0.7%), besides kaempferol (0.1 to 0.3%) and quercetin content (0.2 to 0.3%) after hydrolysis of the respective glycosides.

Bioassay-Guided Fractionation of a Leaf Extract from Combretum mucronatum with Anthelmintic Activity: Oligomeric Procyanidins as the Active Principle.

Combretum mucronatum Schumach. & Thonn. is a medicinal plant widely used in West African traditional medicine for wound healing and the treatment of helminth infections. The present study aimed at a phytochemical characterization of a hydroalcoholic leaf extract of this plant and the identification of the anthelmintic compounds by bioassay-guided fractionation. An EtOH-H2O (1:1) extract from defatted leaves was partitioned between EtOAc and H2O. Further fractionation was performed by fast centrifugal partition chromatography, RP18-MPLC and HPLC. Epicatechin (1), oligomeric proanthocyanidins (OPC) 2 to 10 (mainly procyanidins) and flavonoids 11 to 13 were identified as main components of the extract. The hydroalcoholic extract, fractions and purified compounds were tested in vitro for their anthelmintic activity using the model nematode Caenorhabditis elegans. The bioassay-guided fractionation led to the identification of OPCs as the active compounds with a dose-dependent anthelmintic activity ranging from 1 to 1000 µM. Using OPC-clusters with a defined degree of polymerization (DP) revealed that a DP ≥ 3 is necessary for an anthelmintic activity, whereas a DP > 4 does not lead to a further increased inhibitory effect against the helminths. In summary, the findings rationalize the traditional use of C. mucronatum and provide further insight into the anthelmintic activity of condensed tannins.

Traditionally used medicinal plants against uncomplicated urinary tract infections: Are unusual, flavan-4-ol- and derhamnosylmaysin derivatives responsible for the antiadhesive activity of extracts obtained from stigmata of Zea mays L. against uropathogenic E. coli and Benzethonium chloride as frequent contaminant faking potential antibacterial activities?

The dried stigmata from Zea mays L. are used traditionally for the treatment of uncomplicated urinary tract infections. A recent screening has indicated that hydroalcoholic extract of the herbal material inhibits the adhesion of uropathogenic Escherichia coli (UPEC) to T24 bladder cells. For verification of these data EtOH-water (1:1) extracts from 4 different batches of Maydis stigmata were investigated. Within an in vitro adhesion assay (UPEC strain 2980 and human T24 bladder cells) a dose-dependent antiadhesive activity against UPEC was verified (IC50 1040µg/mL). Bioassay guided fractionation of M. stigmata, batch S1, by EtOH-water extraction, followed by chromatography on Sephadex LH20 revealed two active fractions (I and XI). Further purification of fraction I and structure elucidation of the isolated compound revealed the presence of significant amounts of the biocide benzethonium chloride as contaminant. Benzethonium chloride was also identified in subsequent investigations in 2 different batches of M. stigmata. The presence of such nondeclared and illegal contaminants in the herbal raw material market has to be discussed intensively. From benzethonium-free raw material (batch S2) as well as from batch S1 fraction XI was further fractionated by MPLC and preparative HPLC, leading to a still complex subfraction XIG, which was analyzed by UHPLC/+ESI-QTOF-MS analysis. Advanced data processing and species-metabolite relationship database revealed the tentatively existence of the unusual C-glycosidic flavones derhamnosylmaysin (6), 3'-deoxyrhamnosylmaysin (4), 3'-O-methylderhamnosylmaysin (3), apiferol (2) and alternanthin (8) which might be related to the antiadhesive activity of this subfraction against UPEC.

Isolation and Quantification of Oligomeric and Polymeric Procyanidins in the Aerial Parts of St. John's Wort (Hypericum perforatum).

Proanthocyanidins (condensed tannins) constitute a class of oligomeric and polymeric polyphenols with flavan-3-ols as monomeric building blocks. Despite the high impact of proanthocyanidins, these polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of the flavan-3-ols as cleavage products or low molecular oligomers as lead compounds. For St. John's wort (Hyperici herba) from Hypericum perforatum, a protocol for preparative isolation of oligomeric and polymeric proanthocyanidins from an acetone-water extract by chromatography on Sephadex®LH20 in combination with preparative high-performance liquid chromatography on the diol stationary phase was developed, yielding procyanidin reference clusters with a defined degree of polymerization from 3 to 10. Identity and purity of these clusters was proven by high-performance liquid chromatography (RP18 and diol phase) and mass spectrometry. For identification and quantification of proanthocyanidin clusters from St. John's wort, an ICH-Q2 (International harmonized guideline for analytical validation) validated high-performance liquid chromatography method with fluorimetric detection was developed using an acetone-water extract of the herbal material, purified by solid-phase extraction for the removal of naphthodianthrones. The method enabled the quantification of procyanidin clusters with a degree of polymerization from 2 to 10. Analysis of nine batches of Hyperici herba from different sources indicated a high variability of proanthocyanidin content in the range from 8 to 37 mg/g. In all of the batches investigated, the trimer cluster DP3 was the dominant proanthocyanidin (about 40 %), followed by DP 4 (about 15 %) and DP5 (about 12 %). Monitoring of procyanidin distribution during seasonal growth of fresh plants of H. perforatum indicated the highest proanthocyanidin content in young plants (about 50 mg/g) and a time-dependent decrease during the growing season to about 16 mg/g. The highest proanthocyanidin content was found in young leaves and flowers, while the fruits were proanthocyanidin-free; older parts of the stem and the herb had a lower proanthocyanidin content. From these data, it can be concluded that proanthocyanidins serve as part of the plant defense system in the reproductive organs.

Hydrolyzable tannins from hydroalcoholic extract from Poincianella pluviosa stem bark and its wound-healing properties: phytochemical investigations and influence on in vitro cell physiology of human keratinocytes and dermal fibroblasts.

Extracts from Poincianella pluviosa stem bark are used in traditional medicine of South America for its wound healing properties. For validation of this traditional use and for rationalizing a potential pharmaceutical development towards standardized preparations bioassay-guided fractionation of EtOH-water (1:1v/v) extract (crude extract, CE) of P. pluviosa bark was performed. HaCaT keratinocytes cell line and human primary dermal fibroblasts (pNHDF) were used as in vitro systems. Significant stimulation of mitochondrial activity was found for CE on both cell types, which caused a strong increase of cell proliferation of keratinocytes. Fractionation of CE over Sephadex LH20 revealed two inactive fractions (FA and FB) and an active fraction FC, which was further fractionated by MPLC into 4 subfractions. Subfraction FC1 increased mitochondrial activity and proliferation of keratinocytes and dermal fibroblasts in a dose dependent manner (10 to 100 µg/mL) and did not show necrotic cytotoxicity on keratinocytes (LDH release assay). FC1 was investigated by ESI-MS/MS and solid-state (13)C NMR which confirmed the presence of various polyphenols and hydrolyzable tannins. MS studies suggest the presence of pyrogallol (1), gallic acid (2), gallic acid methyl ester (3), ellagic acid (4), corilagin (5), 1,4,6-tri-O-galloyl-glucose (6), tellimagrandin I (7), 1,2,3,6-tetra-O-galloyl-glucose (8), mallotinic acid (9), tellimagrandin II (10), 1,2,3,4,6-penta-O-galloyl-glucose (11), geraniin (12), and mallotusinic acid (13).

Concentrated green tea extract induces severe acute hepatitis in a 63-year-old woman--a case report with pharmaceutical analysis.

UNLABELLED: ETNOPHARMACOLOGICAL RELEVANCE: The popularity of concentrated green tea extracts as dietary supplements for a wide range of applications is increasing due to their health-promoting effects attributed to the high amounts of catechins they contain. The most important of the green tea catechins is (-)-epigallocatechin-3-O-gallate (EGCG). While their beneficiary effects have been studied extensively, a small number of adverse events have been reported in the medical literature. Here we present a typical reversible course of severe hepatitis after green tea consumption. MATERIALS AND METHODS: The case study describes in a 63-year old woman during treatment with green tea-capsules upon recommendation of a cancer support group. RESULTS: The histological finding was consistent with drug induced hepatitis, and other possible causes of hepatitis were excluded. According to the CIOMS/RUCAM score the causality was assessed as "probable". After discontinuation of medication, followed by extracorporal albumin dialysis, rapid and sustained recovery occurred. Pharmaceutically analysis (HPLC) of the green tea capsules did not give evidence for contaminants but revealed the two typical compounds of green tea, namely (-)-epigallocatechin-3-O-gallate (EGCG, 93.2%) and epicatechin (EC, 6.8%) at a very high dose level. CONCLUSION: The present case highlights the fact that such concentrated herbal extracts from green tea may not be free of adverse effects under certain circumstances. There is still a lack of a uniform European Union-wide surveillance system for adverse drug reactions of herbal products. Therefore this case underlines the importance of public awareness in the potential risks in use of herbal products.

Wound-healing plants from TCM: in vitro investigations on selected TCM plants and their influence on human dermal fibroblasts and keratinocytes.

Wound-healing plants from Traditional Chinese Medicine and described for wound healing in the Pharmacopoeia of People's Republic of China (2005 ed.) were investigated by in vitro bioassay on human skin cells. Therefore water and EtOH-water extracts (6:4, v/v) from 12 plants were tested on human primary dermal fibroblasts (pNHDF) and human HaCaT keratinocyte cell line by quantification of cell viability (MTT assay) and cellular proliferation (BrdU incorporation ELISA). No functional activity was found for extracts from Achyranthis bidentatae rhizoma, Cimicifugae rhizoma, Corydalis rhizoma, Gardeniae fructus, Houttuyniae herba, Lonicerae japonicae caulis, Paeoniae rubrae radix and Rehmanniae radix. Extracts from Notoginseng radix et rhizoma, Angelicae sinensis radix and Lonicerae japonicae flos showed moderate activity, while extracts from Moutan cortex (the root bark of Paeonia suffruticosa Andr., Ranunculaceae) increased cell viability of HaCaT keratinocytes and pNHDF in a dose-dependent manner significantly. Bioassay-guided fractionation yielded paeonol 1, the flavan-3-ols catechin 2 and epicatechin-3-O-gallate 3, the dimeric proanthocyanidin epicatechin-(4ß→8)-catechin 4, a mixture of trigalloyl-glucoses 5 and 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) 6. The proanthocyanidin-containing fractions as well as PGG-containing fractions contributed substantially to the stimulating effects. Especially PGG-containing fractions enhanced cell viability and cellular proliferation of HaCaT keratinocytes at concentration of 100nM. From these data we conclude that indication claims for TCM herbal materials must be carefully investigated in order to establish evidence-driven use of such plants. In case of Moutan cortex skin cell stimulating effects have clearly been proven. These effects can be related to the polyphenol fractions of condensed and hydrolysable tannins.

Phenylpropanoid-substituted procyanidins and tentatively identified procyanidin glycosides from hawthorn (Crataegus spp.).

The rational use of hawthorn leafs and flowers from Crataegus spp. for declining cardiac performance is mainly due to flavon-C-glycosides and oligomeric procyanidins (OPC). From OPC-enriched extracts from different batches, a dimeric phenylpropanoid-substituted procyanidin (cinchonain II b, 1) was isolated and characterized by MS, CD, and NMR. Also the presence of higher oligomeric cinchonains (degree of polymerization 3 to 8) in hawthorn extracts was shown by a specific ultrahigh-pressure liquid chromatography-ESI-qTOF-MS method. Interestingly, strong evidence for the occurrence of oligomeric procyanidin hexosides was found by ultrahigh-pressure liquid chromatography-ESI-qTOF-MS analysis which additionally revealed the presence of peaks indicative of dimeric procyanidin hexosides by their exact mass, which were clearly distinguishable from the cinchonain II type peaks.

Antiadhesion as a functional concept for protection against uropathogenic Escherichia coli: in vitro studies with traditionally used plants with antiadhesive activity against uropathognic Escherichia coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Investigation of medicinal plant extracts traditionally used against uncomplicated urinary tract infections (UTI) and identification of antiadhesive effects under in vitro conditions against binding of uropathogenic Escherichia coli (UPEC) on bladder cell surface. MATERIALS AND METHODS: Literature search on traditionally used medicinal plants for UTI was performed by online data bases and standard herbal monographs. For further identification shortlisting was done by intensive evaluation of results by plausibility and phytochemical aspects. Plant material with documented antibacterial effects was not considered for further investigations. Direct cytotoxicity of EtOH-water (1:1; v/v) extracts of the shortlisted plants was investigated against UPEC strain 2980 and bladder cell line T24. Inhibition of UPEC adhesion to T24 cells was monitored either after pretreatment of bacteria or eukaryotic cells by flow cytometry. RESULTS: Literature search on traditionally used medicinal plants for UTI resulted in 275 plant species, from which 20 were shortlisted by a validated selection process for experimental testing. While direct cytotoxicity of the extracts (1-2000 µg/mL) against UPEC and T24 cells was excluded significant antiadhesive effects were monitored for five plant extracts. Two of them, prepared from the rhizome of Agropyron repens L. and the stigmata of Zea mays L. decreased bacterial adhesion (IC(25) 630 µg/mL, IC(50) 1040 µg/mL, resp.) by interacting with bacterial outer membrane proteins, which was shown by pretreatment of UPEC. Preparations of three plant extracts from the leaves of Betula spp. (according to European pharmacopoeia 7.0), Orthosiphon stamineus BENTH. and Urtica spp. showed antiadhesive effects by interacting with T24 cells (IC(50) 415, 1330 µg/mL, resp. IC(25) 580 µg/mL). Combination of two extracts, one interacting with the bacterial surface (Zea mays L., Agropyron repens L.) and one with the eukaryotic target (Orthosiphon stamineus BENTH.) revealed synergistic effects, as shown by strongly decreased IC(50) values (131 µg/mL, 511 µg/mL, resp.). CONCLUSIONS: Different plant extracts, traditionally used for UTI, exhibit antiadhesive effects against UPEC under in vitro conditions. Molecular targets can be different, either on the bacterial or on the host cell surface. Combination of these medicinal plants with different targets, as observed often in phytotherapy, results in synergistic effects.

Eupatorium perfoliatum L.: phytochemistry, traditional use and current applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Eupatorium perfoliatum L. originates from North America, where it has been widely used since centuries by native Indians. Additionally extracts are used also in Europe as immunostimulating agent for treatment of fever and cold. The following review summarizes published data on phytochemistry, ethnopharmacological use, as well as clinical and preclinical data. MATERIALS AND METHODS: Literature survey was performed via SciFinder(®) on papers and patents and by systematic research in ethnopharmacological literature at various university libraries. RESULTS: The phytochemical composition of Eupatorium perfoliatum is described in detail for volatile oil, caffeic acid derivatives, flavonoids, sesquiterpene lactones, tannins, polysaccharides. Methods for analytical quality control, as well as specification for relevant lead structures can be deduced from published batch analysis. Preclinical studies indicate anti-inflammatory effects of ethanolic extracts, which can be correlated on a molecular level to eupafolin and sesquiterpen lactones. Antiplasmodial, antioxidative and immunomodulating activities are additionally discussed. Clinical data on the use of Eupatorium perfoliatum do not meet modern GCP requirements, but do indicate positive tendencies for use of ethanolic extracts for treatment of common colds. CONCLUSION: While the postulated immunostimulating properties of Eupatorium perfoliatum have not been confirmed by in vitro data, animal-studies and in vitro experiments with plant extracts both indicate antiinflammatory effects beside antiplasmodial effect against Plasmodium falciparum. Such an antiinflammation caused by the ethanolic extracts can be correlated well with clinical symptoms related to diseases as common cold, rheumatism, athritis etc. These data also support the plausibility of the plant's traditional use by the North American indigenous population and early European settlers. In principle quality aspects of the herbal material have to be affirmed by establishing modern pharmacopoeial control methods to guarantee constant and reliable quality.

Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: Geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts.

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-ß-D-glucoside (isoquercitrin), kaempferol-3-O-ß-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract. Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol). Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 µM, and of keratinocytes at 50-100 µM. Furosin stimulated NHDF at about 50 µM, keratinocytes at about 150-200 µM. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 µM for NHDF and 150 µM for keratinocytes. Toxicity of furosin--less than that of geraniin--was observed at > 400 µM. Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 µM and 5-10 µM respectively. Geraniin at 105 µM significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.

Phytochemical characterization of Rhododendron ferrugineum and in vitro assessment of an aqueous extract on cell toxicity.

Within a systematic phytochemical investigation of the leaves of RHODODENDRON FERRUGINEUM L. (Ericaceae), the volatile oil was isolated (0.7 %) and its constituents were characterized. Eleven flavonoids were isolated and characterized, with quercetin 3- O-[6''- O-(2-methyl-1-oxobutyl)]-ß- D-glucopyranoside and 2 R,3 R-dihydromyricetin 3- O- ß- L-arabinopyranoside as new natural products. Beside monomeric flavan-3-ols (catechin, epicatechin, gallocatechin, epigallocatechin) from the tannin fraction (about 3.5 % calculated as pyrogallol), the dimeric procyanidins B1 to B7 were identified, as well as the trimeric compounds procyanidin C1, epicatechin-(4 ß → 8)-epicatechin-(4 ß → 8)-catechin and the trimeric A type-linked cinnamtannin B1. Additionally, phloroacetophenon 4- O- ß- D-glucopyranoside and chlorogenic acid were isolated. Water-soluble carbohydrates comprised about 13.5 % of the dried leaves, including fructans (3 %), polysaccharides (1 %) (mainly type II arabinogalactans), glucose, fructose, sucrose, stachyose and raffinose. The IN VITRO effects on cellular vitality (MTT test), proliferation (BrdU incorporation) and necrosis (LDH release) of an aqueous extract were investigated. The extract did not exert any toxic effects, while the vitality and the proliferation rates of epithelial HaCaT keratinocytes were significantly increased at 100 µg/mL, indicating that the aqueous extract does not have negative effects against cellular activity.

Cinchonain Ib isolated from Eriobotrya japonica induces insulin secretion in vitro and in vivo.

AIMS OF THE STUDY: Eriobotrya japonica leaves had been used traditionally for the treatment of diabetes mellitus by immersing the dried leaves in a hot water drink. Few studies have shown the hypoglycemic effect of Eriobotrya japonica using crude alcoholic extract and isolated methanolic compounds. These studies proposed that the mechanism of action could be by stimulating the beta-islets of Langerhans to secrete insulin, however with no scientific evidence. METHODS: Eriobotrya japonica water extract (EJWE) and the compounds derived from it: cinchonain Ib, procyanidin B-2, chlorogenic acid and epicatechin, were tested for their effects on insulin secretion from INS-1 cells and following oral administration in rats. RESULTS: The present study showed that EJWE increased significantly (p<0.05) insulin secretion from INS-1 cells in dose-dependent manner. Oral administration of EJWE at 230 mg/kg to rats, however, decreased plasma insulin level for as long as 240 min post-administration and caused a transient drop of blood glucose at 15 and 30 min post-administration. On the other hand, cinchonain Ib enhanced significantly (p<0.05) insulin secretion from INS-1 cells, whereas epicatechin inhibited significantly (p<0.05) insulin secretion from INS-1 cells. In addition, cinchonain Ib enhanced significantly (150%: p<0.05) plasma insulin level in rats for as long as 240 min after 108 mg/kg oral administration but did not induce any change in blood glucose level. CONCLUSION: These data indicate that cinchonain Ib has an insulinotropic effect and suggest the possible use of cinchonain Ib for managing type 2 diabetes.

Trans-Aconitic acid, glucosylflavones and hydroxycinnamoyltartaric acids from the leaves of Echinodorus grandiflorus ssp. aureus, a Brazilian medicinal plant

The leaves of Echinodorus grandiflorus are used traditionally in Brazil for their diuretic and antirheumatic activities. In order to obtain more information about its phenolic constituents, an ethanol leaf extract was fractionated. Five glucosylflavones, five hydroxycinnamoyltartaric acids and trans-aconitic acid were isolated and their structures identified by NMR data and chiral HPLC. Quantitative determination of compounds 1 - 11 by HPLC indicated caffeoylferuloyltartaric acid with 0.13 percent as the main representative of the hydroxycinnamoyltartaric acids and swertiajaponin with 0.31 percent of the glucosylflavones; the content of trans-aconitic acid in the investigated material was 0.98 percent.

As folhas de Echinodorus grandiflorus são tradicionalmente utilizadas no Brasil devido as suas atividades diuréticas e anti-reumáticas. Com a finalidade de obter mais informações acerca de seus constituintes fenólicos, um extrato etanólico das folhas foi fracionado. Foram isolados cinco glicosilflavonas, cinco ácidos hidroxicinamoiltartáricos e o ácido trans-aconítico e suas estruturas foram identificadas através de dados de RMN e CLAE quiral. A determinação quantitativa dos compostos 1 - 11 através de HPLC indicou o ácido cafeoilferuloiltartárico com 0,13 por cento como o principal representante dos ácidos hidroxicinamoiltartáricos e a swertiajaponina com 0,31 por cento das glicosilflavonas; a quantidade de ácido trans-aconítico no material investigado foi de 0.98 por cento.

Phenolic esters from the rhizomes of Cimicifuga racemosa do not cause proliferation effects in MCF-7 cells.

Five phenylpropanoid esters, caffeoylglycolic acid, 2-caffeoylpiscidic acid (cimicifugic acid D), 3,4-dihydroxyphenacyl caffeate (petasiphenone), 3,4-dihydroxyphenyl-2-oxopropyl isoferulate (cimiciphenol) and 3,4-dihydroxyphenacyl isoferulate (cimiciphenone) were isolated from a commercially available extract of the rhizomes of Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L.) for the first time; the known cimicifugic acids A, B, E, F, fukinolic acid, fukiic acid and caffeic acid were also obtained. Cimiciphenone and caffeoylglycolic acid are new natural products. The structures were elucidated by means of spectroscopic data (ESI-MS, 1H-, 13C-NMR, COSY, HMQC, HMBC and NOE experiments). Ferulic acid and isoferulic acid were detected by HPLC analysis in comparison to standards. The estrogenic activity of the isolated compounds was tested in an estrogen-dependent MCF-7 mamma carcinoma cell line; 17beta-estradiol (10(-11) M) and the phytoestrogen coumestrol (10(-7) - 10(-5) M) were used as references. The results suggest that, in contrast to an earlier report, the phenolic esters do not exert a proliferative (estrogenic) effect in this test system.

New fatty acid esters originate during storage by the interaction of components in prasaplai, a Thai traditional medicine.

Prasaplai is a Thai traditional preparation composed of twelve ingredients; during storage three newly emerging compounds were detected by HPLC. Their structures were established as the new (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl linoleate, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl oleate and the known (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl palmitate on the basis of spectral data and chemical evidence. The artificial esters were already observable after one day of storage of the freshly prepared dry mixture, and steadily increased during a storage period of 30 days and more. A systematic search indicated that the three esters arose from the interaction of compounds contained in the rhizomes of Zingiber cassumunar and the seeds of Nigella sativa.