Aqueous extract from Orthosiphon stamineus leaves prevents bladder and kidney infection in mice.

BACKGROUND: Extracts from the leaves of Orthosiphon stamineus are used in phytotherapy for treatment of uncomplicated urinary tract infections. PURPOSES: Evaluation of an aqueous extract against infection with uropathogenic Escherichia coli in vivo; investigation of underlying microbiological mechanisms. STUDY DESIGN: In vivo studies in mice and in vitro investigations on cytotoxicity, antiadhesive potential, influence on bacterial gene expression and quorum sensing. METHODS: Extract OWE was prepared by hot water extraction. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of OWE on bacterial load in bladder/kidney tissue was monitored in pre- and posttreatment. Cytotoxicity of OWE against different UPEC strains, T24 bladder/A498 kidney cells, gene expression analysis, monitoring of phenotypic motility and quorum sensing was investigated by standard methods of microbiology. RESULTS: OWE was quantified (UHPLC) according to the content of rosmarinic acid, cichoric acid, caffeic acid. Three- and 5-day treatment of animals with OWE (750mg/kg) after transurethral infection with UPEC CFT073 reduced the bacterial load in bladder and kidney, similar to norfloxacin. Four- and 7-day pretreatment of mice prior to the infection with UPEC NU14 reduced bacterial bladder colonization. In vitro investigations indicated that OWE (≤2mg/ml) has no cytotoxic or proliferation-inhibiting activity against different UPEC strains as well as against T24 bladder and A498 kidney cells. OWE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. OWE reduced gene expression of fimH, but evoked increase of the expression of motility/fitness gene fliC. Increase of bacterial motility on gene level was confirmed by a changed bacterial phenotype by an increased bacterial motility in soft agar assay. OWE inhibited in a concentration-dependent manner bacterial quorum sensing. CONCLUSION: OWE is assessed as a strong antiadhesive plant extract for which the traditional use in phytotherapy for UTI might be justified.

Phytochemical characterization and in vitro wound healing activity of leaf extracts from Combretum mucronatum Schum. & Thonn.: Oligomeric procyanidins as strong inductors of cellular differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Combretum mucronatum Schum. & Thonn. are traditionally used for wound healing in Western Africa. Aqueous and hydroalcoholic extracts of the dried leaves recently have been shown to stimulate viability of human keratinocytes and dermal fibroblasts. AIM OF THE STUDY: Phytochemical characterization of the herbal material, development of a validated HPLC methodology for quality control, and pinpointing the underlying pharmacological mechanism under in vitro conditions to understand the impact of C. mucronatum extracts on human skin cells. MATERIALS AND METHODS: Extracts obtained from the leaves from C. mucronatum by using solvents with different polarities (petrol ether, dichloromethane, ethanol-water 50%, water) were investigated concerning phytochemical composition by GC-MS, LC-MS and in part after fractionation and isolation of purified compounds. For quality control of the herbal material an ICH-2 validated UHPLC method was developed for quantification of the lead compounds epicatechin, procyanidin B2, vitexin and isovitexin. In vitro studies were performed using HaCaT keratinocyte cell line, primary keratinocytes and primary skin fibroblasts with determination of viability (MTT assay), cell proliferation (BrdU incorporation ELISA), cell toxicity (LDH release) and keratinocyte differentiation, using involucrin and keratin K10 as differentiation marker (confocal laser scanning microscopy, Western blot). RESULTS: A detailed phytochemical composition analysis of the extracts from the leaves from C. mucronatum was performed (compounds 1-34) and epicatechin, procyanidin B2, vitexin and isovitexin are assessed to be the lead compounds of the polar extract. Quantitative UHPLC investigations indicated mature leaves to have higher polyphenol content in comparison to young leaves. The drying process of the plant material was shown to have great influence on the content of the lead compounds. The aqueous extract (0.1-100µg/mL) did not change cell viability of dermal fibroblasts and keratinocytes but inhibited cellular proliferation rates significantly at 100µg/mL. The extract stimulated cellular differentiation of primary keratinocytes significantly at 1 and 10µg/mL. Procyanidin B2 at 1 and 10µM was shown to be responsible for the induction of this cellular differentiation, while epicatechin, and procyanidins B5, C1 and D1 were inactive. CONCLUSION: The in vitro effects of the aqueous extract on the skin cells rationalized the remedial effect in wound healing and possibly accounts for the reason why this plant may be widely used for this purpose. On the basis of this study extracts from the leaves of C. mucronatum therefore have potential for the use in wound healing.

Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.).

Proanthocyanidins (PAs) constitute a class of polyphenols with flavan-3-ols as monomeric building blocks. These polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of monomeric flavan-3-ols or low molecular oligomers as lead compounds. No reliable analytical methods are available for unambiguous identification of the homologues series of oligo- and polymeric PAs. For Hawthorn leaf and flower (Crataegi folium cum flore) from Crataegus spp. (Rosaceae) a protocol for preparative isolation of oligomeric and polymeric PAs from an acetone-water extract was developed, yielding procyanidin reference clusters with defined degree of polymerization (DP) from 2 to 10 besides a procyanidin-polymer. Identity and purity of these clusters were proven by HPLC, MS and in part NMR studies. For identification and quantification from Hawthorn an ICH-Q2 validated UHPLC method with fluorimetric detection and less than 10min runtime was developed. The method enabled quantification of procyanidin clusters with DP from 2 to 10 besides the polymer fraction. Batch analysis revealed procyanidin contents of about 20 to 45mg/g from a homologues series of oligomeric PAs and about 50% of polymer fraction. Monitoring of procyanidin distribution during seasonal growth of fresh plants of Crataegus monogyna showed more or less constant contents between 20 and 55mg/g dry weight of oligomeric procyanidins during the growing season in the different plant organs with strong accumulation in the flowers and fruits (55mg/g dry weight). From these data it can be speculated that procyanidins serve as part of the plants defense system in the reproductive organs of the plant.

Is the alkaloid pipermethystine connected with the claimed liver toxicity of Kava products?

The pyridone alkaloid pipermethystine has been considered to be responsible for alleged hepatoxicity of Kava products. Investigation of a series of retain samples of finished products from the German market and self-produced extracts from root and stem material of Piper methysticum clearly showed that pipermethystine (1) is absent from all root and retain samples and extracts, with a limit of quantification of 45 ppm. As a positive control, leaves of P. methysticum showed an amount of 0.2% of 1. Thus, if there is any hepatotoxicity, compound 1 should not be the responsible constituent in the case reports with ethanolic extracts produced in Germany.

Evaluation of analytical markers characterising different drying methods of parsley leaves (Petroselinum crispum L.).

Drying process of parsley leaves from Petroselinum crispum L. can influence the sensory qualities and aromatic taste of this herbal product. Beside oven-dried material, freeze-dried parsley is getting increasingly into the market. In the course of a search for analytical tools to differentiate oven-dried and lyophilised parsley, a HPLC determination of the 6"-O-malonylapiin to apiin ratio was shown to be a suitable marker system. While the ratio is high for fresh and lyophilised leave material, oven-drying leads to demalonylation and, subsequently, to a low malonylapiin--apiin ratio. Additionally, L*a*b colour measurement can be used for quality control to differentiate between different dried parsley raw materials.

Cyanoglucosides from Osmaronia cerasiformis (Rosaceae).

Three nitrile glucosides have been isolated from a methanolic extract of the leaves of Osmaronia cerasiformis (Torr. & Gray) Greene (Rosaceae, Prunoideae). One is the known sutherlandin (Z-4-beta-D-glucopyranosyloxy-3-hydroxymethylbut-2-ene nitrile); the second is Z-4-beta-glucopyranosyloxy-3-methylbut-2-ene nitrile, which has recently been characterized but whose stereochemistry was not previously determined; the Z-isomer identified here was named osmaronin. The third is the new 4-beta-D-glucopyranosyloxy-2R,3R-epoxy-3-methylbutyronitrile (osmaronin epoxide). The nitrile glucosides occur in the leaves and flowers of the title plant; their aglycone is apparently derived from the aliphatic amino acid L-leucine.