Biological investigations of essential oils extracted from three Juniperus species and evaluation of their antimicrobial, antioxidant and cytotoxic activities.

AIMS: To conduct biological investigations and to evaluate the antimicrobial and antioxidant activities of the essential oils (EOs) extracted from Juniperus communis, J. scopulorum and J. horizontalis; to screen their mechanisms of action by conducting the cell membrane permeability assay (CMP); and to determine the possible cytotoxicity of the three EOs against human neuroblastoma cells (SH-SY5Y). METHODS AND RESULTS: The antifungal activity was tested against four phytopathogenic fungi (Monilinia fructicola, Aspergillus niger, Penicillium expansum and Botrytis cinerea). The antibacterial activity was evaluated against two Gram-positive (G+ve) (Bacillus megaterium and Clavibacter michiganensis) and three Gram-negative (G-ve) bacterial strains (Pseudomonas fluorescens, P. syringae pv. phaseolicola and Xanthomonas campestris). Results showed that the three tested EOs have antifungal activity against M. fructicola and P. expansum and effective antibacterial activity against P. syringae pv. phaseolicola and B. megaterium. Moreover, the three EOs were evaluated for their ability to inhibit the growth of SH-SY5Y cells with MTT assay. J. communis EO was the more effective with an IC50 of 53·7 µg ml-1 . The antioxidant capacity of the three EO did not differ as measured by the DPPH assay. CONCLUSIONS: The three tested juniper EOs showed promising antimicrobial and antioxidant activity and cytotoxic effects against human neuroblastoma cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: The outfindings from this research showed promising antimicrobial effects of the three oils against the majority of the tested phytopathogens with a potential to utilize them as natural alternatives to synthetic drugs, the cause of global environmental problems, pathogen resistance and difficulty to control many post-harvest plant diseases.

Bronchodilator, vasodilator and spasmolytic activities of Cymbopogon martinii.

Cymbopogon martinii (Cm.Cr) is traditionally used in south Asian communities for the management of multiple ailments including gastrointestinal, respiratory and vascular disorders and the present study was undertaken to validate these folkloric uses. The application of a methanol extract of the plant (Cm.Cr) to isolated rabbit jejunum preparation exhibited relaxation through decrease in magnitude and frequency of spontaneous contractions. The Cm.Cr also exerted relaxant effect on high K(+) (80 mM) induced contractions in isolated rabbit jejunum preparations. The Cm.Cr and its dichloromethane (Cm.Dcm) and aqueous (Cm.Aq) fractions also caused concentration-dependent relaxation in spontaneous and K(+) (80 mM) induced contractions which are comparables to effects produced by verapamil. Cm.Cr caused shifting of the Ca(2+)-curves toward right, suggesting the presence of a Ca(2+) channel blocking activity. Subsequently, Cm.Cr, Cm.Dcm and Cm.Aq caused relaxation of CCh (1 µM) and K(+) (80 mM) induced contractions in isolated rabbit tracheal preparations, suggesting that the observed relaxant effect can be mediated through antimuscarinic and/or Ca(2+) channel blocking activities. Cm.Cr tested against phenylephrine (PE; 1 µM) and K(+) (80 mM) induced contractions exhibited partial relaxation of isolated rabbit aortic preparations. The above-mentioned studies provided a scientific basis for the folkloric use of Cymbopogon martini in the management of multiple ailments in traditional systems of medicines.

Bronchodilator, vasodilator and spasmolytic activities of methanolic extract of Myrtus communis L.

The present study was undertaken to validate some of the folkloric claims about the effectiveness of the use of a Myrtus communis L. crude methanol extract (Mc.Cr) in gastrointestinal, respiratory and vascular diseases. Mc.Cr caused complete relaxation of spontaneous and K⁺ (80 mM)-induced contractions in isolated rabbit jejunum. It caused right ward parallel shift of calcium concentration response curves. Mc.Cr exhibited relaxant effect on CCh- and K⁺ (80 mM)-induced contractions in isolated rabbit tracheal preparations. Furthermore, Mc.Cr caused relaxation of phenylephrine (1 µM)- and K⁺ (80 mM)-induced contractions in isolated rabbit aorta preparations. These effects were similar to verapamil, a standard calcium channel blocker. These findings could be the basis for explaining the spasmolytic, bronchodilator and vasodilator activities of the extract, through a possible calcium channel blocking activity.

Regulatory T-cell modulation by green tea in chronic lymphocytic leukemia.

Regulatory T cells (Tregs) are considered to be key immunomodulatory cells of the immune system and are increased in chronic lymphocytic leukemia (CLL). Rai stage 0 identifies patients with early stage CLL for which there is no effective intervention at the present time and a "wait and see" policy is usually adopted. Some biological and clinical studies have reported that green tea constituents, such as epigallocatechin-gallate (EGCG), have antitumor effects on hematologic malignancies including CLL. We report data on a clinical trial in which green tea extracts were given orally to 12 patients with stage 0 CLL and 12 healthy subjects. Ten patients and 10 controls completed the 6-month scheduled therapy. Two patients and 2 controls stopped therapy within 1 month because of tachycardia and epigastralgia. Eight out 10 evaluable patients (80 percent) showed a reduction of lymphocytosis and absolute number of circulating Tregs, as well. One patient (10 percent) had a stabilization of lymphocytosis and a reduction of Tregs, and 1 patient (10 percent) showed an increase of both lymphocytosis and Tregs. Only the non-responding patient progressed after 5 months from the end of green tea administration and chemotherapy was given. Interestingly, both IL-10 and TGF-beta serum levels declined throughout the green tea intake period, in both patients and controls. These data seem to indicate that green tea is able to modulate circulating Tregs in CLL patients with early stage of the disease. This can result in the control of lymphocytosis as well as in the prevention of disease progression.

Natural compounds in anti-leukaemic therapy: a review.

Human leukemia results from multiple mutations that lead to abnormalities in the expressions and functions of genes that maintain the delicate balance between proliferation, differentiation and apoptosis. Continued research on the molecular aspects of leukemia cells has resulted in the developments of several potentially useful therapeutic agents. Discovery of new cellular and/or molecular pathways enabling innate or acquired resistance of cancers to current chemotherapeutics to be overcome is therefore of crucial importance if one wants to efficiently combat those cancers associated with dismal prognoses. In this concern, natural compounds are regarded as new chemical entities for the development of drugs against various pharmacological targets, including cancer, and, above all, leukemia.

Synthesis and cytotoxic activity of new ß-carboline derivatives.

On the basis of harmine and 1-methoxy-canthin-6-one chemical structures, a series of novel 1,4-disubstituted and 1,4,9-trisubstituted ß-carbolines and tetracyclic derivatives were designed and synthesized. Cytotoxic activities of these compounds in vitro were investigated in a human tumor cell line panel. Almost all compounds demonstrated interesting cytotoxic activities in particular against prostate cancer cells PC-3 with IC50 in the low micromolar range. Compound X was found to be the most potent one with IC50 value of 8.0 µM; this suggests further studies with models of prostate cancer.

Targeting regulatory T cells for anticancer therapy.

Regulatory T-cells (Tregs) comprise a group of either thymically derived or peripherally induced suppressor CD4+ cells involved in the control of effector T-cells against both self- and foreign-antigens. They are found increased in tumor tissues and are thought to be involved in pathogenesis of cancer by providing tumors with a mechanism to evade immune detection and destruction. Despite the fact that mechanisms of Tregs regulation are still in progress, efforts are made aiming to develop approaches to deplete or inhibit tumor-associated Tregs function. This could lead to restore antitumor immunity and emerging strategies for therapeutic vaccination, and immunotherapeutic targeting of Tregs with specific drugs are underway.

Circulating regulatory T cells in "clinical" monoclonal B-cell lymphocytosis.

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with "clinical" MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to "clinical" MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.

Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia.

We evaluated the pro-apoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater pro-apoptotic ability was shown by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents.

The antiproliferative effects of Uncaria tomentosa extracts and fractions on the growth of breast cancer cell line.

Uncaria tomentosa, also known as "Uña de gato", is a Rubiaceae species widely used in South-American folk medicine for the treatment of cancer, arthritis, gastritis and epidemic diseases. Extracts of the plant have been shown to possess cytostatic and anti-inflammatory activity as well as mutagenic and antimutagenic properties. However, to date no studies have been carried out to verify the direct antitumor activity of the extracts. The present study investigates the effects of some extracts and their chromatographic fractions from the bark of U. tomentosa on the growth of a human breast cancer cell line (MCF7). Our data indicated that, in addition to the antimutagenic activity, U. tomentosa extracts and fractions exert a direct antiproliferative activity on MCF7. The bioassay-directed fractionation from barks and leaves resulted in the isolation of two active fractions, which displayed an IC50 of 10 mg/ml and 20 mg/ml, respectively and an antiproliferative effect, with about 90% of inhibition at a concentration of 100 mg/ml.

Carica candicans Gray (Mito), an alimentary resource from Peruvian flora.

In addition to some histological observations, the chemical composition of Carica candicans Gray (Caricaceae) fruit and seeds, a plant common in Peruvian nutritional habits, was determined. The fruit contains high amounts of total proteins (8.2% on dry weight basis) and carbohydrates (70.1%) and appreciable contents of vitamin C and minerals. The oil extracted from seeds is in high amount (41. 6%). The fatty acid composition, with a prevalence of oleic, palmitic, and linoleic acids, suggests a possible use of this oil in alimentation.

Croton ruizianus: platelet proaggregating activity of two new pregnane glycosides.

The MeOH extract of the aerial parts of Croton ruizianus afforded two new pregnane glycosides 1 and 2, together with the morphinandienone alkaloids flavinantine (3) and O-methylflavinantine (4). Their structures were elucidated by NMR experiments including 1H-1H (1D TOCSY and 2D DQF-COSY) and 1H-13C (HSQC, HMBC) spectroscopy. The proaggregating activity of the MeOH extract and the isolates were evaluated. Although the MeOH extract and pregnane glycosides (at different doses) were found to promote platelet aggregation, flavinantine (3) and O-methylflavinantine (4) showed only slight activity. The ability of the MeOH extract and the four compounds to act synergistically with thrombin was also evaluated. All the tested compounds were successful in augmenting the aggregating effect of thrombin, although to different degrees.

Porrigenins A and B, novel cytotoxic and antiproliferative sapogenins isolated from Allium porrum.

Four new sapogenins, porrigenins A (2a) and B (3a), identified as (25R)-5 alpha-spirostan-2 beta,3 beta,6 beta-triol and (25R)-2-oxo-5 alpha-spirostan-3 beta,6 beta-diol, respectively, and neoporrigenins A (2b) and B (3b) were also isolated from Allium porrum. In addition, the known agigenin (1a) and its 25S epimer, neoagigenin (1b), were also identified. Their structure elucidation was provided by comprehensive spectroscopic analyses. Compounds 1a, 2a, and 3a exhibited cytotoxicity and high antiproliferative activity on four different tumor cell lines in vitro.

The flavonoids of Allium neapolitanum.

An investigation of the extracts from Allium neapolitanum has led to the isolation of 13 flavonoid glycosides, based on kaempferol, quercetin and isorhamnetin. Four of them are new compounds and have been identified as: kaempferol 3-O-[[2-O-alpha-L-rhamnopyranosyl-4-O-beta-D-glucopyranosyl]-beta-D- glucopyranoside]], isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta- D-glucopyranosyl]-beta-D-glucopyranoside], isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta-D-glycopyranosyl] beta-D-glucopyranoside]-7-O-beta-D-glucopyranoside and isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta-D-gentiobiosyl]- beta-D-glucopyranoside]]. The isolated compounds were evaluated for their anti-aggregation human platelet activity.

The flavonoids of Allium ursinum.

From wild garlic Allium ursinum three new flavonoid glycosides were identified as kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)]-beta -D- glucopyranoside, kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-feruloyl)]-beta-D- glucopyranoside, kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)-3-O-b eta-D- glucopyranosyl]-beta-D-glucopyranoside and characterized as the peracetates. Additionally, two known flavonoid glycosides kaempferol 3-O-beta-glucopyranoside and kaempferol 3-O-beta-neohesperidoside were isolated. The isolated compounds showed an inhibition of human platelet aggregation.

Studies on the inhibitory effects of caffeoylquinic acids on monocyte migration and superoxide ion production.

Three caffeoylquinic acids, isolated from the Peruvian plants Tessaria integrifolia and Mikania cordifolia that are used medicinally as anti-inflammatory agents, were tested for their activities on monocyte migration and superoxide anion production. 3,5-Di-O-caffeoylquinic and 4,5-di-O-caffeoylquinic acids exhibited an appreciable anti-inflammatory activity in vitro, while the tricaffeoyl derivative was inactive.

Studies of migration of potentially genotoxic compounds into water stored in pet bottles.

Coloured polyethylene terephthalate (PET) bottles for carbonated beverages were studied for potential migration of genotoxic compounds. A combined approach, using physicochemical methods and a bacterial short-term mutagenicity test (Ames test) was followed. Standard EEC and US FDA tests for total migration of non-volatile migrant compounds into distilled water were performed, together with modified tests, where freeze-drying instead of evaporation of water was used, in order to measure both volatile and non-volatile chemicals. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on these residues. PET bottles filled with naturally carbonated mineral water were also used for long-term total organic carbon (TOC) and mutagenicity migration studies (up to 6 months' storage). Total migration results for PET bottles were within the EEC and US FDA limits. The use of freeze-drying for the elimination of water enabled much higher total migration data (higher than the limits) to be revealed. Some potentially genotoxic compounds (acetaldehyde, dimethyl terephthalate, terephthalic acid) were identified in these migrant compounds by GC-MS analysis. The tests for TOC migration gave a maximum value after 2 wk storage and the mutagenicity tests on non-volatile migrant compounds gave always negative results.

Potential allelochemicals fromRuta graveolens L. and their action on radish seeds.

An aqueous extract ofRuta graveolens L. (250 g/liter) was tested for its allelopathic activity in vitro on radish germination and radicle growth in light and darkness. It caused a delay in the onset and a decrease in the rate of germination (40%) in the light. The photoinhibition of germination was accompanied by an inhibition of water uptake into the seed. Furthermore, the inhibition of radicle growth was slightly higher in the light than in darkness. Three potential allelochemicals, biologically active in the light, were isolated from the extract: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and 4-hydroxy-coumarin at concentrations of 10(-4) M, 2×10(-4) M, and 0.4 ×10(-5) M respectively. At a concentration of 2×10(-4) M, 5-MOP was the most potent inhibitor, decreasing radish germination to 32% and radicle growth to 17% with respect to control. Microscopic observations of radish seeds treated with 5-MOP suggest that this substance changes the swelling of the seed coat and aleurone layer, which precedes radicle protrusion.