Phytochemical and functional analysis of horseradish (Armoracia rusticana) fermented and non-fermented root extracts.

BACKGROUND: The roots of horseradish (Armoracia rusticana) are used for infections of respiratory airway and for urinary tract infections due to isothiocyanates (ITC), enzymatically formed during fermentation of glucosinolates by myrosinase. HYPOTHESIS/PURPOSE: The present study aims to present a comprehensive overview on the phytochemical composition of A. rusticana roots, especially concerning isothiocyanates and respective glucosinolates. The complex flavonoid spectrum of the herbal material is reviewed. Published data on in vitro activity of horseradish extracts and isolated compounds are summarized. These data indicate well-established use of horseradish as an antibacterial remedy against bacterial infections of the airway and urinary tract. STUDY DESIGN: To answer the question if other compounds from A. rusticana beside ITC contribute to the antibacterial activity, non-targeted LC-MS studies were performed with fermented and non-fermented horseradish extracts, and detailed phytochemical profiles were established. RESULTS: Comparative investigations on the antibacterial activity indicated that only ITC-containing extracts and fractions exert antibacterial activity. The huge variety of non-ITC compounds do not significantly contribute to the antibacterial activity, but can be used for analytical characterisation and quality control of the herbal material. Detailed phytochemical analysis additionally revealed a variety of compounds, not described until now for horseradish roots: the flavonol glycosides kaempferol-3-O-ß-d-xylopyranosyl-(1''' â†’ 2'')-ß-d-galactopyranoside, kaempferol-3-O-α-l-rhamnopyranosyl-(1''' â†’ 6'')-ß-d-glucopyranoside, kaempferol-3-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-7-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-(1'''' â†’ 2''')-ß-d-galactopyranoside-7-O-ß-d-glucopyranoside, the oxo-indole derivative spirobrassinin, the phenylthiazole 2-methylsulfanyl-4-phenyl-4,5-dihydro-1,3-thiazole, a series of lysophophatidylethanolamine and 13 different N-phenylpropenoyl-L-amino acids. CONCLUSION: The antibacterial effects of horseradish are only due to the presence of glucosinolates resp. the corresponding ITC, and the detailed overall composition of horseradish extracts has been reported.

Aqueous extract from Equisetum arvense stimulates the secretion of Tamm-Horsfall protein in human urine after oral intake.

BACKGROUND: Within European traditional phytotherapy, extracts from different herbal plants are used for prevention and therapy of uncomplicated urinary tract infections and for flushing out of kidney grits. Besides increased urine flow by slight diuretic effects, also stimulation of Tamm-Horsfall protein (syn. THP, uromodulin) in the distal part of the kidney could explain reduced kidney gravel and anti-virulent activity against uropathogenic E. coli. PURPOSES: Evaluation of THP-inducing activity of extracts from Equisetum arvense, Levisticum officinalis, Ilex paraguariensis, Juniperus communis, Urtica dioica, and Taraxacum officinale by quantification of THP in urine samples after oral application to humans. STUDY DESIGN: 7 days p.o. application of the test intervention to healthy volunteers (n = 10 per intervention group) and analysis of urine samples at day 1 (untreated control values), and days 3, 6 and 8 on THP content by validated ELISA. Antiadhesive activity of urine samples was monitored by flow cytometry using UPEC strain NU14 against human T24 bladder cells. RESULTS: An aqueous extract from E. arvense, fully characterized by a specific LC-MS method, induced THP concentration in urine samples significantly during a 7-day p.o. application up to 300%, related to the untreated controls. Ex vivo investigation of the individual and pooled urine samples with elevated THP concentrations showed good correlation to antiadhesive effects against UPEC NU14 to T24 cells. Urine samples of the Equisetum treated volunteers had no effect on the proliferation and on biofilm formation of UPEC NU14. Silica excretion in the urine samples had no correlation to the respective THP levels. Monitoring of electrolyte content in the urine samples indicat ed diuretic effects of the intervention with Equisetum extract. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 80 compounds from the extract by MS evaluations and 18 compounds by UV detection. This protocol will provide a valuable tool for future quality control of Equisetum extract. CONCLUSION: Aqueous extract from E. arvense significantly stimulates THP secretion in urine samples after 7 days of oral intake and inhibits the interplay between UPEC and bladder host cells. This could explain the therapeutic use of this herbal material for urinary tract infections and kidney gravel. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 82% of all eluted compounds. This protocol will provide a valuable tool for future quality control of Equisetum extract.

Cyanogenesis in Aralia spinosa (Araliaceae).

A systematic survey of Aralia spinosa (Araliaceae), covering an entire growing season and including aboveground organs at various developmental stages, revealed that only about half of all samples collected showed cyanogenesis. Cyanogenesis was detected in inflorescences and leaves but is apparently restricted to certain harvest times or developmental stages. The structurally unusual triglochinin, characterized by a hex-2-enedioic acid partial structure, was the only cyanogenic glycoside detected. This is the first description of triglochinin in this species and in the family of Araliaceae. Triglochinin is biogenetically derived from tyrosine, which is in good agreement with the few cyanogenic glycosides previously detected in members of the Araliaceae family. Triglochinin was identified, characterized, and quantified by modern chromatographic methods, and the amount of enzymatically releasable hydrocyanic acid was determined qualitatively and quantitatively. Two isomers of triglochinin were detected chromatographically at minor levels. The isomeric pattern agreed well with literature data from other triglochinin-containing plants. This was confirmed in the two species, Triglochin maritima and Thalictrum aquilegiifolium, which were comparatively studied. In the case of A. spinosa, inflorescence buds harvested in July showed the highest content of triglochinin, just under 0.2% on a dry weight basis. The detection of triglochinin adds to the knowledge of toxicological properties and the dereplication of U(H)PLC/MS² data provides a comprehensive phytochemical profile of A. spinosa.

Cytotoxicity, genotoxicity, and gene expression changes induced by methanolic extract of Moringa stenopetala leaf with LC-qTOF-MS metabolic profile.

Moringa stenopetala (Baker f.) Cuf.and other Moringa species have traditionally been used to treat various diseases. The purpose of this study was to determine the cytotoxic and genotoxic effects of the methanolic extract of M. stenopetala leaf and its fractions on selected tumor cells. Cytotoxicity was determined by MTT assay. The comet assay was used toassess DNA damage, and gel electrophoresis was used to determine DNA fragmentation. Gene expression was analyzed by qPCR using two specific genes for each cancer cell line. Fractionation of the methanolic extract (E-1) on Diaion HP-20 yielded five fractions (Fr-2 to Fr-6); only Fr-4 and Fr-6 were cytotoxic to breast cancer cells (MCF-7; IC50 = 58.3 ± 0.93 and 35.8 ± 2.44 µg/mL, respectively), human hepatocellular carcinoma cells (HepG2; IC50 = 57.8 ± 1.57 and 39.3 ± 1.90 µg/mL, respectively), and Fr-4 was cytotoxic to human colon cancer cells (HCT-116; IC50 = 94.2 ± 4.9 µg/mL). In addition, exposure of the cancer cells to Fr-4 and Fr-6 resulted in a high level of DNA damage. Moreover, relative expression of MTAP and CDKN2A in MCF-7 were increased, whereas expression of p21 and p53 in HCT-116, and APC and TERT in HepG2 were decreased, similar to that of doxorubicin. LC-qTOF-MS was used to identify metabolites in E-1, the majority of which were enriched in Fr-4. Two terpenes (loliolide and dihydroactinidiolide), the majority of the flavonoids, and niazirin were about two fold enriched in Fr-4, whereas the majority of the lipids were 4-10 fold enriched. However, Fr-6 hardly showed compounds other than the two terpenes that were enriched 1.5 and 7 fold. The findings suggest that Fr-4 and Fr-6 are promising sources of compounds possessing cytotoxic and genotoxic properties.

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.

Root Extracts From Ononis spinosa Inhibit IL-8 Release via Interactions With Toll-Like Receptor 4 and Lipopolysaccharide.

Extracts from the roots of Ononis spinosa L. (restharrow roots) are traditionally used for the treatment of patients with urinary tract infections due to its mild diuretic activity, caused by the inhibition of renal human hyaluronidase-1 by isoflavonoids. Preliminary studies also indicated anti-inflammatory effects. The following study aimed at investigating potential anti-inflammatory effects of restharrow extracts, prepared with solvents of different polarity. A dichloromethane extract (OS1), mainly composed of isoflavonoids and triterpenes as characterized by LC-MS, showed a concentration-dependent (25-100 µg/ml) inhibition of IL-8 and TNF-α release from LPS-stimulated human neutrophils. Significant inhibition was also found for the triterpene α-onocerin and the norneolignan clitorienolactone B, isolated from OS1. Further, OS1 and both compounds significantly decreased the expression of the adhesion molecules CD11b/CD18 and conversely increased the expression of CD62L in LPS-stimulated human neutrophils. This finding corresponds to a reduced inflammatory response by the inhibition of adhesion and migration of immune cells. As all of the observed effects are potentially mediated via Toll-like receptor 4 (TLR4) signaling, TLR4 transfected HEK293 cells were incubated with OS1. LPS-induced IL-8 secretion was significantly inhibited in a concentration-dependent manner, confirming TLR4 antagonism. This inhibition, however, was in part caused by an interaction of OS1 with LPS. In addition, also an aqueous extract containing high amounts of isoflavonoid glycosides and saponins from the roots of O. spinosa showed anti-inflammatory effects by interacting with the TLR4 signaling pathway. This study rationalizes the traditional use of extracts from O. spinosa for therapy of urinary tract infections, due to its potential anti-inflammatory effects that are mediated via TLR4 receptor antagonism.

Antiadhesive natural products against uropathogenic E. coli: What can we learn from cranberry extract?

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from Cranberry fruits (Vaccinium macrocarpon) are traditionally used against urinary tract infections, mainly due to antiadhesive activity against uropathogenic E. coli (UPEC), but the exact mode of action and compounds, responsible for the activity, are unknown. AIM OF THE STUDY: i. To investigate if cranberry extract acts only by a single component or must be assessed as a multi-active-compound preparation; ii to screen isolated cranberry-related natural products under in vitro conditions to pinpoint natural products with antiadhesive effects against UPEC, followed by in silico calculations (QSAR) to predict potential antiadhesive compounds; iii. investigations by using urine samples from cranberry treated volunteers for evaluation on the bacterial transcriptome and the mannose-binding side of FimH, iv. to investigate if besides Tamm Horsfall Protein induction in the kidney, the extract acts also directly against UPEC. MATERIAL AND METHODS: Antiadhesive activity of 105 compounds was determined by flow cytometric adhesion assay (UPEC UTI89 on T24 bladder cells). Urine samples from 16 volunteers treated with cranberry extract (p.o., 7 days, 900 mg/day) were used for ex vivo testing concerning influence on the bacterial transcriptome (Illumina RNA-seq) and interaction with the mannose binding domain of type-1 fimbriae. RESULTS: i. The antiadhesive effect of cranberry extract cannot be attributed to a single compound or to a single fraction. ii. Unglycosylated flavones and flavonols with bulky substitution of the B ring contribute to the antiadhesive activity. 3'-8″-biflavones and flavolignans (not related to cranberry fruits) were identified as potent antiadhesive compounds against UPEC. iii. QSAR yielded a model with good statistical performance and sufficient internal and external predictive ability. iv. Urine samples from male cranberry-treated volunteers indicated significant interaction with the mannose binding domain of type-1 fimbriae, which correlated with the amount of Tamm-Horsfall Protein in the test samples. v Cranberry extract did not influence the UPEC transcriptome; gene expression of bacterial adhesins (P-, S-fimbrae, curli) was not influenced by the urine samples, while a slight, but non-significant upregulation of type 1 fimbriae was observed. CONCLUSIONS: B-ring substituted flavones and flavonols from cranberry contribute to the antiadhesive activity against UPEC by inhibition of the FimH-mediated interaction with the host cell bladder epithelium.

Aqueous Root Extract from Ononis spinosa Exerts Anti-adhesive Activity against Uropathogenic Escherichia coli.

Extracts from Ononis spinosa are traditionally used for urinary tract infections due to diuretic and anti-inflammatory activity. A potential influence on the virulence of uropathogenic Escherichia coli has not been investigated until now. The following study aimed to investigate the influence of an aqueous O. spinosa extract on uropathogenic E. coli and uropathogenic E. coli host cell interaction. A hot water extract from the roots of O. spinosa (O. spinosa extract) was characterized by LC-qTOF-MS. The influence of O. spinosa extract on the proliferation of uropathogenic E. coli UTI89 and on cell viability against human T24 bladder cells was investigated. Anti-adhesive activity of O. spinosa extract was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled UTI89 to T24 bladder cells. Internalization of uropathogenic E. coli into T24 cells was monitored by an invasion assay. O. spinosa extract was characterized by the presence of isoflavones, isoflavanones, licoagrosides, pterocarpans, tartaric acid derivatives, and saponines. O. spinosa extract had no influence on the proliferation of uropathogenic E. coli (125 - 1000 µg/mL) and did not influence the cell viability of T24 cells. Bacterial adhesion to T24 cells was significantly (p > 0.001) inhibited by O. spinosa extract in a concentration-dependent manner (125 - 1000 µg/mL) during coincubation. Preincubation of uropathogenic E. coli or T24 cells with O. spinosa extract reduced bacterial adhesion, but to a lower extent than during coincubation. Consequently, the reduced bacterial adhesion also leads to a reduced internalization of uropathogenic E. coli uropathogenic E. coli into the host cell. O. spinosa extract does not interact with FimH-mediated uropathogenic E. coli adhesion to host cells. From these data, the traditional use of O. spinosa extracts for urinary tract infections seems to be rationalized.

An Unsuccessful Attempt to Confirm the Occurrence of 4'-O-ß- d-Glucosyl-9-O-(6″-deoxysaccharosyl)olivil in Valerian Root.

The lignan 4'-O-ß- d-glucosyl-9-O-(6″-deoxysaccharosyl)olivil had previously been discovered in a methanolic extract of valerian root (Valeriana officinalis agg.) and characterized as a potent partial agonist at the A1 adenosine receptors. Today, countless scientific sources, webpages, and press articles mention this compound and discuss it as an active constituent for the sedative effect of this herbal drug. As no second report confirmed the occurrence of this lignan in valerian root during the 20 years since its first description in 1998, we intended to re-prove its presence by means of LCMS using other genuine or added lignans as a quantitative benchmark. Whilst those lignans were clearly detectable in methanolic valerian extracts of all six investigated batches of valerian root, no positive proof of 4'-O-ß- d-glucosyl-9-O-(6″-deoxysaccharosyl)olivil was achieved. Our result suggests that this compound does not occur regularly in valerian root in the amounts expected from the single report on the occurrence of this compound.

Crataegus Extract WS®1442 Stimulates Cardiomyogenesis and Angiogenesis From Stem Cells: A Possible New Pharmacology for Hawthorn?

Extracts from the leaves and flowers of Crataegus spp. (i.e., hawthorn species) have been traditionally used with documented preclinical and clinical activities in cardiovascular medicine. Based on reported positive effects on heart muscle after ischemic injury and the overall cardioprotective profile, the present study addressed potential contributions of Crataegus extracts to cardiopoietic differentiation from stem cells. The quantified Crataegus extract WS®1442 stimulated cardiomyogenesis from murine and human embryonic stem cells (ESCs). Mechanistically, this effect was found to be induced by promoting differentiation of cardiovascular progenitor cell populations but not by proliferation. Bioassay-guided fractionation, phytochemical and analytical profiling suggested high-molecular weight ingredients as the active principle with at least part of the activity due to oligomeric procyanidines (OPCs) with a degree of polymerization between 3 and 6 (DP3-6). Transcriptome profiling in mESCs suggested two main, plausible mechanisms: These were early, stress-associated cellular events along with the modulation of distinct developmental pathways, including the upregulation of brain-derived neurotrophic factor (BDNF) and retinoic acid as well as the inhibition of transforming growth factor ß/bone morphogenetic protein (TGFß/BMP) and fibroblast growth factor (FGF) signaling. In addition, WS®1442 stimulated angiogenesis ex vivo in Sca-1+ progenitor cells from adult mice hearts. These in vitro data provide evidence for a differentiation promoting activity of WS®1442 on distinct cardiovascular stem/progenitor cells that could be valuable for therapeutic heart regeneration after myocardial infarction. However, the in vivo relevance of this new pharmacological activity of Crataegus spp. remains to be investigated and active ingredients from bioactive fractions will have to be further characterized.

Lignans and sesquiterpene lactones from Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae).

Four undescribed lignans and two undescribed sesquiterpenic acids, together with three known compounds (hypochoeroside C, hypochoeroside D, and 5-O-caffeoylshikimic acid) were isolated from the roots of Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae). The lignans were identified as 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranosyl-2'-O-methacrylate, (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one, and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid. The two sesquiterpenic acids were identified as the ring open precursors of hypochoerosides C and D. Structures were elucidated using NMR and HRMS. Absolute configurations of (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid were determined using electronic circular dichroism (ECD) spectroscopy. 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside was evaluated for its anti-proliferative activity against myeloma cell lines MM1S, U266, and NCI-H929 and showed cytotoxicity at 100 mM against MM1S strain. No neurotoxicity was observed for major compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, and hypochoeroside D in a fluorescence assay measuring neurite outgrowth in dorsal root ganglion (DRG) neurons. Additionally, compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, hypochoeroside D, and hypochoerosidic acid D were quantified in unstressed and drought-stressed plants using HPLC-DAD. Drought-stressed plants were found to contain lower concentrations of the lignan 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside and sesquiterpene lactone hypochoeroside C.

Influence of Cranberry Extract on Tamm-Horsfall Protein in Human Urine and its Antiadhesive Activity Against Uropathogenic Escherichia coli.

LC-MS characterized cranberry extract from the fruits of Vaccinium macrocarpon inhibited under in vitro conditions the bacterial adhesion of Escherichia coli strain 2980 uropathogenic E. coli (UPEC strains UTI89, NU14) to T24 bladder cells and adhesion of UPEC strain CFT073 to A498 kidney cells in a concentration-dependent manner. Within a biomedical study, urine samples from 16 volunteers (8 male, 8 female) consuming cranberry extract for 7 d (900 mg/d) were analyzed for potential antiadhesive activity against UPEC by ex vivo experiments. Results indicated inhibition of adhesion of UPEC strain UTI89 to human T24 bladder cells. Subgroup analysis proved significant inhibition of bacterial adhesion in case of urine samples obtained from male volunteers while female urine did not influence the bacterial attachment. Differences between antiadhesive capacity of urine samples from male/female volunteers were significant. Protein analysis of the urine samples indicated increased amounts of Tamm-Horsfall protein (THP, syn. uromodulin) in the active samples. Inhibition of bacterial adhesion by the urine samples was correlated to the respective amount of THP. As it is known that THP, a highly mannosylated glycoprotein, strongly interacts with FimH of UPEC, this will lead to a decreased interaction with uroplakin, a FimH-binding transmembrane protein of urothelial lining cells. From these data it can be concluded that the antiadhesive effect of cranberry after oral intake is not only related to the direct inhibition of bacterial adhesins by extract compounds but is additionally due to an induction of antiadhesive THP in the kidney.

Antidiabetic Activities of an LC/MS Fingerprinted Aqueous Extract of Fagonia cretica L. in Preclinical Models.

Diabetes mellitus is a chronic disease and one of the most important public health challenges facing mankind. Fagonia cretica is a medicinal plant used widely in the Punjab in Pakistan. A recent survey has demonstrated that traditional healers and herbalists frequently use this plant to treat diabetes. In the current study, the traditional medicine was prepared as a tea, and the profile of the main metabolites present in the traditional medicine was analysed via LC/MS/MS. The extract was shown to contain a number of phenolic glycosides including quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glycoside, kaempferol-3(6'-malonylglucoside), isorhamnetin-3-O-rutinoside, and isorhamnetin 3-(6″-malonylglucoside) in addition to two unidentified sulphonated saponins. The traditional medicine inhibits α-glucosidase in vitro with an IC50 of 4.62 µg/mL. The hypoglycaemic effect of the traditional medicine was evaluated in normoglycaemic and streptozotocin-treated diabetic rats, using glibenclamide as an internal control. The preparation (250 or 500 mg/kg body weight) was administered once a day for 21 consecutive days. The dose of 500 mg/kg was effective in the management of the disease, causing a 45 % decrease in the plasma glucose level at the end of the experimental period. Histological analysis of pancreatic sections confirmed that streptozotocin/nictotinamide treatment caused destruction of pancreatic islet cells, while pancreatic sections from the treatment groups showed that both the extract and glibenclamide partially prevented this deterioration. The mechanism of this protective effect is unclear. However, such a finding suggests that ingestion of the tea could confer additional benefits and should be investigated further.

Phytochemical Characterization of Low Molecular Weight Constituents from Marshmallow Roots (Althaea officinalis) and Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase-1.

Extract RE was obtained from the roots of Althaea officinalis in a yield of 8.1%, related to the dried plant material, by extraction with MeOH-H2O (1:1), followed by precipitation with EtOH to remove high molecular weight constituents. Phytochemical investigation of RE revealed the presence of N-phenylpropenoyl-l-amino acid amides 1-5, 8% glycine betaine 6, about 9% total amino acids with proline as the main compound, and about 61% mono- and oligomeric carbohydrates with sucrose as the main compound. Further fractionation revealed the presence of a hypolaetin diglycoside (12) and four hypolaetin glycosides (7-9 and 11) with O-sulfocarbohydrate moieties; additionally, 4'-O-methylisoscutellarein-8-O-ß-d-(3″-O-sulfo)glucuronopyranoside (10) and the diglycosylated coumarin haploperoside D (13) were identified. The hypolaetin-O-sulfoglycosides 7-10 are new natural products. RE inhibited the enzymatic activity of surface-displayed human hyaluronidase-1 on Escherichia coli F470 cells with an IC50 of 7.7 mg/mL. RE downregulated mRNA expression of hyal-1 in HaCaT keratinocytes at 125 and 250 µg/mL, respectively. These data contribute to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy of irritated and inflamed buccal tissue and cough.

Traditionally used medicinal plants against uncomplicated urinary tract infections: Hexadecyl coumaric acid ester from the rhizomes of Agropyron repens (L.) P. Beauv. with antiadhesive activity against uropathogenic E. coli.

The rhizomes from Agropyron repens are traditionally used for the treatment of uncomplicated urinary tract infections. Extracts prepared with solvents of different polarity did not show any cytotoxic effects against different strains of uropathogenic E. coli (UPEC) and human T24 bladder cells under in vitro conditions. Significant antiadhesive activity against the bacterial attachment to human T24 bladder cells was found for an acetone extract (AAE) at concentrations >250µg/mL. More hydrophilic extracts did not influence the bacterial attachment to the eukaryotic host cells. Bioassay guided fractionation of AAE led to the identification of (E)-hexadecyl-3-(4-hydroxyphenyl)-acrylate (hexadecyl-coumaric acid ester) 1 as the compound responsible for inhibiting the UPEC adhesion to T24 bladder cells. 1 reduced the bacterial invasion into the bladder cells as shown by a specific invasion assay. Additionally, 1 was obtained by chemical synthesis, and also the synthetic structural analogs 2 and 3 were tested for their potential antiadhesive activity, indicating that a shorter alkyl chain at the ester function as well as the lack of hydroxylation of the phenyl moiety will abolish the antiadhesive activity.

Occurrence of Benzoic Acid Esters as Putative Catabolites of Prunasin in Senescent Leaves of Prunus laurocerasus.

Prunus laurocerasus is an evergreen shrub containing large quantities of the cyanogenic glycoside prunasin (1) in its leaves, which decomposes to prunasin amide (2) or glucose-1-benzoate (4) when the leaves become chlorotic as a result of senescence or pseudosenescence. This study was aimed at the systematic identification of senescence-associated metabolites to contribute further insight into the catabolism of 1. LC-ESIMS profiles of senescent and green leaves were analyzed by principal component analysis. In senescent leaves, the concentrations of 36 compounds were increased significantly including several benzoic acid derivatives, of which prunasin amide-6'-benzoate (5) and prunasin acid-6'-benzoate (6) were isolated and identified. The observed metabolic changes were also induced by treatment of P. laurocerasus shrubs with exogenous ethylene. The data presented support an oxidative catabolism of 1 without release of hydrogen cyanide and the remobilization of its nitrogen in the course of senescence. The results are discussed in the context of functional diversification and drug discovery, where senescent plant material represents a widely unexplored source for the discovery of natural products.

Antiviral activity of hydroalcoholic extract from Eupatorium perfoliatum L. against the attachment of influenza A virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Eupatorium perfoliatum have been traditionally used by American natives as a treatment for fever and infections. Also modern phytotherapy in Europe documents the use of hydroalcoholic extracts of this herbal material for the treatment of infections of the upper respiratory tract. AIM OF THE STUDY: The aim of the present study was to characterize the anti-influenza A virus (IAV) potential of extracts derived from the aerial parts of E. perfoliatum and to identify their antiviral mode of action and potential active fraction's compounds of the extract. MATERIALS AND METHODS: The inhibitory effects of extracts obtained by different organic solvents with different polarities on the cytopathic effect induced by IAV replication was determined in a Madin-Darby Canine Kidney Epithelial (MDCK II) cell-based assay measuring cell viability by MTT stain (MTTIAV assay). Plaque reduction assays were used for investigation of antiviral activity. The mode of action was investigated by different incubation and treatment cycles as well as hemagglutination inhibition assays. Influence of the test extract on tumor necrosis factor (TNF-α) and epidermal growth factor (EGF)-induced cell signaling was analyzed in human lung epithelial (A549) cells. Analytical characterization of extract and fractions obtained from the extract was performed by UHPLC-MS. RESULTS: Hydroalcoholic extracts from the aerial parts of E. perfoliatum were shown to inhibit growth of a clinical isolate of IAV(H1N1)pdm09 I1 and the influenza virus A/Puerto Rico/8/34 (PR8; H1N1) with a half-maximal inhibitory concentration (IC50) of 7µg/mL and 14µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 52 and 26, respectively. Extracts prepared with dichloromethane and methanol were inactive. At concentrations >1-10µg/mL of the hydroalcoholic extract plaque formation of IAV(H1N1)pdm09 was abrogated. The extract was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. The extract blocked attachment of IAV and interfered with virus-induced hemagglutination. TNF-α-induced signal transduction in A549 cells was not affected, while the EGF-induced signaling to phosphorylated ERK was slightly upregulated by the extract. Bioassay-guided fractionation and subsequent LC-MS analysis indicated that the antiviral activity might be due to polyphenolic compounds with higher molecular weights, which strongly interact with stationary phases of different chromatographic systems. These still unknown compounds with probably high molecular weight could not be isolated in the present study. A variety of different flavonoid glycosides and caffeoyl quinic acids obtained from E. perfoliatum did definitely not contribute to the antiviral effect of the extract and its respective fractions. CONCLUSION: Hydroalcoholic extracts from the aerial parts of E. perfoliatum and its main active polyphenolic constituents protect cells from IAV infection by inhibiting viral attachment to the host cells. The extract appears to be a promising expansion of the currently available anti-influenza agents.

Flavonoid glycosides from Olax mannii: Structure elucidation and effect on the nuclear factor kappa B pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Olax mannii Oliv. (Olacaceae) is among the many medicinal plants used in Nigeria for the ethnomedicinal management of both cancer and inflammation. Such plants represent potential sources of innovative therapeutic agents for the treatment of cancer and other malignant disorders. While the majority of medicinal plants exert their anticancer effects by direct cytotoxicity on tumor cells, it is important that other mechanisms through which these plants can exhibit anticancer effects are investigated. Preliminary studies indicated that Olax mannii leaves are rich sources of novel flavonoid glycosides. The detailed chemistry as well the mechanisms through which these flavonoid constituents may exert their cancer chemo-preventive and therapeutic effects are, however, not yet investigated. AIM OF THE STUDY: The aim of this study is to carry out a detailed chemical investigation of Olax mannii leaves and the effects of the isolated constituents on the nuclear factor kappa B (NF-κB) pathway. MATERIALS AND METHODS: A methanol leaf extract was subjected to various chromatographic separations to achieve isolation of flavonoid glycosides and the structures of the isolated compounds were elucidated by a combination of 1D and 2D NMR and high resolution mass spectrometry. Biological activities were assessed by measurement of cellular viability and proliferation using quantitative IncuCyte videomicroscopy, trypan blue staining and by quantification of the number of metabolically active K562 cells based on quantitation of ATP. The effect of the compounds on the inhibition of the NF-κB pathway as well as toxicity towards peripheral blood mononuclear cells to evaluate differential toxicity was also assayed. RESULTS: Chemical investigation of the methanol leaf extract of the plant material led to the isolation of three new flavonoid triglycosides, kaempferol 3-O-[α-D-apiofuranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (1), kaempferol 3-O-[ß-D-glucopyranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (2), kaempferol 3-O-[ß-D-arabinopyranosyl-(1→4)-α-L-rhamnopyranoside]-7-O-α-L-rhamnopyranoside (3), in addition to fourteen known flavonoid glycosides (4-17). Of all the tested compounds, only compound 9 (kaempferol 3-O-α-L-rhamnopyranoside) exhibited promising and specific antiproliferative activity on human K562 chronic myelogenous leukemia cells and dose-dependently inhibited NF-κB transactivation. CONCLUSION: The presence of this flavonoid glycoside and derivatives may account for the reported efficacy of Olax mannii leaf extract in the ethnomedicinal management of cancer and inflammation.