Rein Bos

Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

ABSTRACT The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

Analytical aspects of phytotherapeutic valerian preparations

A high performance liquid chromatographic method combined with diode array detection is described by which the valerian constituents valtrate, isovaltrate, acevaltrate, didrovaltrate, isovaleroxyhydroxydidrovaltrate, valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid, as well as the valepotriate decomposition products baldrinal and homobaldrinal, can be separated and identified simultaneously. Using this procedure, roots of Valeriana officinalis, which are used for the production of phytomedicines, were analysed. The influence of different ethanol:water mixtures, used as extraction liquid, on the composition of extracts of V. officinalis is reported. The analytical procedure was also applied to a number of valerian-containing phytomedicines available on the Dutch market. In order to study the stability of the valepotriates and the formation of their decomposition product(s), samples of freshly prepared valerian tinctures were analysed after being stored at 4, 20, and 36 degrees C for up to one month.

The essential oil of Tanacetum parthenium (L.) Schultz-Bip.

The composition of the essential oil of Tanacetum parthenium (L.) Schultz-Bip. (feverfew; Asteraceae) of various origins was investigated using GC and GC-MS. Camphor and chrysanthenyl acetate were the main constituents of the samples originating from England and The Netherlands. No infraspecific variation in the composition of the oil was found. Furthermore, the essential oil content and composition of Dutch feverfew during a vegetative period was studied. The young herb, before the formation of the stems, yielded a relatively high percentage of oil (0.53%, v/w), calculated on the dry weight. After a sharp decline at the beginning of the formation of the stems (0.30%, v/w), the percentage of the oil increased until full bloom (0.83%, v/w). During the development of the plant the percentage of camphor rose from 28% to 48%, whereas the amount of chrysanthenyl acetate decreased from 30% to 22%. In none of the oil samples investigated could the potentially toxic monoterpenes α- or β-thuojone be detected. This is important, because feverfew is used over long periods of time as a migraine prophylactic agent. The sesquiterpene lactone parthenolide is held responsible for this biological activity. In addition, chrysanthenyl acetate may display an analgesic effect by inhibiting the enzyme prostaglandin synthetase. Based on the results of our investigations and literature data, a number of recommendations are proposed with respect to the essential oil of T. parthenium.

Composition of the essential oil from roots and rhizomes of Valeriana wallichii DC.

The volatile constituents isolated from roots and rhizomes of Valeriana wallichii DC. ( = V. jatamansi Jones) were investigated by GC and GC–MS (EI and NICI) analysis. The plant material was obtained from different sources, namely from a local market in Kathmandu (Nepal), from plants grown in The Netherlands and in Germany, and from commercially available material from several German importers. The various roots and rhizomes yielded between 0.09 and 1.30% (v/w) essential oil on a dry weight basis. The main constituents of the oils from the plants grown in Europe were an unidentified sesquiterpene hydrocarbon, α-santalene, ar-curcumene and xanthorrhizol. In the oils from the Nepalese material and from the commercially available roots, except in one sample, patchouli alcohol was the main component.

Isolation and identification of valerenane sesquiterpenoids from Valeriana officinalis

Abstract Seven new valerenane sesquiterpenoids were isolated from a dichloromethane extract of Valeriana officinalis . The NMR, IR and mass spectral data of the isolated compounds, including the valerenic acids, are given in this paper.

Determination of valepotriates

In this paper an overview is given of qualitative and quantitative methods of analysis used for valepotriates. Methods like spectophotometry, titrimetry, TLC, GC, HPLC, MS, CE as well as p-SFC have been applied. Today HPLC is the method of choice. The usefulness of the individual methods are discussed.

Composition of the essential oils from underground parts of Valeriana officinalis L. s.l. and several closely related taxa

The volatile constituents from roots and rhizomes of Valeriana officinalis L. s.l. and of several closely related Valeriana taxa were investigated by GC and GCMS (EI and NICI) analysis. Seeds were obtained from different botanical gardens in Europe, and the plants investigated were grown in an experimental field in The Netherlands. In addition, commercially available plant material of Dutch origin was investigated. Four subspecies of the collective species V. officinalis were included in the study. The roots and rhizomes of 16 V. officinalis ssp. officinalis samples yielded 0.22-1.55% (v/w) essential oil on a dry weight basis. Oil components present in all samples were bornyl acetate (2-36%), myrtenyl acetate (trace-9%), nojigiku acetate (trace-0.8%), valerenic acid (0.3-3%), and one as yet unidentified tertiary sesquiterpene alcohol. The oil yield for the two samples of V. officinalis ssp. collina (Wallr.) Nyman was 0.78-0.85% (v/w). The main components were bornyl acetate (22-24%), myrtenyl acetate (6-8%), camphene (4-7%), kessane (3-6%), beta-eudesmol (4-5%) and a tertiary sesquiterpene alcohol (RI=1622) amounting to 20%, which was present in one sample only. For seven samples of V. officinalis ssp. sambucifolia (Mikan f.) Celak the yield of oil was 0.191.57% (v/w), with main components bornyl acetate (4-25%) and valerianol (3-34%). The only sample of V. repens Host. yielded 0.34% (v/w) oil, with bornyl acetate (13%), valerianol (19%) and kessane (8%) as the main components. Among the other (sub)species investigated in this study, the oil of V. celtica L. ssp. norica Vierh. contained bornyl acetate (22%) and patchouli alcohol (5%). Patchouli alcohol was also present in the oil of the two samples of V. phu L. together with gamma-patchoulene, patchoulyl acetate and a valerenal isomer (15-19%). Valeranone (16%) was the main constituent of the oil of V. exaltata Mikan. Bornyl acetate and valerianol were present in the essential oils of the other related valerian taxa studied

Essential Oil Content and Constituents of Black Zira (Bunium persicum [Boiss.] B. Fedtsch.) from Iran During Field Cultivation (Domestication)

Abstract Bunium persicum fruit oils from wild type (WT), first (CY1) and second year (CY2) cultivars (fourth and fifth year plants) were analyzed by GC and GC/MS. The essential oil content of the WT (9.1% v/w) was higher than the oil content of the CY1 (6.2% v/w) and CY2 (5.1% v/w). No significant differences were found with respect to the oil constituents. The main constituents were γ-terpinene (WT: 44.2%, CY1: 40.8%, and CY2: 36.8%), associated with cuminaldehyde (WT: 16.9, CY1: 14.1, and CY2: 11.8%), and γ-terpinen-7-al (WT: 10.5, CY1: 10.6, and CY2: 18.7%). In total 35 components could be identified covering 95.4% (WT), 94.9% (CY1) and 96.3% (CY2) of the oil content. We also studied the phenology of the plants during the period 1999–2004.

Volatile components from Anthriscus sylvestris (L.) Hoffm

The volatile components of fresh leaves and roots from Anthriscus sylvestris (L.) Hoffm., obtained through hydrodistillation, were analysed by GC and GC-MS. This was compared to dichloromethane extracts of both fresh and dried leaf and root material. The monoterpene fraction (69-70%) dominated, while beta-phellandrene (39-45%) was the main component in both the leaf and the root oil. Other components in the leaf oil were beta-myrcene (17%), sabinene (6.2%), Z-beta-ocimene (5.4%) and benzene acetaldehyde (4.1%). In the roots we found Z-beta-ocimene (16.9%) and alpha-pinene (4.6%) as other major components. These principle constituents of both essential oils were also present in the dichloromethane extracts of the fresh and dried leaves and the roots, although in much smaller percentages. Comparing hydrodistillation of fresh plant material with a dichloromethane extract, the latter yielded a considerably lower amount of constituents. In addition, air drying and freeze drying resulted in a significant loss of volatile constituents as compared to fresh material (dichloromethane extract).

The origin of mentha — gracilis (Lamiaceae). II. essential oils

Essential oils were eamined in nine clones of Mentha arvensis, four clones of M. spicata, and 20 clones of M. gracilis. An F1 hybrid of M. arvensis M. spicata, selected on the basis of morphology and chromosome number, was matched with one clone of M. Gracilis. Genes for the inheritance of limonene, 1,8-cineole, linalool, isomenthone, carvone, and piperitenone oide were identified in one clone of M. arvensis and two clones of M. spicata. The range of essential oil compounds detected indicates that no one character can be used to identify M. gracilis, but the critical compounds of the oil of M. gracilis can be derived from crosses of M. arvensis M. spicata.ResumenDe Samenstelling van de vluchtige olie van negen klonen van Mentha arvensis, vier klonen van M. spicata en 20 klonen van M. gracilis werd onderzocht. Een F1 hybride van M. arvensis M. spicata, geselecteerd op basis van morphologische kenmerken en Chromosomen aantal, werd gekruist met een kloon van M. gracilis.De genen voor de overrevingvanlimoneen, 1,8-cineole, linalool, isomenthone, carvone en piperitenone oide werden geidentificeerd in een kloon van M. arvensis en in twee klonen van M. spicata. De reeks van verbindingen, die zijn aangetoond in de vluchtige olie wijst er op dat geen enkele verbinding kan worden gebruikt om M. gracilis te identificeren maar dat de kritische verbindingen in de olie van M. gracilis kunnen worden verkregen uit kruisingen van M. arvensis M. spicata.