Franco Matías Escobar

Safety assessment of essential oil from Minthostachys verticillata (Griseb.) Epling (peperina): 90-days oral subchronic toxicity study in rats.

Minthostachys verticillata (Lamiaceae), popularly known as peperina is largely used in popular medicine for its digestive, carminative, antispasmodic and antirheumatic properties. There are no reports of repeated exposure toxicity to guarantee their safety. The present study investigated the chemical composition, analyzed by GC-FID, and the 90-day toxicity and genotoxicity effect of M. verticillata essential oil (Mv-EO), using Wistar rats as test animals. The rats were divided into four groups (5 rats/sex/group) and Mv-EO was administered on diet at doses of 0, 1, 4 and 7 g/kg feed. The main components of Mv-EO were pulegone (64.65%) and menthone (23.92%). There was no mortality, adverse effects on general conditions or changes in body weight, food consumption and feed conversion efficiency throughout the study in male and female rats. Subchronic administration of Mv-EO did not alter the weights, morphological and histopathological analyses of liver, kidney and intestine. Genotoxicity was tested by micronucleus and comet assays. Mv-EO up to a concentration of 7 g/kg feed for 90 days did not exert a cyto-genotoxic effect on the bone marrow and cells blood of Wistar rats. These results suggest that Mv-EO appears to be safe and could be devoid of any toxic risk.

Evaluation of antiviral activity of aqueous extracts from Achyrocline satureioides against Western equine encephalitis virus

Achyrocline satureioides (Asteraceae) is a medicinal plant traditionally used in Argentina for the treatment of intestinal infections and various digestive disorders. Its infusion is widely utilised for respiratory problems and viral infections. The objective of this study was to investigate cytotoxicity, virucidal and antiviral properties of the cold aqueous extract (CAE) and hot aqueous extract (HAE) of this plant against Western equine encephalitis virus (WEEV). Cytotoxicity in Vero cells was evaluated by maximum non-cytotoxic concentration (MNCC), neutral red (NR) uptake and MTT reduction methods. To study the antiviral activity of aqueous extracts, plaque reduction assay was performed after pre-treatment of host cells, adsorption, penetration and post-penetration of the virus. Extracellular virus inactivation was also analysed by the same method. Extracts showed strong inhibitory activity after virus penetration with selective index values of 32 (NR) and 63.3 (MTT) for the CAE, and 16.2 (NR) and 24.3 (MTT) for the HAE. Both extracts exhibited virucidal action with lower efficacy than their antiviral properties. The present results demonstrate that aqueous extracts of A. satureioides are active against WEEV. Further studies are needed in order to identify which compounds could be responsible for this effect, and how they exert antiviral action.

Apoptosis and mutagenicity induction by a characterized aqueous extract of Baccharis articulata (Lam.) Pers. (Asteraceae) on normal cells

In a previous study we have demonstrated that cold aqueous extract of Baccharis articulata (Ba-CAE) induced the death of human peripheral blood mononuclear cells (PBMCs) and exerted low mutagenic effects on mice at 6h after administration. The aim of this work was to investigate whether the PBMCs death induced by Ba-CAE is due to apoptosis, and whether this extract exerts mutagenic effects on mice at 24 and 48h after administration. In addition, Ba-CAE was chemically characterized. PBMCs from healthy volunteers were exposed to extract (10, 20, 40, 80, 160, 320, 640 and 1280μg/mL) for 18-24h. Cell viability was determined by staining of trypan blue dye exclusion method. Apoptosis was determined by Hoechst 33258 staining, TUNEL, and DNA fragmentation analysis by agarose gel electrophoresis. BALB/c mice were injected with extract (1800, 900 and 450mg/kg) and sacrificed at 24 and 48h postinjection. Bone marrow samples were used to assess chromosome mutations by the micronucleus test. The extract induced PBMCs death by apoptosis and increased the frequency of micronuclei in bone marrow. The phytochemical study of Ba-CAE showed the presence of flavones as luteolin and acacetin, caffeoylquinic acids as chlorogenic acid, and tannins.

Monoterpenes isolated from Minthostachys verticillata (Griseb.) Epling essential oil modulates immediate-type hypersensitivity responses in vitro and in vivo.

In a previous work, we have demonstrated that Minthostachys verticillata essential oil has immunomodulatory effects in vitro on cells from allergic patients. Here we characterized main components of M. verticillata essential oil and also tested if these compounds modulate In vitro and in vivo the immediate-type allergic reaction. Gas chromatography was used to identify main components of the essential oil. Pulegone (63.4 %), menthone (15.9 %), and limonene (2.1 %) were found as main classes. IL-13 levels were evaluated from lymphocytes cultures stimulated with allergen alone or combined with monoterpenes. All compounds stimulated cell proliferation but, interestingly, promoted a reduction of IL-13 values, limonene and the mixture of the three compounds being the most active. β-Hexosaminidase release was determined from basophils to which essential oil or monoterpenes were added. We observed that, whichever combination of monoterpenes was used, β-hexosaminidase release was diminished in all cases. Obtained values were even lower than those of antiallergic drug desloratadine. Essential oil and limonene inhibited mast cell activation and degranulation in the skin when testing passive cutaneous anaphylaxis, limonene being the most active. In conclusion, limonene was the compound that showed the most potent immunomodulatory activity. This fact suggests that it constitutes a promising natural alternative for a novel treatment of allergic diseases.

Evaluation of the cytotoxicity, genotoxicity and apoptotic induction of an aqueous extract of Achyrocline satureioides (Lam.) DC

Achyrocline satureioides is widely consumed as infusion or aperitif and shows important therapeutic properties. Previously, we reported absence of genotoxicity of cold aqueous extract (CAE) of A. satureioides by Allium test. However, one test cannot predict the genotoxic effects of a substance. Thus, the aim of this work was to investigate cytotoxicity, genotoxicity and apoptotic ability of CAE of A. satureioides. In addition, CAE was chemically characterized. The cytotoxicity was evaluated by Trypan blue and MTT assays. The apoptotic capacity was evaluated by Hoechst staining and DNA fragmentation-analysis. The genotoxicity was studied by comet assay (CA) and micronucleus test. The identification and quantification of flavonoids were performed by HPLC-ESI-MS/MS. The cytotoxicity studies indicated low toxicity of CAE. In addition, CAE did not induce apoptotic effects on human PBMCs. CAE did not show genotoxicity in vitro against Vero cells, at 10-50 μg/mL. CAE did not induce in vivo genotoxic effects, but it showed at high concentrations cytotoxicity by micronucleus assay. CAE presented flavonoids such as quercetin, 3-O-methylquercetin and luteolin. In conclusion, A. satureioides at popularly concentrations used, in aperitif or infusion, can be consumed safely because did not show any cytotoxic or genotoxic effects.

Lack of cytotoxic and genotoxic effects of Minthostachys verticillata essential oil: Studies in vitro and in vivo

Minthostachys verticillata (peperina) is an aromatic and medicinal plant with several uses and ethnobotanical properties. Numerous studies have demonstrated that its essential oil (Mv-EO) presents antimicrobial capacity and shows immunomodulating and anti-allergic properties in human cell lines. Thus, the goal of this study was to investigate the main chemical composition, analyzed by GC-FID, and the cyto-genotoxic effects of Mv-EO, using Vero cells, human PBMCs and mice bone marrow cells. The Mv-EO was rich in pulegone 60.5% and menthone 18.2%. Our results clearly show that Mv-EO is not cyto-genotoxic in vitro nor in vivo. It not induced cytotoxic effects, as indicated by trypan blue dye exclusion and NRU assays both in Vero cells and human PBMCs. In addition, Mv-EO (100-1000 μg/mL) not induced apoptotic effects on human PBMCs, as indicated by Hoechst staining and DNA fragmentation analysis by agarose gel electrophoresis. The in vivo assay showed that Mv-EO (25-500 mg/kg) not increased the frequency of micronucleus in bone marrow cells of mice. Further, the ratio of polychromatic/normochromatic erythrocytes was not modified. These findings suggest that Mv-EO appears to be safe as a therapeutic agent.

In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.

Antiviral effect and mode of action of methanolic extract of Verbascum thapsus L. on pseudorabies virus (strain RC/79).

The methanolic extract of Verbascum thapsus was evaluated for its antiviral activity against the pseudorabies virus strain RC/79 (PrV), and also for its cytotoxic activity on Vero cells. The extract showed CC50 values of 1100 µg mL−1 and 1426 µg mL−1 by NRU and MTT assays, respectively. The 50% inhibitory concentration of the extract for PrV plaque formation was determined at 35 µg mL−1, and selectivity indices were 31.4 (NRU) and 40.7 (MTT). When cells were pre-treated with the extract prior to virus infection, the inhibition in plaque formation was 70%. PrV was highly inhibited when it was incubated with plant extract or when the extract was added during the adsorption phase (99%). However, no inhibitory effect was observed when the extract was added to the cells after the adsorption period. Thus, these results suggest that the methanolic extract of Verbascum thapsus may contain bioactive compound(s) that affect PrV mostly in the adsorption phase.