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Continued examination of the stem bark of Newbouldia laevis afforded three minor phenylethanoid glycosides, designated as newbouldiosides D – F. Their structures were elucidated by spectroscopic methods as β-(3,4-dihydroxyphenyl)ethyl 5-O-syringoyl-β-D-apiofuranosyloxy-(1 → 2)-O-[α-L-rhamnopyranosyl-(1 → 3)]-6-O-E-sinapoyl-β-D-glucopyranoside, β-(3,4-dihydroxyphenyl)ethyl β-D-apiofuranosyloxy-(1 → 2)-O-[α-L-rhamnopyranosyl-(1 → 3)]-6-O-E-sinapoyl-β-D-glucopyranoside, and β-(3,4-dihydroxyphenyl)ethyl β-D-apiofuranosyloxy-(1 → 2)-O-α-L-rhamnopyranosyl-(1 → 2)-6-O-E-sinapoyl-β-D-glucopyranoside, respectively. These metabolites are the first members possessing a sinapoyl structural element. In addition, the series of naturally occurring flavonoids is extended by the identification of 3′,4′,5,7-tetrahydroxy-5′-methoxyflavanone and apigenin-5-O-α-L-rhamnopyranosyl-7-O-β-D-glucopyranoside obtained from leaf extracts of Markhamia zanzibarica and aromadendrin-7-O-(2″-O-formyl)-β-D-glucopyranoside isolated from Spathodea campanulata. The latter compound is the first example of a flavonoid possessing a formylated glucosyl moiety.

Resveratrol has been shown to be a potential chemopreventive and anticancer agent, inducing apoptosis in a variety of cancer cells. The present study was performed to evaluate the effect of resveratrol on A549 human lung adenocarcinoma epithelial cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide evaluation demonstrated that the exposure of cells to increasing concentrations of resveratrol (0–175 µM) for 24 h resulted in a decrease in cell viability (IC 50 85.5 µM). Annexin V/propidium iodide double stain verified apoptosis in A549 cells, while negligible cell cytotoxity (≥ 0.5 %) was observed in all untreated incubations. Using colorimetric assay kits, induction of caspase-3, but not of caspase-8, activity was detected in response to resveratrol (> 130 µM). Confirmatory evidence of this finding was provided by Western blotting, indicating expression of cleaved caspase-3 levels in a concentration-dependent manner with a minimum resveratrol concentration of 65 µM required for activation of this protease, while that of caspase-8 remained unaffected. The apoptotic process was associated with reactive oxygen species production in a concentration-dependent manner, evidenced by microscopic examination and fluorescence-activated cell sorting analysis using the 2′,7′-dichlorofluorescein diacetate assay. In the presence of the mitochondrial electron transport chain inhibitor rotenone, reactive oxygen species production and the concomitant apoptotic cell population were significantly reduced. This finding suggested that the resveratrol-induced apoptosis was mediated via a mitochondrial pathway alignment in human A549 cells. Although effective levels were observed at high concentrations, the outcome may well differ under in vivo conditions. Finally, experiments reaffirmed the chemical instability of trans -resveratrol, suggesting the need for protection of the solutions from extended exposure to light.

In addition to a range of beneficial pharmacological activities, resveratrol is recently reported to have potential antileishmanial activities in vitro. The present study was conducted to evaluate the effect of resveratrol on promastigotes and amastigotes of transgenic Leishmania major expressing green fluorescent protein in comparison with its direct cytotoxic effects on host cells (bone marrow-derived and J774-G8 macrophages, respectively). As assessed by FACS analysis, resveratrol showed moderate antipromastigote activity at35 µg/mL (153.2 µM) and promising effects at higher sample concentrations. In contrast, the green fluorescent protein signal as a measure of the intracellular parasites' viability was reduced in a concentration-dependent manner. Resveratrol strongly inhibited NO production, but did not display direct NO-scavenging activity in sodium nitroprusside solution. Western blotting indicated that resveratrol reduced recombinant interferon-γ/LPS-induced expression of iNOS protein. Microscopic studies, MTT evaluation, and FACS analysis showed significant cytotoxic effects on host cells in a concentration-dependent manner. This finding suggests that the in vitro antileishmanial activity of resveratrol is due to cytotoxic effects on host cells rather than attributable to a specific antiparasitic potential.

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.

EPs ® 7630 is an aqueous-ethanolic extract of the roots of Pelargonium sidoides, employed in the treatment of upper respiratory tract infections. Its anti-infective activity is supposed to be associated with the activation of the nonspecific immune system. Using Leishmania major GFP-infected murine BMMΦ, the NO production of EPs ® 7630-activated macrophages was correlated with the reduction of the GFP signal measured at single cell levels using flow cytometry. The anti-infectious effect of EPs ® 7630 (3-10 μg/mL) on its own (NO production: 4-13 μM; signal reduction: 25-73%) was less prominent than that in combination with IFN-γ (100U/mL) (NO production: 20-27μM; signal reduction: 35-78%). Furthermore, supernatants of EPs ® 7630-stimulated AMMΦ (10 μg/mL) significantly reduced the cytopathic effect of EMCV on L929 fibroblasts (antiviral activity 80 U/mL) when compared with an IFN-γ standard (100U/mL). Direct addition of EPs ® 7630 to L929 did not mediate cytoprotective effects. The antiviral components induced in BMMΦ by EPs ® 7630 remain to be identified. Detection of any IFNs by ELISA was unsuccessful, which may be due to their very low concentrations in cell supernatants. The current data provide convincing support for the induction of anti-infectious responses by EPs ® 7630.

A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote forms of L. donovani residing within murine macrophages. The most active aurone (6-hydroxy-2-[phenylmethylene]-3(2H)-benzofuranone) had an EC50 of 0.45 microgram/ml in the extra-, and an EC50 of 1.40 micrograms/ml in the intracellular assay. Other aurones were active between 0.06-12.50 micrograms/ml and 0.04-7.81 micrograms/ml, respectively. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, the compounds showed only moderate cytotoxicity (EC50 2.32 to > 25.0 micrograms/ml). This is the first report on aurones as a new class of natural products with leishmanicidal activity.

In the present study, the effects of tannins obtained from various plant sources on the activity of 5-LOX and AT were examined. With IC50 values ranging from 1.0 to 18.7 muM, hamamelitannin and the galloylated proanthocyanidins isolated from Hamamelis virginiana L. were found to be most potent inhibitors of 5-LOX. Unlike the 5-LOX study, hamamelitannin proved to be ineffective in the AT assay. Potent candidates are represented by the group of B-type proanthocyanidins. Structure-activity relationships regarding the in vitro inhibitory potency of the polyphenols in the biological assays are discussed.

Giardia duodenalis is a parasite that colonizes the small intestine of various mammalian hosts especially in humans. The common treatment for giardiasis includes metronidazole, furazolidone and benzimidazole-based drugs which cause many side effects besides an increasing resistance problem [1,2]. Having in mind that Boswellia serrata Roxb. is used for the treatment of chronic inflammatory disorders and that this parasite is known to facilitate these conditions [3], the gum resin of this plant source was tested for antigiardial effects. A crude extract standardized to 85% boswellic acids reduced the viability of the parasite by ca. 65% at a concentration of 20µg/mL. At the highest concentration tested (80µg/mL), the antigiardial effect was ca. 80% based on the metabolic conversion of resazurin [4]. Metronidazole (50µg/mL) served as a positive control. In search for the active principle, the extract was subjected to HPLC separation showing two major peaks at Rt 12.3 and 14.7min, respectively. The former, comprising a complex mixture of boswellic acids, exhibited pronounced antigiardial activity at 20µg/mL, as evident from a ca. 80% reduction in parasite viability. HPLC analysis showed also the presence of oleanolic acid. Preliminary analyses proved this triterpenoid only moderately active (parasite viability ca. 65% at 45µM corresponding to 20µg/mL). This finding suggested boswellic acids as the active principle. Owing to the complexity of the fractions, the isolation of distinct boswellic acid members for antigiardial activity studies is still in progress. This is the first report on antigiardial effects of boswellic acids. Keywords: Boswellia serrata, antigiardial, boswellic acids, oleanolic acid References: 1. Gardner B and Hill D (2001) Clin Microbiol Rev 14: 114–128 2. Upcroft P and Upcroft J (2001) Clin Microbiol Rev 14: 150–164 3. Layton MA et al. (1998) Brit J Rheumatol 37: 581–583 4. Benere E et al. (2007)J Microbiol Methods 71: 101–106

Clinical trials have shown that EPs 7630, an aqueous ethanolic extract from the roots of Pelargonium sidoides, is an efficacious treatment for respiratory tract infections. A large body of in vitro studies has provided evidence for an anti-infective principle associated with activation of the non-specific immune system. However, the mode of action at the cellular and molecular level is still insufficiently defined. This study, therefore, aimed to provide further insight into the underlying principles of the therapeutic benefits of EPs 7630 under these conditions. Using BMM phi experimentally infected with intracellular bacteria, Listeria monocytogenes, incubation with EPs 7630 (1 - 30 micro increased release of NO, production of membrane bound/intra- and extracellular IL-1, IL-12 and TNF-alpha and changed the expressions of the surface markers CD40 and CD119 at an early time point post infection (6 h) in a concentration-dependent manner in most experiments. Compared with non-infected cells, the effects were more pronounced. LPS + IFN-gamma served as positive and untreated cells as negative controls. Analyses were carried out at single cell levels using flow cytometry, while ELISA was additionally utilized for monitoring secreted cytokines. Although the current data provide additional valuable information for understanding the anti-infective effects of EPs 7630, the triggered signalling pathways associated with host immune responses appear even more complex than anticipated and are evidently not shared by 'classical' immunomodulators to this extent.

Among the PELARGONIUM-based herbal remedies that are widely used in traditional medical systems in the Southern African region is a highly valued root medicine (commonly termed UMCKALOABO) of initially unknown botanical origin for the treatment of infectious conditions of the respiratory tract including tuberculosis. Nowadays, a modern aqueous-ethanolic formulation of the roots of Pelargonium sidoides (EPs 7630), developed from this traditional medicine, is successfully employed for the treatment of bronchitis. The article summarizes the fascinating story of this herbal medicine including its way to Europe, identification of the botanical origin, and provides background information of the many profound anti-infectious actions and clinical studies. In spite of considerable effort, the underlying chemical principle is still not clear.

The volatile fractions, obtained by water distillation from the leaves and bark of Hamamelis virginiana L. were analysed in detail by GC-MS. About 175 (leaves) and 168 (bark) compounds have been identified or at least partly characterized on the basis of a computerized database (SeKoMS). The dominating substances were represented by a homologous series of alkanes, alkenes, aliphatic alcohols, related aldehydes, ketones, and fatty acid esters. Importantly, significant differences in the terpenoid and phenylpropanoid patterns of the products obtained from the bark and leaves are apparent: whereas the product of bark distillation was found to typically contain phenylpropanoids and mainly sesquiterpenoids, that obtained from the leaves included some distinct monoterpenoids detected in comparably higher amounts. The chemical composition of the volatiles, when taken together with the absence of specific accumulation sites of lipophilics, emphasizes the definition "volatile fraction" rather than "essential oil".

The effects of gallic acid on the gene expressions of inducible nitric oxide synthase (iNOS) and the cytokines interleukin (IL)-1, IL-10, IL-12, IL-18, TNF-alpha, and interferon (IFN)-gamma were investigated by reverse-transcription polymerase chain reaction (RT-PCR). The experiments were performed in parallel in non-infected and in L. major-infected RAW 264.7 cells and the expression profiles were compared with those mediated by IFN-gamma plus lipopolysaccharide (LPS). The infection per se induced the expression first of IL-1 and TNF-alpha mRNA, later that of IL-10 mRNA. Gallic acid induced low and transient levels of TNF-alpha and IL-10 in non-infected cells, and it clearly enhanced and prolonged iNOS and cytokine mRNA expressions in Leishmania-parasitised cells. Interestingly, and in contrast to activation by IFN-gamma/LPS, gallic acid also stimulated Leishmania-infected cells to produce IFN-gamma mRNA. For IFN-alpha, a sandwich immunoassay was performed to determine its amount present in the supernatant of gallic acid-stimulated RAW 264.7 cells. In showing predominant stimulation of infected cells and the induction especially of IFN-gamma, a cytokine that plays a central role in antimicrobial macrophage and T cell regulation, these data provide the basis for an immunological concept of gallic acid and possibly other plant polyphenols for their beneficial effects in various infectious conditions.

The range of natural galloylhamameloses is extended by the recognition of the highly unstable novel analogues, the 1- O-(4-hydroxybenzoyl)-2',5-di- O-galloyl-alpha- D-hamamelofuranose and its 1- O-beta-anomer. The observed facile elimination of the C-1 acyl moiety is discussed and a mechanism proposed. These compounds are accompanied in the bark of HAMAMELIS VIRGINIANA by the "conventional" hamamelitannin, the related new 1,2',5-tri- O-galloyl-alpha- D-hamamelofuranose, and a tentatively characterized di- O-galloyl- D-hamamelopyranose, the first pyranose analogue, shown to be also present in commercially available hamamelitannin. The structures of these compounds were established from spectroscopic evidence of their acetate derivatives.

This special issue contains selected contributions from the plenary and keynote lectures as well as from the special workshops given at the 7th Tannin Conference (presymposium) and the 58th International Congress and Meeting of the Society for Medicinal Plant and Natural Product Research (GA), Berlin, Germany. Tannins have been the subject of extensive research for many decades. Significant advances in the elucidation of their constituents and conformations, as well as in the broad recognition of their fascinating biological, pharmacological, and physiological activities have led to increasing attention to these metabolites. Since all tannin actions very probably derive from some of their chemical features, and molecular recognition is a key step in many biological processes, conformational analyses and studies on the interaction with biological macromolecules have attracted the interest of scientists from many disciplines. This focus is reflected in contributions on the stereochemical structure determination of polyphenol complexes with caffeine (T. Ishizu et al.), the molecular interpretation of astringency (I. Pianet et al.), and in the field of biological activities, the antioxidant activity of metabolites of ellagitannins (H. Ito). The Polyphenol Community of Scientists proudly honoured Prof. Daneel Ferreira at the 7th Tannin Conference through the presentation of the 2010 Tannin Conference Award. This appreciation was in recognition of his life-long seminal contributions to the field of tannin chemistry. His comprehensive review (Ferreira and Coleman) deals with the synthesis of proanthocyanidins at a scale that permits assessment of their pharmacological activities. The isolation of chemically defined tannins in their free phenolic forms from natural sources often relies on tedious processes that have low yields. Lack of pure defined materials has considerably limited biological activity studies. The availability of effective synthetic procedures alleviates these restrictions and will significantly contribute to the research on proanthocyanidins, the compounds credited for the profound health-promoting effects of many herbal medicines and foodstuff. The use of herbal medicines for the treatment of illness and disease may be contemplated to be as old as mankind itself. There is a continuing effort to investigate the chemical and biological basis for the activities of indigenous medicinal plants. The picture that emerges from numerous scientific

The antimicrobial activity of a total of 27 tannins and related compounds was evaluated against 8 microorganisms, including 2 Gram-positive (Bacillus subtilis, Staphylococcus aureus), 4 Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis), and 2 yeasts (Candida albicans, Cryptococcus neoformans). The compounds tested were generally found to possess only weak to moderate antibacterial, but fairly high anticryptococcal activities. Attention is given to structure-activity relationships with emphasis on simple galloyl esters, hydrolyzable tannins and proanthocyanidins among this class of secondary products.

The antibacterial activity of extracts and isolated constituents (scopoletin, umckalin, 5,6,7-trimethoxycoumarin, 6,8-dihydroxy-5,7-dimethoxycoumarin, (+)-catechin, gallic acid and its methyl ester) of Pelargonium sidoides and Pelargonium reniforme (Geraniaceae), plant species used in folk medicine by the Southern African native population, was evaluated against 8 microorganisms, including 3 Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, and beta-hemolytic Streptococcus 1451) and 5 Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Haemophilus influenzae). Minimum inhibitory concentrations (MICs) varied with the preparation of the extracts and microorganisms tested, from about 0.6 mg/ml for aqueous phases to over 10 mg/ml for crude Pelargonium extracts. With the exception of the ineffective (+)-catechin, all the potentially active compounds exhibited antibacterial activities with MICs of 200-1000 micrograms/ml. The results provide for a rational basis of the traditional use of the titled Pelargonium species.

In the present study, the effects of tannins obtained from various plant sources on the activity of 5-LOX and AT were examined. With IC50 values ranging from 1.0 to 18.7 muM, hamamelitannin and the galloylated proanthocyanidins isolated from Hamamelis virginiana L. were found to be most potent inhibitors of 5-LOX. Unlike the 5-LOX study, hamamelitannin proved to be ineffective in the AT assay. Potent candidates are represented by the group of B-type proanthocyanidins. Structure-activity relationships regarding the in vitro inhibitory potency of the polyphenols in the biological assays are discussed.