Investigation of transport of genistein, daidzein and their inclusion complexes prepared with different cyclodextrins on Caco-2 cell line.

Isoflavonoids are widespread constituents in medical plants especially in legumes (Fabaceae), but occur in other different plant families as well (Rosaceae, Iridaceae, Amaranthaceae). Their antioxidant, estrogen-like, anti-inflammatory and analgesic effects make them promising compounds in therapy of important disorders especially in estrogen related diseases. Poor solubility in aqueous system of genistein and daidzein needs a solubility enhancement for pharmaceutical use. These compounds are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs) considering matching their size and polarity. The molecular encapsulation with beta-cyclodextrin (ß-CD), gamma-cyclodextrin (γ-CD), hydroxypropyl-beta-cyclodextrin (HP-ß-CD) and random methyl-beta cyclodextrin (RAMEB-CD) results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of genistein and daidzein. Determining enhancement in solubility and bioavailability we investigated the transport of these inclusion complexes across Caco-2 cell line comparing that of the pure compounds and found significant improving effect of the different CD derivatives on membrane permeation of the two isoflavone aglycons.

Applicability of preparative overpressured layer chromatography and direct bioautography in search of antibacterial chamomile compounds.

In situ sample preparation and preparative overpressured layer chromatography (OPLC) fractionation on a 0.5 mm thick adsorbent layer of chamomile flower methanol extract prepurified by conventional gravitation accelerated column chromatography were applied in searching for bioactive components. Sample cleanup in situ on the adsorbent layer subsequent to sample application was performed using mobile phase flow in the opposite direction (the input and output of the eluent was exchanged). The antibacterial effect of the fractions obtained from the stepwise gradient OPLC separation with the flow in the normal direction was evaluated by direct bioautography against two Gram-negative bacteria: the luminescence gene tagged plant pathogenic Pseudomonas syringae pv. maculicola, and the naturally luminescent marine bacterium Vibrio fischeri. The fractions having strong activity were analyzed by SPME-GC/MS and HPLC/MS/MS. Mainly essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were tentatively identified in the fractions.

Composition and antipseudomonal effect of essential oils isolated from different lavender species.

The aims of our research include the phytochemical characterization of the essential oils extracted from different lavender species (Lavandula vera L., L. intermedia L., L. pyrenaica DC., and L. stoechas subsp. stoechas) that are important from therapeutic and economic aspects, as well as the optimization of the tube dilution method for microbiological investigation of the effect of essential oils on the selected Pseudomonas strain. The chemical and percentage compositions of the essential oils were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The main component of the essential oils in the majority of lavender species was linalool, while L-fencone was identified in the largest amount in L. stoechas subsp. stoechas. Tube dilution is one of the most frequently used methods for microbiological investigation. Its advantages are that it is easy to perform and provides information about minimum bactericidal concentration (MBC). Because of the lipophilic character of essential oils, the tube dilution method should be optimized for the detection of antibacterial activity of these plant extracts, and, therefore, emulsions containing 0.2% polysorbate 80 were prepared from the samples. In each of the eight investigated essential oils an inhibitory effect was detected, and MBC values were in the range 12.5-50 microL/mL in seven cases.

Investigation of dietary important components in selected red fleshed apples by GC-MS and LC-MS.

Three red-fleshed apple cultivars (Malus 'Geneva': GFV-03, Hungarian hybrid: GFV-04, Malus pumila Niedwetzkyana: GFV-05) were investigated for their chemical composition by sHS-SPME-GC-MS and HPLC-DAD-MS/MS analytical techniques. In cultivars GFV-03 and GFV-05 sesquiterpene α-farnesene were dominant while the alphatic esters were present mostly in traces. In GFV-04 - the new disease resistant advanced selection of the Hungarian apple breeding program - hexanol and hexyl 2-methylbutanoate were present in larger amounts while the amount of α-farnesene was lower than the other two cultivars. Using HPLC-DAD-MS/MS four phenolic acid derivatives, four anthocyanins, six flavonoids of quercetin derivatives and two dihydrochalcone phloretin glycosides were identified or characterized among the detected sixteen phenolic compounds.

Role of direct bioautographic method for detection of antistaphylococcal activity of essential oils.

The aim of the present study was the chemical characterization of some traditionally used and therapeutically relevant essential oils (thyme, eucalyptus, cinnamon bark, clove, and tea tree) and the optimized microbiological investigation of the effect of these oils on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). The chemical composition of the oils was analyzed by TLC, and controlled by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The antibacterial effect was investigated using a TLC-bioautographic method. Antibacterial activity of thyme, clove and cinnamon oils, as well as their main components (thymol, carvacrol, eugenol, and cinnamic aldehyde) was observed against all the bacterial strains used in this study. The essential oils of eucalyptus and tea tree showed weak activity in the bioautographic system. On the whole, the antibacterial activity of the essential oils could be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and better in comparison with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique).

Chemical and genetic relationships among sage ( Salvia officinalis L.) cultivars and Judean sage ( Salvia judaica Boiss.).

The essential oil composition and genetic variability of common sage ( Salvia officinalis L.) and its three ornamental cultivars ('Purpurascens', 'Tricolor', and 'Kew Gold') as well as Judean sage ( Salvia judaica Boiss.) were analyzed by GC-FID, GC-MS, and random amplified polymorphic DNA (RAPD). Common sage and its cultivars contained the same volatile compounds; only the ratio of compounds differed. The main compounds were the sesquiterpene alpha-humulene and the monoterpenes beta-pinene, eucalyptol, and camphor. Judean sage contained mainly the sesquiterpenes beta-cubebene and ledol. All of the samples exhibited characteristic RAPD patterns that allowed their identification. Cluster analyses based on oil composition and RAPD markers corresponded very well to each other, suggesting that there is a strong relationship between the chemical profile and the genetic variability.

Utility of cyclodextrins in the formulation of genistein part 1. Preparation and physicochemical properties of genistein complexes with native cyclodextrins.

Isoflavones are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs). The molecular encapsulation with CDs results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of isoflavones. Genistein, a key isoflavone constituent of Ononidis spinosae radix was found to form a supramolecular, non-covalent inclusion complex with both beta-cyclodextrin (beta-CD) and gamma-cyclodextrin (gamma-CD), while it did not form a stable complex with alpha-CD. The guest genistein was found to spatially located in the less polar cavity of cyclodextrin. The isolated binary genistein/CD complexes appeared novel crystalline lattices. The in vitro dissolution of genistein entrapped into both beta- and gamma-CD, significantly surpassed that of the plain isoflavone.

[Gas chromatography for analysis of essential oils. Characteristics of essential oil of Dracocephalum species and the influence of extraction method on its composition]. / Gáizkromatográfia alkalmazása illóolajok analízisére Dracocephalum fajok illóolaj jellemzoi és az extrakciós mód illóolajösszetételt befolyásoló hatása.

In this work the essential oil composition of some less known Dracocephalum species was studied and compared the effectiveness, selectivity and influence of different extraction methods (hydrodistillation, Soxhlet extraction with organic solvents and supercritical fluid extraction) on essential oils. For investigations in Hungary and Transylvania cultivated plant material was used. The analysis of essential oils was carried out by GC and GC-MS methods. The components were identified by standard addition, retention factors and mass spectra. The percentile evaluation of each volatile constituents was made on basis of GC-FID chromatograms. The accuracy of measurements was characterized by relative standard deviation. In the essential oil of D. renati Emb. (studied firstly by us) 18.3% of limonene was measured and carvone, citrals and linalyl acetate monoterpenes, methyl chavicol and some sesquiterpene (e.g. bicyclovetivenol) determined in lower quantities. We established that more than 50% of essential oil of D. grandiflorum L. was formed by sesquiterpenes (beta-caryophyllene and- oxide, beta-bourbonene, beta-cubebene, aromadendrene) and the essential oil of D. ruyschiana L. contained pinocamphone isomers in more than 60%. The oxygenated acyclic monoterpenes, the characteristic constituents of Moldavian dragonhead were present in some tenth percent only in D. renati oil. We found significant differences in the composition of the SFE extract and traditional essential oil of D. moldavica L. The supercritical fractions collected at the beginning of the extraction process were richer in valuable ester component (geranyl acetate) than the essential oil obtained by hydrodistillation. The fractions collected at the end of supercritical were poor in oxygenated monoterpenes but rich in minor compounds of traditional oil, e.g. palmitic acid.

Beta-eudesmol, a new sesquiterpene component in intact and organized root of chamomile (Chamomilla recutita).

Gas chromatography (GC) and GC-mass spectrometry are used to identify a new sesqiterpene, beta-eudesmol, which seems to be a characteristic essential oil component of the intact and in vitro organized root of chamomile [Chamomilla recutita (L.) Rauschert]. It is identified on three types of stationary phases by GC. The confirmation of identity is carried out by comparison of mass spectra with those reported in the literature and measured from a reference compound. The percentage evaluation of the oil component is made by area normalization, on the basis of three parallel measurements. Among the cultivated and wild chamomile species examined, the wild species from the areas of Szeghalom contain the highest quantity of beta-eudesmol (9.25% in the total essential oil).

New terpenoids in cultivated and wild chamomile (in vivo and in vitro).

The effect of Chamomilla recutita (L.) Rauschert is made up by several groups of active substances, among which terpenoids in the inflorescences are of greatest importance. Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its (-)-alpha-bisabolol. Both components have important antiinflammatory activities. Among wild chamomile populations in Hungary, a population was found in the area of Szabadkigyós containing significant amounts-on average 48%-of (-)-alpha-bisabolol in its inflorescence oil. In vitro cultures were made from this population to obtain propagation material containing a high number of active substances. The intact roots contained no (-)-alpha-bisabolol but the sesquiterpene alcohol beta-eudesmol as new compound was identified by our group. Sterile plantlets, cultured in vitro, were multiplied for phytochemical investigations. Pharmacologically important compounds of the essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. These volatile compounds were identified by comparing their retention times with those of authentic standards, essential oils of known composition and peak enrichment. The confirmation of identity was done by comparison of their mass spectra with those reported in the literature and reference compounds. The percentage evaluation of each component was made by area normalisation. Gas chromatography (GC) and mass spectrometry (MS) showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristic of the parent plant. We identified germacrene-D, berkheyaradulene, 4-(2', 4', 4'-trimethyl-bicyclo[4.1.0]hept-2'-en-3'-yl)-3-buten-2-one, geranyl-isovalerate and cedrol as new components in these sterile cultures.

[Influence of extraction methods on the composition of essential oils]. / Extrakciós módszerek hatása az illóolajok osszetételére.

The aim of this work is to demonstrate our results on comparison of composition of essential oil fractions obtained by traditional steam distillation and supercritical fluid extraction. The plant materials for the various extraction methods were selected from the Lamiaceae, Apiaceae and Asteraceae families. For the supercritical fluid extraction (SFE) carbon dioxide was used as supercritical solvent. The extracts were collected by stage wise precipitation in two separators. The waxy product and extract rich in essential oil were collected in the 1st and in the 2nd separator respectively. The traditional water steam distillation (SD) was carried out in the special apparatus of the Hungarian Pharmacopoea (7th ed.). GC analysis was carried out on capillary silica fused columns coated with DB-1701 and the specific chiral columns coated with Rt-beta DEX m or Rt-beta DEX sm. Comparing the composition of steam distilled oils with that of volatile SFE fractions the following general characteristics were established. The SFE fractions were richer in monoterpene-esters and poorer in alcohols than the traditional essential oils (clary sage, lavander, moldavian dragonhead). Regarding the distribution of the monoterpene and sesquiterpene compounds, the SFE fractions contained sesquiterpenes in higher percentage than the distilled oils (Salvia fruticosa). Furthermore, the proportion of sesquiterpenes increased in SFE fractions collected successively with time (Salvia officinalis) similar to the ratio of oxygenated monoterpenes to monoterpene hydrocarbons (Rosmarinus officinalis). The phtalides of lovage (Satureja hortensis) did not show regular change during the supercritical extraction. In other cases it was verified that part of the mono- and sesquiterpenes were present originally in bound form (glycosides) in plants. Thus they appeared only in essential oil fractions after previous acidic treatment (Thymus, Origanum, Satureja species). During the super-critical extraction the azulenogene sesquiterpene lactones did not transform to azulenes (chamomile, yarrow), but SFE fractions of some Asteraceae plants contained sesquiterpene-gamma-lactones of unchanged structure.

Effect of magnesium on essential oil formation of genetically transformed and non-transformed chamomile cultures.

OBJECTIVE: The importance of chamomile (Chamomilla recutita) is widely known in classical and folk medicine, with the largest group of its effective substances forming the essential oil (chamazulene, alpha-bisabolol, trans-beta-farnesene, spathulenol, cis/trans-en-in-dicycloethers). The increasing need for plant-derived high quality drugs cannot be provided by their collection in the wilderness. METHOD A: To preserve the genome of Szabadkigyo. wild type having high (-)-alpha-bisabolol content, we used biotechnological methods. RESULTS: The roots of organized culture contained beta-eudesmol, which we have identified in the intact roots. Our gas-chromatographic and mass-spectroscopic studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the intact plant. We identified berkheyaradulene, geranyl-isovalerate and cedrol, as new components in these cultures. Magnesium (Mg) (370 and 740 mg/l MgSO4) has a positive effect on the growth of organized cultures and also on the quality and quantity of essential oil production. METHOD B: Another possible source of variants is available by the genetic transformation of organized cultures by infection with Agrobacterium rhisogenes. With this method, we cultivated chamomile infected by A4-Y clone and investigated the essential oil production by hairy root cultures cultivated on solid and liquid MS B-5 media. The main component of the essential oil of hairy root cultures was trans-beta-farnesene. RESULTS: We identified alpha-selinene, as a new component in these hairy roots. We studied the growth rate of A4-Y clone on the cited media, containing MgSO4 concentrations: 0; 185; 370 and 740 mg/l. The cultures grew most in medium containing 740 mg/l of MgSO4. Essential oil content was compared from hairy root cultures of different Mg containing media and measured by GC and GC-MS methods. Mg has a similar effect on hairy roots as on organized cultures.