Mycochemicals against Cancer Stem Cells.

Since ancient times, mushrooms have been considered valuable allies of human well-being both from a dietary and medicinal point of view. Their essential role in several traditional medicines is explained today by the discovery of the plethora of biomolecules that have shown proven efficacy for treating various diseases, including cancer. Numerous studies have already been conducted to explore the antitumoural properties of mushroom extracts against cancer. Still, very few have reported the anticancer properties of mushroom polysaccharides and mycochemicals against the specific population of cancer stem cells (CSCs). In this context, ß-glucans are relevant in modulating immunological surveillance against this subpopulation of cancer cells within tumours. Small molecules, less studied despite their spread and assortment, could exhibit the same importance. In this review, we discuss several pieces of evidence of the association between ß-glucans and small mycochemicals in modulating biological mechanisms which are proven to be involved with CSCs development. Experimental evidence and an in silico approach are evaluated with the hope of contributing to future strategies aimed at the direct study of the action of these mycochemicals on this subpopulation of cancer cells.

Convolvulus pluricaulis Choisy's Extraction, Chemical Characterization and Evaluation of the Potential Effects on Glycaemic Balance in a 3T3-L1 Adipocyte Cell Model.

Convolvulus pluricaulis (CP) is a common Indian herb, largely employed in Ayurvedic medicine and known for its neuroprotective and neuroinflammatory action. Its effectiveness against several pathologic/sub-pathologic conditions is widely accepted, but it is not yet completely chemically characterized. In recent years, several researchers have pointed out the involvement of CP and other Convolvulaceae in lipidic and glucidic metabolism, particularly in the control of hyperlipidaemia and diabetic conditions. In this scenario, the aim of the study was to chemically characterize the medium polarity part of the CP whole plant and its fractions and to shed light on their biological activity in adipocyte differentiation using the 3T3-L1 cell model. Our results demonstrated that the CP extract and fractions could upregulate the adipocyte differentiation through the modulation of the nuclear receptor PPARγ (Peroxisome Proliferator-Activated Receptor γ), broadly recognized as a key regulator of adipocyte differentiation, and the glucose transporter GLUT-4, which is fundamental for cellular glucose uptake and for metabolism control. CP also showed the ability to exert an anti-inflammatory effect, downregulating cytokines such as Rantes, MCP-1, KC, eotaxin, and GM-CSF, which are deeply involved in insulin resistance and glucose intolerance. Taken together, these data suggest that CP could exert a potential beneficial effect on glycemia and could be employed as an anti-diabetic adjuvant or, in any case, a means to better control glucose homeostasis.

A Comparative Study on Chemical Compositions and Biological Activities of Four Amazonian Ecuador Essential Oils: Curcuma longa L. (Zingiberaceae), Cymbopogon citratus (DC.) Stapf, (Poaceae), Ocimum campechianum Mill. (Lamiaceae), and Zingiber officinale Roscoe (Zingiberaceae).

Essential oils (EOs) and their vapour phase of Curcuma longa (Zingiberaceae), Cymbopogon citratus (Poaceae), Ocimum campechianum (Lamiaceae), and Zingiber officinale (Zingiberaceae) of cultivated plants grown in an Amazonian Ecuador area were chemically characterised by Gas Chromatography-Flame Ionization Detector (GC-FID), Gas Chromatography-Mass Spectrometry (GC-MS), and Head Space-Gas Chromatograph-Flame Ionization Detector-Mass Spectrometry (HS-GC-FID-MS).figure The EOs analyses led to the identification of 25 compounds for C. longa (99.46% of the total; ar-turmerone: 23.35%), 18 compounds for C. citratus (99.59% of the total; geraniol: 39.43%), 19 compounds for O. campechianum (96.24% of the total; eugenol: 50.97%), and 28 for Z. officinale (98.04% of the total; α-Zingiberene: 15.45%). The Head Space fractions (HS) revealed C. longa mainly characterised by limonene and 1,8-cineole (37.35%) and α-phellandrene (32.33%); Z. officinale and C. citratus showed camphene (50.39%) and cis-Isocitral (15.27%) as the most abundant compounds, respectively. O. campechianum EO revealed a higher amount of sesquiterpenes (10.08%), mainly characterised by E-caryophyllene (4.95%), but monoterpene fraction remained the most abundant (89.94%). The EOs were tested for antioxidant, antimicrobial, and mutagen-protective properties and compared to the Thymus vulgaris EO as a positive reference. O. campechianum EO was the most effective in all the bioactivities checked. Similar results emerged from assaying the bioactivity of the vapour phase of O. campechianum EO. The antioxidant and antimicrobial activity evaluation of O. campechianum EO were repeated through HP-TLC bioautography assay, pointing out eugenol as the lead compound for bioactivity. The mutagen-protective evaluation checked through Ames's test properly modified evidenced a better capacity of O. campechianum EO compared with the other EOs, reducing the induced mutagenicity at 0.1 mg/plate. However, even with differences in efficacy, the overall results suggest important perspectives for the functional use of the four studied EOs.

Perillaldehyde is a new ferroptosis inducer with a relevant clinical potential for acute myeloid leukemia therapy.

Ferroptosis induction is an emerging strategy to treat cancer and contrast the tricky issue of chemoresistance, which can arise towards apoptosis. This work elucidates the anticancer mechanisms evoked by perillaldehyde, a monoterpenoid isolated from Ammodaucus leucotrichus Coss. & Dur. We investigated and characterized its antileukemic potential in vitro, disclosing its ability to trigger ferroptosis. Specifically, perillaldehyde induced lipid peroxidation, decreased glutathione peroxidase 4 protein expression, and depleted intracellular glutathione on HL-60 promyelocytic leukemia cells. Besides, it stimulated the active secretion of ATP, one of the most crucial events in the induction of efficient anticancer response, prompting further studies to disclose its possible nature as an immunogenic cell death inducer. To preliminarily assess the clinical relevance of perillaldehyde, we tested its ability to induce cell death on patient-derived acute myeloid leukemia biopsies, recording a similar mechanism of action and potency compared to HL-60 cells. To round the study off, we tested its selectivity towards tumor cells and disclosed lower toxicity on normal cells compared to both HL-60 and acute myeloid leukemia biopsies. Altogether, these data depict a favorable risk-benefit profile for perillaldehyde and reveal its peculiar antileukemic potential, which qualifies this natural product to proceed further through the drug development pipeline.

Wild Italian Hyssopus officinalis subsp. aristatus (Godr.) Nyman: From Morphological and Phytochemical Evidences to Biological Activities.

Three specimens of H. officinalis subsp. aristatus were collected in three areas of the Abruzzo region (Italy) and subjected to macroscopic and microscopic observation to support their botanical identification. The essential oils (EOs) obtained from the aerial parts of the samples were characterized with the object to define their phytochemical and pharmaceutical biology profile. They highlight three different chemotypes, including one never seen in previous literature (CIV17-EO, distilled from sample harvested in 2017 at Civitaretenga), that showed a fingerprinting with the predominance of (-)-limonen-10-yl-acetate (67.9%). In 2017 European Food Safety Authority (EFSA) reported the genotoxicity of similar compounds, therefore, to dismiss any safety concern for the CIV17-EO use as flavouring substance, the Ames test was performed with no evidence of mutagenic activity. Safety of use coupled with chemical characterization of this new chemotype set the stage for a better standardization of H. officinalis EOs. The ethanolic extracts, on the other hand, with qualitatively similar chemical profiles in which caftaric, chlorogenic and rosmarinic acid were the main molecules, showed interesting antioxidant activity and a slight cytotoxicity towards the A549 cell line that could indicate a starting point for the evaluation of an additional preventive tool for maintaining health status.

Cancer stem cells and nanomedicine: new opportunities to combat multidrug resistance?

'Multidrug resistance' (MDR) is a difficult challenge for cancer treatment. The combined role of cytochrome P450 enzymes (CYPs) and active efflux transporters (AETs) in cancer cells appears relevant in inducing MDR. Chemotherapeutic drugs can be substrates of both CYPs and AETs and CYP inducers or inhibitors can produce the same effects on AETs. In addition, a small subpopulation of cancer stem-like cells (CSCs) appears to survive conventional chemotherapy, leading to recurrent disease. Natural products appear efficacious against CSCs; their combinational treatments with standard chemotherapy are promising for cancer eradication, in particular when supported by nanotechnologies.

Hemidesmus indicus induces apoptosis via proteasome inhibition and generation of reactive oxygen species.

Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H. indicus's phytochemicals on leukemia cells and their intestinal absorption on a human intestinal epithelium model consisting of a monolayer of differentiated Caco2 cells. We observed a potent antileukemic effect for H. indicus, imputable to the modulation of different critical targets at protein and mRNA levels and the reduction of the 26S proteasome expression. We found that some phytomarkers of H. indicus decoction passed through the enterocyte monolayer. Overall, our study supports the pharmacological potential of H. indicus, which can represent an interesting botanical drug in the oncological area.

Rediscovering Medicinal Amazonian Aromatic Plants: Piper carpunya (Piperaceae) Essential Oil as Paradigmatic Study.

Piper carpunya Ruiz & Pav. (Piperaceae) is a perennial aromatic shrub of Amazonian area of Ecuador and Peru, belonging to the ethnomedicine of these countries. The traditional preparations of the crude drug (fresh leaves used topically as is, and dried leaves in infusions or decoctions) are known for anti-inflammatory, antiulcer, antidiarrheal, antiparasitic effects, and wound healing properties. In light of this traditional evidence, chemical composition (GC-MS) and biological activity, i.e., antioxidant, antifungal (yeast) capacities, and genotoxicity, of Amazonian P. carpunya leaf essential oil (EO) have been investigated in order to valorize some of the putative ethnomedical effects. The EO was obtained through steam distillation of fresh leaves (yield: 7.6 g/kg [0.76%]; refractive index at 20°C: 1.49; density: 0.928 g/mL). Chemical characterization performed through GC-MS evidenced the presence of 21 compounds (96.2% of the total). The most abundant constituents were piperitone (26.2%), limonene (9.5%), elemicin (7.2%), and ß-phellandrene (5.6%). In vitro DPPH antioxidant assay showed a weak radical scavenging ability (IC50) if compared to positive control. Low bioactivity of the EO was also demonstrated against yeast, but it showed an interesting synergistic activity (FIC index of EO+fluconazole) against Candida sp. strains. Ames test evidenced the safety of the EO concerning genotoxicity.

In Vitro Study of the Cytotoxic, Cytostatic, and Antigenotoxic Profile of Hemidesmus indicus (L.) R.Br. (Apocynaceae) Crude Drug Extract on T Lymphoblastic Cells.

In traditional Indian medicine, the crude drug Hemidesmus indicus root-commonly known as Indian sarsaparilla-is used alone or in poly-herbal preparations for the treatment of a wide range of diseases. The present study focuses on the cancer chemopreventive and therapeutic potential of H. indicus extracts on an acute lymphoblastic leukemia cell line (CCRF-CEM). With this aim in mind, we subjected H. indicus roots to two subsequent extractions (hydro-alcoholic extraction and soxhlet extraction). As DNA damage is an important prerequisite for the induction of mutations/cancer by genotoxic carcinogens, cancer chemoprevention may be achieved by preventing genotoxicity. Through an integrated experimental approach, we explored the genoprotective potential of the soxhlet H. indicus extract against different mutagenic compounds and its cytotoxic, proapoptotic, and cytostatic properties. In our experimental conditions, H. indicus induced a cytotoxic effect involving the activation of both intrinsic and extrinsic apoptotic pathways and blocked the cell cycle in the S phase. Moreover, the antigenotoxicity results showed that the extract was able to mitigate DNA damage, an essential mechanism for its applicability as a chemopreventive agent, via either the modulation of extracellular and intracellular events involved in DNA damage. These data add to the growing body of evidence that H. indicus can represent a noteworthy strategy to target early and late stages of cancer.

Multidrug resistance in cancer or inefficacy of neuroactive agents: innovative strategies to inhibit or circumvent the active efflux transporters selectively.

Multidrug resistance (MDR) is a crucial issue in the treatment of cancer cells that protect themselves by overexpression of active efflux transporters (AETs). AET expression maintains the homeostasis in healthy tissues and in the blood-brain barrier it often prevents drugs from reaching the brain. Inhibition of AETs could therefore be a valuable solution for preventing MDR; but nonselective long-term AET blocking can be harmful toward healthy tissues and, in particular, the brain. This review looks at the development of innovative formulations suitable for selectively blocking or avoiding AETs as promising ways to overcome the challenges of MDR and inefficacy of neuroactive agents.

Brain uptake of a Zidovudine prodrug after nasal administration of solid lipid microparticles.

Our previous results demonstrated that a prodrug obtained by the conjugation of the antiretroviral drug zidovudine (AZT) with ursodeoxycholic acid (UDCA) represents a potential carrier for AZT in the central nervous system, thus possibly increasing AZT efficiency as an anti-HIV drug. Based on these results and in order to enhance AZT brain targeting, the present study focuses on solid lipid microparticles (SLMs) as a carrier system for the nasal administration of UDCA-AZT prodrug. SLMs were produced by the hot emulsion technique, using tristearin and stearic acid as lipidic carriers, whose mean diameters were 16 and 7 µm, respectively. SLMs were of spherical shape, and their prodrug loading was 0.57 ± 0.03% (w/w, tristearin based) and 1.84 ± 0.02% (w/w, stearic acid based). The tristearin SLMs were able to control the prodrug release, whereas the stearic acid SLMs induced a significant increase of the dissolution rate of the free prodrug. The free prodrug was rapidly hydrolyzed in rat liver homogenates with a half-life of 2.7 ± 0.14 min (process completed within 30 min). The tristearin SLMs markedly enhanced the stability of the prodrug (75% of the prodrug still present after 30 min), whereas the stabilization effect of the stearic acid SLMs was lower (14% of the prodrug still present after 30 min). No AZT and UDCA-AZT were detected in the rat cerebrospinal fluid (CSF) after an intravenous prodrug administration (200 µg). Conversely, the nasal administration of stearic acid based SLMs induced the uptake of the prodrug in the CSF, demonstrating the existence of a direct nose-CNS pathway. In the presence of chitosan, the CSF prodrug uptake increased six times, up to 1.5 µg/mL within 150 min after nasal administration. The loaded SLMs appear therefore as a promising nasal formulation for selective zidovudine brain uptake.

Development and characterization of PLGA nanoparticles as delivery systems of a prodrug of zidovudine obtained by its conjugation with ursodeoxycholic acid.

CONTEXT: Zidovudine (AZT) is employed against AIDS and hepatitis; its use is limited by active efflux transporters (AETs) that induce multidrug resistance for intracellular therapies and hamper AZT to reach the brain. Ursodeoxycholic acid (UDCA) conjugation with AZT (prodrug UDCA-AZT) allows to elude the AET systems. OBJECTIVE: To investigate the effect of the Pluronic F68 coating on the loading, release and stability of poly(D,L lactide-co-glicolide) nanoparticles (NPs) embedded with UDCA-AZT. MATERIALS AND METHODS: The mean diameter of the NP prepared by nanoprecipitation or emulsion/solvent evaporation methods was determined using both photon correlation spectroscopy and sedimentation field-flow fractionation; particle morphology was detected by scanning electron microscope. The stability of the free and encapsulated UDCA-AZT was evaluated in rat liver homogenates by high-performance liquid chromatography analysis. RESULTS AND DISCUSSION: The mean diameter of the NPs was found to be ∼ 600 nm with a relatively high polydispersity. The NPs obtained by emulsion/solvent evaporation were not able to control the prodrug release, differently from NPs obtained by nanoprecipitation. The presence of the Pluronic coating did not substantially modify the kinetics of the drug release, or the extent of the burst effect that were instead only influenced by the preparation parameters. UDCA-AZT incorporated in the NPs was more stable in the rat liver homogenates than the free prodrug and no influence of the Pluronic coating was observed. CONCLUSIONS: Considering the different potential applications of nanoparticles coated and uncoated with Pluronic (brain and macrophage targeting, respectively), both of these nanoparticle systems could be useful in the therapies against HIV.

Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil as possible mutagen-protective food ingredient against heterocyclic amines from cooked food.

The Amazonian Croton lechleri stem bark essential oil was tested for its anti-mutagenic potential by performing the Ames test against heterocyclic amines (HCAs), in continuing research on applicative functional profile of this phytocomplex as food ingredient (Rossi et al., 2011). Salmonella typhimurium strain TA98 was used with and without metabolic activation (S9 mix). The anti-mutagenic properties was assayed with the following HCAs: 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx), the imidazoles 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-aminodipirydo-[1,2-a:3',2'-d]imidazole (Glu-P-2). All HCAs with S9 induced mutagenicity at 10(-10) mol/plate. Without S9, IQ and MeIQ showed mutagenicity at 10(-8) mol/plate, MeIQx and Glu-P-1 at 10(-5) mol/plate, while Glu-P-2 was inactive. In presence of HACs (10(-9) mol/plate), C. lechleri essential oil was tested for mutagen-protective properties (concentration range: 0.01-0.10 mg/plate) taking the Highest Uneffective Dose (HUD) as threshold reference. With S9 mix, C. lechleri essential oil displayed a significant reduction of revertants at 0.05 mg/plate, from 21% to 34%. The essential oil showed mutagen-protective efficacy against IQ and MeIQ tested as direct mutagens (10(-7) mol/plate), with a revertants percentage reduction of 39% and 40%, respectively. No anti-mutagen capacity was noted for MeIQx and Glu-P-1 (10(-5) mol/plate). Since HACs are known as possible colon and liver cancer inducers, C. lechleri essential oil was tested for its cytotoxicity and anti-proliferative capacity against LoVo and HepG2 cancer cell lines showing IC50 of 74.95±0.05 µg/ml (LoVo) and 82.28±0.03 µg/ml (HepG2), displaying a promising role of this essential oil as a functional food ingredient with interesting mutagen preventing properties.

Zidovudine and ursodeoxycholic acid conjugation: design of a new prodrug potentially able to bypass the active efflux transport systems of the central nervous system.

We have synthesized a new prodrug obtained by the 5'-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA-AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half-life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA-AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA-AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 µM AZT across this monolayer was regulated by apparent permeability coefficients (P(E)) higher from the apical to basolateral compartments (P(E) = 209 ± 4 × 10⁻5 cm/min) than in the opposite way (P(E) = 133 ± 8 × 10⁻5 cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P(E) = 39.1 ± 1.2 × 10⁻5 cm/min) and efflux (P(E) = 31.3 ± 3.6 × 10⁻5 cm/min) permeability coefficients of 30 µM UDCA-AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P(E) values of UDCA-AZT were associated with a partial hydrolysis during its permeation across the cell monolayer.

Dopamine-sensitive adenylyl cyclases in neuronal development: physiopathological and pharmacological implications.

Pharmacological studies of molecular mechanisms leading to the differentiation of neurons with retained dopaminergic fate and function suggest that such differentiation could be a form of treatment of neurodegenerative disorders, such as Parkinson's disease (PD) and schizophrenia. This goal could be achieved by neuronal replacement therapies based upon the manipulation of endogenous precursors in situ or by transplantation-based approaches. Signals conveyed by the adenylyl cyclase (AC) pathway appear to be crucial for the suitable differentiation of neurons. Here, we discuss dopamine (DA)-sensitive isoforms of AC as key cues for dopaminergic neuronal patterning and as interesting therapeutic targets for the induction of regenerative processes or to drive correct neuronal development.

Chemical characterization (GC/MS and NMR Fingerprinting) and bioactivities of South-African Pelargonium capitatum (L.) L' Her. (Geraniaceae) essential oil.

Chemical fingerprinting of commercial Pelargonium capitatum (Geraniaceae) essential oil samples of south African origin was performed by GC, GC/MS, and (13) C- and (1) H-NMR. Thirty-seven compounds were identified, among which citronellol (32.71%) and geraniol (19.58%) were the most abundant. NMR Spectra of characteristic chemicals were provided. Broad-spectrum bioactivity properties of the oil were evaluated and compared with those of commercial Thymus vulgaris essential oil with the aim to obtain a functional profile in terms of efficacy and safety. P. capitatum essential oil provides a good performance as antimicrobial, with particular efficacy against Candida albicans strains. Antifungal activity performed against dermatophyte and phytopathogen strains revealed the latter as more sensitive, while antibacterial activity was not remarkable against both Gram-positive and Gram-negative bacteria. P. capitatum oil provided a lower antioxidant activity (IC(50) ) than that expressed by thyme essential oil, both in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ß-carotene bleaching tests. Results in photochemiluminescence (PCL) assay were negligible. To test the safety aspects of P. capitatum essential oil, mutagenic and toxicity properties were assayed by Ames test, with and without metabolic activation. Possible efficacy of P. capitatum essential oil as mutagenic protective agent against NaN(3) , 2-nitrofluorene, and 2-aminoanthracene was also assayed, providing interesting and significant antigenotoxic properties.

Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells.

AIMS: Human amnion-derived cells have been used as in vitro models to test the release of inflammatory mediators, such as arachidonic acid (AA) and prostaglandin E(2) (PGE(2)). We compared estrogen metabolites for their ability to induce AA release, to influence PGE(2) production and to interact toward intracellular estrogen receptors (ERs). MAIN METHODS: Metabolite effects on AA and PGE(2) release were examined by radiolabelled substrate incorporation and by colorimetric enzyme immunoassays, respectively. [(3)H]17-ß-estradiol binding displacements were performed on Ro-20-1724 treated whole cells. KEY FINDINGS: In WISH cells, estrone, 2-hydroxy-estrone and estriol induced a rapid dose dependent release of AA that was not inhibited by cycloheximide. Estrone and 2-hydroxy-estrone showed biphasic dose-response curves of PGE(2), whereas estriol and 16-α-hydroxy-estrone increased PGE(2) levels at high concentrations. 2-methoxy-estrone, 4-hydroxy-estradiol and 4-hydroxy-estrone did not significantly affect PGE(2) release. 2-methoxy-estradiol and 2-hydroxy-estradiol decreased the PGE(2) release. Effects of metabolites on PGE(2) were inhibited by cycloheximide and by the ER antagonist tamoxifen. In AV3 cells PGE(2) production was poorly detectable. On Ro-20-1724 treated WISH cells the K(i) of 17-ß-estradiol was 29.2 ± 5.4 nM. Estrone, 2-methoxy-estrone and 2-methoxy-estradiol showed similar affinity values. The hydroxyl substituent at position 2, 4 and 16 decreased or markedly increased the affinity for estradiol or estrone derivatives, respectively. SIGNIFICANCE: The estrogen metabolites induced nongenomic effects on AA release from WISH cells. The influence on PGE(2) release was detectable only on WISH cells. These effects appeared genomic and mediated by intracellular ERs, whose properties seemed strongly dependent on intracellular cAMP levels.

Bioactivities of Piper aduncum L. and Piper obliquum Ruiz & Pavon (Piperaceae) essential oils from Eastern Ecuador.

Essential oils from aerial parts of Piper aduncum (Matico) and Piper obliquum (Anis del Oriente) of ecuadorian origin were analyzed by GC-FID, GC-MS, (13)C NMR and their biological and pharmacological activities were assessed. Chemical composition proved to be unusually different from previous reports for safrole-rich P. obliquum (45.8%), while P. aduncum main constituent was dillapiol (45.9%). No genotoxic activity was found in the Ames/Salmonella typhimurium (TA98 and TA100) assay, either with or without S9 activation. Mutagen-protective properties, evaluated using sodium azide, 2-nitrofluorene and 2-aminoanthracene as mutagens/promutagens, was observed against promutagen 2-aminoanthracene, likely in consequence of microsomial deactivation. Antimicrobial assays have been performed on Gram+/Gram- bacteria, dermatophyte and phytopathogenic fungi and best results were provided by P. aduncum against fungal strains with complete inhibition at 500µg/ml. Preliminary analgesic and antithrombotic activities evidenced the absence of the former in hot plate and edema assays and a limited antiplatelet action against three different agonists (ADP, AA and U46619). Both oils have a very limited antioxidant capacity.

An RPE cell line as a useful in vitro model for studying retinoic acid receptor beta: expression and affinity.

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-beta is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-beta can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-beta-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-beta and none of the other receptor subtypes. In addition, we show that the expression of RAR-beta increases with increasing passage number of the cells. Interestingly, the increase in RAR-beta expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-beta-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-beta. Using [(3)H]CD367 as the ligand, RAR-beta in HRPE cells showed an affinity of 9.6 +/- 0.6 nM and a B(max) of 780 +/- 14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-beta by investigating the ability of certain flavonoids to inhibit the binding of [(3)H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-beta was between approx. 1-30 microM, showing that the flavonoids interact with RAR-beta with low affinity.

Antimutagenic, antioxidant and antimicrobial properties of Maytenus krukovii bark.

The hydroalcoholic extract of Maytenus krukovii bark was investigated for its in vitro mutageno-protective activities by means of the Ames Salmonella/microsome assay. The extract showed an inhibitory effect in both T98 and T100 strains against the mutagenic activity of promutagen 2-aminoanthracene but was not protective against directly acting mutagens sodium azide and 2-nitrofluorene. When tested as a radical scavenger and antioxidant it produced a dose-dependent inhibition. The extract did not show significant antibacterial properties, and was weakly active against dermatophyte and phytopathogenic fungi, but inhibited the growth of phytopathogen Pithyum ultimum.