Oregano demonstrates distinct tumour-suppressive effects in the breast carcinoma model.

PURPOSE: There has been a considerable interest in the identification of natural plant foods for developing effective agents against cancer. Thus, the anti-tumour effects of oregano in the in vivo and in vitro breast cancer model were evaluated. METHODS: Lyophilized oregano (ORE) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration. At autopsy, mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation in MCF-7 cells was carried out. RESULTS: Low-dose ORE suppressed tumour frequency by 55.5 %, tumour incidence by 44 %, and tumour volume by 44.5 % compared to control animals. Analysis of rat tumour cells showed Ki67, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase after low-dose ORE treatment. High-dose ORE lengthened tumour latency by 12.5 days; moreover, Bcl-2, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase in carcinoma cells were observed. Histopathological analysis revealed a decrease in the ratio of high-/low-grade carcinomas in both treated groups. In vitro studies showed that ORE decreased survival and proliferation of MCF-7 cells. In ORE-treated MCF-7 cells, an increase in cells expressing sub-G 0/G 1 DNA content and an increase in the percentage of annexin V/PI positive MCF-7 cells were observed. In vitro, both caspase-dependent and possible non-caspase-dependent apoptotic pathways were found. The deactivation of anti-apoptotic activity of Bcl-2, a decrease in mitochondrial membrane potential, and the activation of mitochondrial apoptosis pathway were observed in the ORE-treated MCF-7 cells. CONCLUSIONS: Our results demonstrate, for the first time, a distinct tumour-suppressive effect of oregano in the breast cancer model.

[Constituents of the leaves of Cornus officinalis Sieb. et Zucc.] / Obsahové látky listov Cornus officinalis Sieb. et Zucc.

The paper deals with the isolation and identification of the constituents of Cornus officinalis Sieb. et Zucc. leaves. There was isolated ß-sitosterol from petroleum ether extract, glucose and saccharose from methanolic extract and gallic acid, quercetin and quercetin-3-O-glucoside (isoquercitrin) from aqueous-methanolic extract. Isolates were identified by physico-chemical data, and by comparing with authentic samples and literature data. The compounds I - V were isolated from the leaves of Cornus officinalis Sieb. et Zucc. for the first time.Key words: Cornus officinalis Sieb. et Zucc. Cornaceae petroleum ether extract methanolic extract aqueous-methanolic extract.

Formulation of sage essential oil (Salvia officinalis, L.) monoterpenes into chitosan hydrogels and permeation study with GC-MS analysis.

This study deals with the formulation of natural drugs into hydrogels. For the first time, compounds from the sage essential oil were formulated into chitosan hydrogels. A sample preparation procedure for hydrophobic volatile analytes present in a hydrophilic water matrix along with an analytical method based on the gas chromatography coupled with the mass spectrometry (GC-MS) was developed and applied for the evaluation of the identity and quantity of essential oil components in the hydrogels and saline samples. The experimental results revealed that the chitosan hydrogels are suitable for the formulation of sage essential oil. The monoterpene release can be effectively controlled by both chitosan and caffeine concentration in the hydrogels. Permeation experiment, based on a hydrogel with the optimized composition [3.5% (w/w) sage essential oil, 2.0% (w/w) caffeine, 2.5% (w/w) chitosan and 0.1% (w/w) Tween-80] in donor compartment, saline solution in acceptor compartment, and semi-permeable cellophane membrane, demonstrated the useful permeation selectivity. Here, (according to lipophilicity) an enhanced permeation of the bicyclic monoterpenes with antiflogistic and antiseptic properties (eucalyptol, camphor and borneol) and, at the same time, suppressed permeation of toxic thujone (not exceeding its permitted applicable concentration) was observed. These properties highlight the pharmaceutical importance of the developed chitosan hydrogel formulating sage essential oil in the dermal applications.

Antiproliferative Activities of Water Infusions from Leaves of Five Cornus L. Species.

Cornaceae plants are known for their edible berries, and their leaves are used as tea. In the present study aqueous leaf extracts from Cornus mas (CM), C. alba (CA), C. flaviramea (CF), C. kousa (CK), and C. officinalis (CO) were tested for their antiproliferative activity in human breast cancer cells (MCF-7). Dose- (50-750 µg/mL) and time (24, 48, 72 h)-dependent antiproliferative effects were measured by WST-1, and correlated with the content of flavonoids (FL), total hydroxycinnamic derivatives (THD), total polyphenols (TP) and tannins (T). Extracts induced time dependent decreases in cell survival; CA, CO and CM were the most effective (11.2%, 10.3% and 11.1%, after 72 h). The ED50 (effective dose) values were similar for all extracts and times tested. The THD and TP were identical in all samples, while a two-fold higher T content was present in CK and CO, and of FL in CF. The maximal effects (% of surviving cells) negatively correlated with the T and TP levels, and positively with FL and THD. The results demonstrate the significant antiproliferative effects of the tested water extracts in MCF-7 cells, in which CA, CO and CM are the most effective; and the effectiveness is related to the T and TP contents.

[Flavonoids - main constituents of the leaves of Philadelphus tenuifolius Rupr. et Maxim]. / Flavonoidy - hlavné obsahové látky listov Philadelphus tenuifolius Rupr. et Maxim.

The paper deals with the isolation and identification of the constituents of the leaves of Philadelphus tenuifolius Rupr. et Maxim. A methanolic extract was used to isolate quercetin-3-O-glucoside (isoquercitrin), and a butanolic extract to isolate kaempferol-3-O-glucoside-7-O-rhamnoside. Isolates were identified by physical-chemical data, comparison with authentic samples and literature data. The above-mentioned compounds were isolated from Philadelphus tenuifolius Rupr. et Maxim. for the first time.

[Formulation of salvia-containing gels]. / Formulácia gélov s obsahom salvie lekárskej.

The trend during the past years is to prefer natural drugs to synthetically prepared medicines. Due to its important antiphlogistic, antiseptic and antihidrotic effects, Salvia officinalis L. has a wide use in medicine, pharmacy and cosmetics in various dosage forms. The aim of this research was the formulation of Salvia officinalis L. in the form of a tincture to hydrogels. Gels were parallelly prepared on the basis of the natural polymer chitosan and the synthetic polymer Carbopol 940. The evaluation was focused on the influence of the anionic polymer and the cationic polymer in different concentrations on hydrogel flow properties. Glycerol functioning as a humectant was used in various percentage ratios. On the basis of the determination of rheological parameters and flow curves lapses the most suitable hydrogel from the application point of view was selected.

Antiproliferative, cytotoxic, antioxidant activity and polyphenols contents in leaves of four Staphylea L. species.

Staphylea has been used for long time in Traditional Chinese Medicine (TCM) and by Native Americans in a number of therapeutical indications. The present study describes in vitro antiproliferative, cytotoxic properties (MTT and LDH test) and antioxidant activities (reduction of DPPH radical and peroxynitrite radical) of Staphylea colchica Stev. (SC), S. elegans Zab. (SC), S. holocarpa Hemsl. (SH) and S. pinnata L. (SP) leave water extracts. Time- (24 and 72 h) and dose- (1-150 microg/mL) dependent effects of the above extracts were tested at the mitochondrial (MTT test) and plasma membrane level (LDH leakage) in A431 human skin carcinoma cells. Screening of these properties has shown time and dose dependent increase of harmful effects, the highest activity was observed for the SE, while the less active was the SH extract. The ED(50) values for the mitochondrial and membrane damage were nearly identical for the SE and very similar for SH extract. These findings indicate simultaneous injury of both cell compartments by SE and SH extracts. The highest antioxidant potential of SE species is accompanied by the highest content of flavones/flavonols and polyphenols. Only flavonoid contents are associated with antiproliferative effects and cell membrane injury, while antioxidant properties are the result of polyphenol content. The data clearly demonstrate that individual Staphylea L. species differ, not only in the amount of biologically active compounds, but also by the extent of harmful and beneficial effects.

Antioxidant activity and total phenols in different extracts of four Staphylea L. Species.

Staphylea L. is a deciduous ornamental shrub that possesses significant cytotoxic and antibacterial activity, although the chemical composition of its extracts and the identity of the structures responsible for these biological activities are not yet known. In this study we have determined the total phenolic content in chloroform and ethyl acetate extracts of four Staphylea species: Staphylea colchica Stev., S. elegans Zab., S. holocarpa Hemsl. and S. pinnata L.. The antioxidant potential (DPPH radical and peroxynitrite scavenging activity) of these extracts was also determined and a correlation between the phenolic content and antioxidant activities of the ethyl acetate extracts has been found. Ethyl acetate extracts were more active and one of them, obtained from S. colchica Stev., possessed the highest activity.

Research on biomodulatory effect of natural compounds.

OBJECTIVES: The purpose of this study was to determine whether the extract isolated from the artichoke Cynara cardunculus L. (ECC) had antimutagenic effect and was able to enhance the therapeutic effect of cytostatic drug cis-platinum (cis-Pt). METHODS: The potential antimutagenic activity of ECC was assayed by a test on sex-linked recessive lethal mutations detection in Drosophila melanogaster males treated with ethylmethane sulfonate (EMS). The possible enhancement of cytostatic/cytotoxic effect of cis-Pt by ECC was evaluated in the cell revitalization assay by measuring cell viability via Trypan blue exclusive assay using mouse leukemia cells L1210. RESULTS: EMS was both toxic and genotoxic in D. melanogaster males. It statistically significantly increased the frequency of sex-linked recessive lethal mutations in comparison to the negative control. Furthermore, ECC statistically significantly reduced the genotoxic effect of EMS. It acted in a desmutagenic manner via EMS inactivation. In the cell revitalization assay, ECC enhanced the cytotoxic/cytostatic effect of cis-Pt. The therapeutic potential of ECC was established on the basis of statistically significantly lowered recovery of cis-Pt pre-treated mouse leukemia cells in the presence of ECC. CONCLUSIONS: The results imply that the extract isolated from artichoke C. cardunculus L. has marked beneficial activities antimutagenic and therapeutic effect enhancing) and its potential biomedical application in the combination therapy of cancer and some neurodegenerative diseases may be suggested.

Free radical scavenging activity of different extracts and some constituents from the leaves of Ligustrum vulgare and L. delavayanum.

The free radical scavenging activity of the water infusions, different organic solvent extracts and some constituents from Ligustrum vulgare and Ligustrum delavayanum leaves was assessed with the aid of DPPH radical. Among the samples screened, water infusions had the strongest free radical scavenging capacity. From the tested compounds scavenging active flavonoid aglycones are present in the most active chloroform fractions from both leaves samples.

Cytotoxicity of water extracts from leaves and branches of Philadelphus coronarius L.

Philadelphus coronarius L. is big, leggy and deciduous old-fashioned shrub known for its fragrant white flowers in the late spring. Some members of genus Philadelphus L. are known for their antibacterial, antiradical and immunomodulatory effects. Therefore, these herbs represent prospective sources for the isolation of active substances with desired effects. We have investigated the cytotoxicity effects of water extracts from leaves and branches of Philadelphus coronarius L. (Hydrangeaceae). A431 cells (human skin carcinoma cell line) and the human breast adenocarcinoma cell line (MCF-7) were treated with various doses of individual extracts (0,1-100 microg dry matter/ml) for 24 h and 72 h. The highest toxic effects of both plant parts extracts were observed on MCF-7 cells regardless the time of treatment. Cells A431 were less sensitive to toxic effects of leaves and branches extracts but the time dependence was present with the tendency of increased toxicity after chronic treatment. There were no differences in the extent of toxic effects between branches and leaves extracts. The results obtained so far will provide the basis for the future studies with isolated active substances from these extracts.

Immunomodulatory Activity of Leaf Infusions of Selected Cornaceae Species on Human Leukocytes.

Particular Cornus species are known for their edible fruits and their leaves are used as a tea in some countries. In the present study, the immunomodulatory activity of water infusions of leaves of Cornus mas (CM), C. kousa (CK), C. alba (CA), C. flaviramea (CF), C. coreana (CC) and C. amomum (CU) was tested on human leukocytes. Phagocytic and antimicrobial activity was determined for concentrations of 0.17 and 0.25 mg x mL(-1). All samples significantly enhanced the phagocytic activity of cells. Samples at the concentration of 0.17 mg x mL(-1) were in all cases, except CK, slightly more effective than those at 0.25 mg x mL(-1). The strongest bactericidal (E. coli) and also candidacidal (C. albicans) activity was exerted by CM at 0.25 mg x mL(-1). The most potent samples at the lower concentration were CF (for E. coli) and CK (for C. albicans). CC and CU were antimicrobially non-effective.

Lycopus europaeus: phenolic fingerprint, antioxidant activity and antimicrobial effect on clinical Staphylococcus aureus strains.

Lycopus europaeus L. leaves water extract (LEL) was subjected to phytochemical analysis, and evaluated for its antibacterial and antioxidant effects. Antibacterial activity testing was performed on Staphylococcus aureus clinical strains from catheter-related and skin infections by broth microdilution test. LEL showed bactericidal activity at concentrations from 2500 to 5000 µg/mL against all, including methicillin resistant and polyresistant nosocomial, strains. Antioxidant activity was examined using DPPH and ABTS (11.3 and 9.8 µg/mL, respectively) and by ferric reducing ability of the plasma method (891 µmol AAE/g dry extract). Phytochemical analysis of LEL was performed by LC-DAD-MS/MS. Ten phenolic compounds were identified; two minor compounds (glucopyranosyl rosmarinic acid and sagerinig acid) have not been described in Lycopus yet. The major compounds, considered to be responsible for biological activities detected in the study, were determined as rosmarinic acid (76 mg/g) and luteolin-7-O-glucuronide (23 mg/g). L. europaeus arises from our study as a promising source of antibacterial agent for topical usage.

Water Extract of Mentha x villosa: Phenolic Fingerprint and Effect on Ischemia-Reperfusion Injury.

Qualitative analysis of the water extract of Mentha x villosa Huds. leaves was performed by liquid chromatography mass spectrometry (LC-MS/MS) and quantitative analysis was made by reverse-phase liquid chromatography coupled with photodiode array detection (LC-DAD). Sixteen phenolic compounds were identified and quantified consisting of 8 phenolic acids/derivatives and 8 flavonoid glycosides (quinic acid, chlorogenic acid, coumaroyl-hexoside, caffeic acid, coumaroylquinic acid, lithospermic acid, rosmarinic acid, salvianolic acid A, luteolin-7-O-glucuronide, luteolin-7-O-glucoside, luteolin-7-O-rutinoside, eriodictyol-7-O-rutinoside, apigenin-7-O-glucuronide, kaempferol-3-O-glucuronide, chrysoeriol-7-O-rutinoside, and hesperetin-7-O-rutinoside). Luteolin-7- O-rutinoside (25.6 ± 0.7 mg/g dry extract) and rosmarinic acid (17.9 ± 0.4 mg/g dry extract) were the most abundant. High antioxidant activity of this phenolic-rich water extract was confirmed in vitro by DPPH and ABTS tests and ex vivo in the ischemia-reperfusion injured rat superior mesenteric artery. Thus, the water extract of M. x villosa leaves seems to be a promising agent in prevention of tissue injury caused by oxidative stress.

Development of LC-MS method for determination of ursolic acid: application to the analysis of ursolic acid in Staphylea holocarpa Hemsl.

Ursolic acid is a hydroxy pentacyclic triterpene, which has a chemoprotective activity in human. A reliable and reproducible liquid chromatography-mass spectrometric assay (LC-MS) was developed for the determination of ursolic acid in laboratory-made mixtures and in leaves and twigs extracts of Staphylea holocarpa Hemsl. The methanolic solution of the extracted ursolic acid was chromatographically analyzed using Shim Pack CLC-CN, C18 (150 x 6 mm, 5 mu) column and a mobile consisting of methanol-1% acetic acid solution (4:1) at a flow rate of 1.0 ml min(-1). The mass spectrometer (LCQ-Finnigan) was programmed in the positive single ion monitoring (SIM) to permit detection and quantitation of ursolic acid in MS-SIM mode at m/z 439.2, 411.2 and 390.9. Linear correlation (r > 0.99) of the peak area and the concentration of ursolic acid over the concentration range 0.25-10 microg ml(-1) was obtained. The relative standard deviation (%R.S.D.) and percentage deviation from the nominal concentrations (%DEV) were found to be 3.03-3.59% and -4.5 to +6.2%, respectively. Analysis of laboratory-made mixtures containing known concentrations of ursolic acid, as quality control samples, gave a mean recovery percentage of 97.8%. Application of the proposed method for the analysis of leaves and twigs extracts of S. holocarpa Hemsl. gave mean percentage contents of ursolic acid of 0.95 and 0.25%, respectively.

Rosmarinic acid interaction with planktonic and biofilm Staphylococcus aureus.

The subject of study was the evaluation of antibacterial activities of rosmarinic acid (RA) on clinical Staphylococcus aureus strains obtained from catheter-related infections. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) of RA were tested by broth microdilution assay. Biofilm-eradication activity was detected on 24-hour biofilm in microtiter plates using a regrowth technique; activity on biofilm formation was measured by a microtiter plate method after RA application to bacterial samples after 0, 1, 3 and 6 hours of biofilm development. RA had antimicrobial activity on all tested strains in concentrations from 625 to 1250 microg.mL-1 (MICs equal to MBCs). No biofilm-eradication activity on 24-hour biofilm was observed in the tested range of concentrations (from 156 to 5000 microg.mL-1). Subinhibitory RA concentrations suppressed the biofilm production, when applied at early stages of its development. Concentrations lower than subinhibitory stimulated the biofilm mass production in a concentration- and time-dependent manner. Considering our results, RA could be a candidate for a topical antimicrobial agent with killing activity on planktonic forms of bacteria and suppressing activity in the early stages of biofilm development, but probably not for the therapy of catheter-related infections as a sole agent.

Anti-inflammatory potential and fatty acid content of lipophilic leaf extracts of four Staphylea L. species.

Staphylea preparations are used in TCM and have been used by native Americans for a number of indications, such as rheumatism. Based on this knowledge, the anti-inflammatory activity of light petroleum extracts of leaves of Staphylea colchica Stev., S. elegans Zab., S. holocarpa Hemsl. and S. pinnata L. has been determined using in vitro assays for inhibition of cyclooxygenase (COX-1 and COX-2) and leukotriene B4 (LTB4) formation by lipoxygenase (5-LOX). All extracts inhibited COX-1 and COX-2, with S. holocarpa and S. elegans performing best. Inhibition of LTB4 formation was less pronounced. As unsaturated fatty acids are known to inhibit arachidonic acid metabolism in vitro, the fatty acid content was determined of the active extracts and set in correlation with their activity. Unsaturated fatty acids were found to contribute to the observed COX-2 and LTB4 formation inhibitory activity to a different extent.

Inhibition activities of natural products on serine proteases.

Proteases play a key role in a variety of pathologies, including cancer, pancreatitis and thrombosis. Low molecular inhibitors can act as drugs to combat these pathologies. Twelve natural phenolic compounds and one alkaloid were evaluated. Quercetin was used as a standard in the in vitro tests on serine proteases (trypsin, thrombin and urokinase). Salicin showed a highly selective effect with a value of IC50 = 11.4 microm for thrombin, suggesting it may be a suitable lead structure for developing thrombin inhibitors and thus for perspective thrombolytics. Interesting results were also observed for hyperoside with IC50 = 8.3 microm for urokinase. The flavonoid skeleton seems to be a suitable structure for investigating urokinase inhibitors as prospective drugs for cancer therapy. A very high inhibitory activity on trypsin was observed for the flavonoid silybin (IC50 = 3.7 microm), indicating a prospective structure on which to base possible polyphenolic trypsin inhibitors.