Proposal for a standardised method for the identification of essential oils by HPTLC.

The European Pharmacopoeia (Ph. Eur.), includes both individual monographs on essential oils and a general monograph that covers all essential oils for pharmaceutical use, whether covered by an individual monograph or not. The individual monographs generally describe gas chromatography as a first identification test, while thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) methods are included in the second identification series. To comply with Ph. Eur. general chapter 2.8.25. High-performance thin-layer chromatography of herbal drugs and herbal drug preparations, HPTLC parameters must be standardised. Currently, 18 of the 32 monographs on essential oils feature the same TLC/HPTLC method, but differ in terms of the other conditions described. A single, standardised chromatographic system with a system suitability test (SST) and intensity markers for all 32 essential oils covered by individual monographs would be desirable, particularly for pharmacies and other users that cannot perform gas chromatography for financial reasons. To this end, this paper describes the development of a general HPTLC method for the identification of essential oils in compliance with general chapter 2.8.25. The method proposes the use of ethyl acetate, toluene (5:95 V/V) as mobile phase, isoeugenol/isoeugenyl acetate for the SST, and a combination of one alcohol (either borneol or linalool) and one ester (either linalyl acetate or bornyl acetate) as intensity markers.

An alternative and simplified approach to identification and test for minimum content of TCM herbal drugs.

Following a decision of the European Pharmacopoeia (Ph. Eur.) Commission, the Traditional Chinese Medicines (TCM) Working Party started a pilot phase to examine the suitability of a high-performance thin-layer chromatography (HPTLC) minimum content test as an alternative to the classical assay in TCM monographs. This approach was evaluated with two TCM herbal drugs: Fritillaria thunbergii bulbs (FTB) and Corydalis rhizome (CYR). Firstly, the existing HPTLC methods were optimised for both drugs. The new methods were applied to the evaluation of multiple samples, and acceptance criteria for the identification, following Ph. Eur. chapter 2.8.25. High-performance thin-layer chromatography of herbal drugs and herbal drug preparations, were set. The HPTLC test for minimum content of markers was then developed and validated. In this test, the intensity of the marker zone in the fingerprint of the sample is compared to the corresponding zone in the reference solution, which has a concentration giving an intensity equivalent to the acceptance criterion. This test gives a pass or fail result rather than a content and can be performed visually (on the images) or by software (using peak profiles from images; PPI). Reproducibility of the HPTLC methods was evaluated in a collaborative trial including six laboratories. In summary, results for FTB from five laboratories were in agreement. The remaining laboratory did not pass the identification of the samples. For CYR, all laboratories presented the same results for identification. In the test for minimum content, one borderline sample passed in four laboratories and failed in two. All laboratories reached similar conclusions for the other seven samples. The HPTLC methods proposed offer a simplified approach to evaluating identity and minimum content of TCM drugs in a single analysis.

Activity of artichoke leaf extract on reactive oxygen species in human leukocytes.

Artichoke leaf extract was studied in human leukocytes for activity against oxidative stress using flow cytometry and dichlorofluorescin diacetate as a fluorescence probe. It produces a concentration-dependent inhibition of oxidative stress when cells are stimulated with agents that generate reactive oxygen species (ROS): hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Cynarin, caffeic acid, chlorogenic acid, and luteolin, constituents of artichoke leaf extract, also show a concentration-dependent inhibitory activity in the above models, contributing to the antioxidant activity of the extract in human neutrophils.

Anti-inflammatory action of Pluchea sagittalis: involvement of an antioxidant mechanism.

Pluchea sagittalis, (Lam.) Cabr., a popular medicinal herb grown in South America, was studied for anti-inflammatory and antioxidant activities. The anti-edema action of P. sagittalis aqueous extract was assayed in different models of inflammation: 1) the mouse ear edema test induced by arachidonic acid and croton oil; 2) the rat hind-paw edema test produced by several inflammatory inductors: carrageenan, dextran, zymosan, platelet-activating factor (PAF) and arachidonic acid; 3) a subacute model based on the rat carrageenan air-pouch granuloma test. Blood leukocyte free radical production was measured by flow cytometry with 2',7'-dichlorofluorescin diacetate (DCFH-DA) in vivo, in rats with induced air-pouch granuloma, and in a model in vitro. stimulating leukocytes with hydrogen peroxide. The aqueous extract of P. sagittalis showed a marked anti-inflammatory effect in both ear edema tests, dextran and carrageenan hind-paw edemas and carrageenan air-pouch model. It also had a potent antioxidant activity in blood leukocytes, both in vivo and in vitro. Our results correlate the reduction of free radical production with the anti-inflammatory effect of this plant.

Chromatographic analysis of polyphenols of some Iberian Thymus.

A comparative chromatographic analysis of polyphenols of some Iberian Thymus species of the sections Pseudothymbra, Thymus and Serpyllum was carried out by means of HPLC and TLC, in order to investigate their pharmacological interest. The purified leaf extracts were analysed and compared with authentic samples of 15 flavones, one flavonol, four flavanones, two dihydroflavonols, and caffeic and rosmarinic acids. The taxa belonging to the section Pseudothymbra and some of those in the section Thymus showed the highest content of methoxylated flavonoids, which are known to have antispasmodic properties.

Antifungal activity of Paraguayan plants used in traditional medicine.

The antifungal activity of aqueous, dichloromethane and methanol extracts from 14 Paraguayan plants used in traditional medicine for the treatment of skin diseases was assayed in vitro by the agar disk diffusion method against 11 fungal strains comprising several filamentous fungi and yeasts. Among them, the dichloromethane extracts of Acanthospermum australe, Calycophyllum multiflorum, Geophila repens and Tabebuia avellanedae, as well as the aqueous and methanol extracts of the latter, showed the highest activity.

Chemical composition of the essential oil from the leaves of Piper fulvescens, a plant traditionally used in Paraguay.

The essential oil from the leaves of the aromatic aniseed smelling plant Piper fulvescens, obtained by hydrodistillation, was investigated by GC and GC-MS, as well as by 13C-NMR after fractionation of the oil by column chromatography. A total of 37 constituents, representing 78.2% of the oil were identified. The main compounds were trans-anethole (26.4%) and ishwarane (12.1%), an unusual sesquiterpene hydrocarbon.

Antiinflammatory activity of Anthurium cerrocampanense Croat in rats and mice.

Aqueous (AE), ethanol (EE) and dichloromethane (DE) extracts from the whole plant of Anthurium cerrocampanense Croat, endemic to Panama, were assayed for antiinflammatory activity by carrageenan-induced rat paw edema and croton oil-induced mouse ear edema tests. The extracts were administered i.p. and topically, respectively. The best antiinflammatory activity in the carrageenan-induced rat paw edema test, was shown by DE (31% inhibition), which did not show any irritant effect in the peritoneal examination 4 h after administration of the extracts. Furthermore, the highest activity in the croton oil-induced mouse ear edema test was also exhibited by DE (93% inhibition). Since DE was the most effective of the extracts in both tests, it's activity was assessed in various inflammation models in rats. At 100 mg/kg, DE markedly inhibited inflammation produced by dextran (66.6% inhibition) and decreased weakly the edema induced by zymosan A, carrageenan and C16-platelet activation factor (C16-PAF) (33.2, 31.6 and 23.8%, respectively). At 50 mg/kg, DE only significantly reduced the swelling caused by dextran (36.2%). No effect was observed at the same doses in arachidonic acid-induced edema. A clear dose-related antiinflammatory activity of DE was seen in the croton oil-induced mouse ear edema test, and ED50 calculated was found to be 25.5 microg/ear. which was much lower than ED50 obtained for indomethacin (56.1 microg/ear).

Composition and chemical polymorphism of the essential oils from Piper lanceaefolium.

Essential oils obtained by hydrodistillation from leaves and spikes of Piper lanceaefolium H.B.K. of Costa Rica were analysed by GC-FID, GC-MS and 13C-NMR methods. Main constituents found in the oil from leaves were sesquiterpene hydrocarbons - especially beta-caryophyllene and germacrene D - and phenylpropanoids, of which elemicin and parsley apiol were the major ones. The volatile oil from spikes showed monoterpene hydrocarbons, namely alpha- and beta-pinene, and the same phenylpropanoids as in the oil from leaves as the major constituents. Results obtained in the analysis by GC-FID and GC-MS of the essential oils from individual plants of different geographic origin were submitted to chemometric cluster analysis and principal component analysis, showing the presence of three different types of oils (i) parsley apiol/elemicin, (ii) elemicin/parsley apiol/dill apiol, and (iii) parsley apiol/dill apiol.

Essential oil composition and variability of Thymus lotocephalus and Thymusxmourae.

The composition of the essential oils of four populations of Thymus lotocephalus G. López and R. Morales and one population of T.xmourae Paiva and Salgueiro, two endemic taxa from Portugal, was investigated mainly by GC and GC-MS. Txmourae is a natural hybrid between T. lotocephalus and T. mastichina (L.) L. subsp. donyanae R. Morales, which essential oil was analysed for the first time. In its oil, it was possible to find compounds of both parents, which could enable us to confirm its intermediate status between those two taxa. 1,8-Cineole and borneol were the main constituents in the essential oil of T.xmourae, whereas linalool, geranyl acetate and 1,8-cineol were the major ones in T. lotocephalus. Intermedeol was also an important constituent in the oils of both taxa. Nevertheless, the volatile oils of the four populations investigated of T. lotocephalus showed important differences among the main constituents. In order to study their infraspecific variability, the results obtained in the analysis of individual plants were submitted to a Principal Component and Chemometric Cluster Analyses. Five types of essential oils were found: linalool, 1,8-cineole, linalool/1,8-cineole, linalyl acetate/linalool and geranyl acetate.

Chemotaxonomic study on Thymus villosus from Portugal.

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.

Activity of taraxasteryl acetate on inflammation and heat shock protein synthesis.

Pluchea sagittalis whole plant dichloromethane extract showed inhibitory activity in several inflammatory models: rat hind paw-edema, mice ear edema, and air-pouch rat granuloma. The extract inhibited the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in stimulated human neutrophils. It also showed inhibitory effect on heat shock protein 72 (hsp72) synthesis in stimulated neutrophils, while it had opposite effects on unstimulated cells. The triterpene taraxasteryl acetate was obtained from the dichloromethane extract by bioassay directed isolation, being active against induced ROS and RNS production in human neutrophils. In mice ear edema (induced by phorbol-12-mirystate-13-acetate, croton oil and arachidonic acid), taraxasteryl acetate showed a topical anti-inflammatory activity similar to the extract, but at 1/20 of the dose. The same ratio was observed for the inhibition of hsp72 production in stimulated human neutrophils. In unstimulated monocytes and neutrophils, taraxasteryl acetate showed a higher stimulating activity of hsp72 production than the extract, involving different mechanisms in each cell type. To our knowledge, taraxasteryl acetate is the first natural product for which a dual effect on the hsp response is reported.

Activity of plant extracts on the respiratory burst and the stress protein synthesis.

Aqueous, methanol and dichloromethane extracts from Artemisia copa, Baccharis grisebachii, Baccharis incarum, Baccharis latifolia, Mutisia kurtzii and Pluchea sagittalis, plants used in the Traditional Medicine of South America, are studied for activity on the respiratory burst and the inducible heat shock protein of 72 kD (hsp72) synthesis. Activity on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as on hsp72 synthesis was measured by flow cytometry in human neutrophils. Cells were stimulated using hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (FMLP) for ROS generation, and sodium nitroprusside (SNP) or PMA in the presence of calmodulin inhibitor W-13 for RNS. The production of hsp72 was induced by heat, PMA, H2O2 and SNP. The best inhibitory activity was shown by the dichloromethane extracts of Baccharis grisebachii and Pluchea sagittalis that were active in all the assays. The aqueous extract of Pluchea sagittalis was also active in most assays. The aqueous extract from Mutisia kurtzii caused a clear increase of the hsp72 production and showed prooxidant activity.

Analysis of Hydrosoluble Polysaccharides from Ramonda myconi Leaves.

The mucilage of RAMONDA MYCONI leaves is composed of several Polysaccharides differing in acidity due to the content of galacturonic acid. RMP-2 and RMP-4 are the most important fractions, where RMP-2 is an arabinogalactan containing galacturonic acid and rhamnose, probably connected to a protein moiety. RMP-4 is the major fraction, containing a Polysaccharide with 90% of galacturonic acid. It has a pectin-like structure and the neutral sugar enriched regions show a structure related to the rhamnogalacturonan II type.

Antifungal principles from Piper fulvescens.

Activity-guided fractionation of the dichloromethane extract from leaves of Piper fulvescens, using an agar overlay bioautographic method, led to the isolation of three antifungal neolignans identified as conocarpan, eupomatenoid 5 and eupomatenoid 6. The minimal inhibitory concentration of these three neolignans against five fungi strains were determined. Conocarpan showed the widest activity, whereas eupomatenoid 6 was the most active against dermatophytes.

Anti-inflammatory and analgesic activity of Baccharis trimera: identification of its active constituents.

The butanolic fraction (BT-II) derived from the aqueous crude extract was prepared from aerial parts of Baccharis trimera and assessed in anti-inflammatory, analgesia, and ulcerogenesis models. Intraperitoneal pretreatment with lyophilized BT-II, at doses ranging from 40 to 100 mg/kg, markedly inhibited carrageenan- and dextran-induced inflammation (70.4-90.8% and 25.7-71.3%, respectively) and weakly decreased C16-paf- and arachidonic acid-induced swelling (24.9-36.7% and 0-30.6%, respectively). No effect was observed, at the same doses, on zymosan-induced edema. The intraperitoneal examination indicates that the anti-phlogistic action of BT-II was not due to an irritating effect at the injection site. Besides, BT-II reduced abdominal constrictions in mice following injection of acetic acid: at 50 mg/kg, it gave 67.4% inhibition and, at 100 mg/kg, 95.1%. The ulcerogenic assay showed that the incidence of ulcers after BT-II i.p. treatment was 2/6 at 50 mg/kg and 6/6 at 100 mg/kg. Ulcerogenic indices were 1.3 +/- 0.5 and 2.7 +/- 0.8, respectively. These results indicate that B.trimera shows strong anti-inflammatory and analgesic properties which seem to be due, at least partly, to the inhibition of prostaglandin biosynthesis. The chromatographic separation of BT-II monitored by bio-assay (carrageenan-induced edema test in mice) was carried out. The active constituents were found to be mainly saponins in which echinocystic acid (or its enantiomer) is the major aglycone, and also rutin.

Composition and antimicrobial activity of the essential oil of Peumus boldus leaves.

The composition and the antimicrobial activity of the essential oil from the leaves of Peumus boldus is investigated. Analyses of the oil obtained by hydrodistillation were carried out by GC and GC-MS using columns of two different stationary phases. Fractionation of the essential oil by column chromatography on silica gel was performed to improve identification of some constituents. More than 90% of the total oil (46 components) was identified, major constituents being monoterpenes (90.5%), among which limonene (17.0%), p-cymene (13.6%), 1.8-cineole (11.8%), and beta-phellandrene (8.4%) reached the highest percentages. Determination of the minimal bactericidal or fungicidal concentration against several microorganisms showed interesting activities towards Streptococcus pyogenes, Micrococcus sp., and Candida sp.

Essential oil composition and antimicrobial activity of Santiria trimera bark.

The composition and the antimicrobial activity of the bark oil of Santiria trimera (Oliv.) Aubrév., a plant widely used by the traditional healers in S. Tomé and Príncipe, especially for wound healing, are reported for the first time. The analysis of the essential oil was carried out by GC and GC-MS. The oil contains a high content of monoterpenes, alpha-pinene (66.6 %) being the major constituent, followed by beta-pinene (20.0 %). The essential oil was active against both bacteria and fungi strains, except Staphylococcus epidermidis and Aspergillus niger. It exhibited significant antimicrobial activity against Proteus vulgaris and Cryptococcus neoformans with MICs values of 1.11 microl/ml and lower than 0.71 microl/ml, respectively.

Anti-inflammatory activity of dichloromethane extract of Heterotheca inuloides in vivo and in vitro.

The dichloromethane extract from the dried flowers of Heterotheca inuloides Cass. was investigated on several pharmacological models of inflammation in vivo and in vitro. It showed anti-inflammatory activity on the croton oil-induced oedema test in mouse ear, at 1 mg/ear. The compound isolated from this extract, 7-hydroxy-3,4-dihydrocadalin, showed anti-inflammatory effect on the same experimental model (ED50 of 0.9 mumol/ear), as well as on COX-1 and COX-2 catalysed prostaglandin biosynthesis assays, with IC50 values of 22 microM and 526 microM, respectively. No effect was observed on carrageenan-induced oedema and on fMLP/PAF-induced exocytosis of human neutrophils. The COX-1 inhibitory effect showed by 7-hydroxy-3,4-dihydrocadalin might be related to the anti-inflammatory activity on the topical oedema induced by croton oil.

Essential oil composition and antimicrobial activity of three Zingiberaceae from S.Tomé e Príncipe.

The essential oil composition of three Zingiberaceae widely used as medicinal aromatic plants from S. Tomé and Príncipe: Aframomum danielli (Hook. f.) K. Schum., Curcuma longa L. and Zingiber officinale Rosc. was studied. Two samples of the essential oils from fruit of A. danielli and from rhizomes of the other two species, were obtained by hydrodistillation and analyzed by GC, GC-MS, and (13)C-NMR. The essential oil from fruits of A. danielli has been studied for the first time and was characterised by its high content of monoterpenes, with 1,8-cineole (25.5 - 34.4 %) the major constituent, followed by beta-pinene (14.1 - 15.2 %) and alpha-terpineol (9.9 - 12.1 %). Essential oils from the rhizomes of C. longa contained a lower content of ar-turmerone (4.0 - 12.8 %) than those reported in the literature for C. longa from other origins (24.7 - 31.4 %), whereas the results for Z. officinale essential oils were in accordance with the literature data. The essential oils of A. danielli and Z. officinale showed antimicrobial activity against all Gram-positive and Gram-negative bacteria tested, as well as against yeasts and filamentous fungi, using the agar diffusion method.