Evaluation of oral supplementation of free and nanoencapsulated Minthostachys verticillata essential oil on immunological, biochemical and antioxidants parameters and gut microbiota in weaned piglets.

Early weaning is an important stressor that impairs the piglet´s health, and essential oils appear as promising candidates to improve it instead of antibiotics. The aim of this study was to evaluate the effect of oral supplementation of free and nanoencapsulated Minthostachys verticillata essential oil (EO and NEO, respectively) on immunological, biochemical and antioxidants parameters as well as on gut microbiota in weaned piglets. EO was extracted by hydrodistillation and nanoencapsulation was performed by high-energy method using Tween 80 and Span 60 as surfactants. EO and NEO were chemically analyzed by gas chromatography-mass spectrometry (GC-MS). The cytotoxic effects of both EO and NEO was evaluated on Caco-2 cell line. For in vivo assay, male weaned piglets (age: 28 days, mean initial body weight: 11.63 ± 0.37 kg) were randomly distributed in six groups of six animals each (n = 6) and received orally EO (10.0 mg/kg/day) or NEO (2.5, 5.0 and 10.0 mg/kg/day), named hereinafter as EO-10, NEO-2.5, NEO-5 and NEO-10, for 30 consecutive days. Animals not treated or treated with surfactants mixture were evaluated as control and vehicle control. Subsequently, histological, hematological and biochemical parameters, cytokines production, oxidative markers, CD4+/CD8+ T cells and gut microbiota were evaluated. GC-MS analysis was similar in both EO and NEO. The NEO was more toxic on Caco-2 cells than EO. Oral supplementation of EO-10 or NEO-10 improved growth performance compared to control group NEO-2.5 or NEO-5 (p < 0.05) groups. NEO-2.5, NEO-5 and NEO-10 did not alter the morpho-physiology of digestive organs and decreased malondialdehyde (MDA) levels in liver compared to control (p < 0.05) or EO-10 groups (p < 0.05, p < 0.01). In addition, NEO-10 showed an increase in CD4+/CD8+ T cells ratio (p < 0.001), and induced the highest serum levels of IL-10 (p < 0.01). Serum triglycerides levels were significantly lower in animals treated with EO-10 or NEO-2.5, NEO-5 and NEO-10 compared to control group (p < 0.001). Gut microbiota analysis showed that NEO-10 favor the development of beneficial intestinal microorganisms to improve parameters related to early weaning of piglets. In conclusion, EO and NEO improved parameters altered by early weaning in piglets however, NEO was safer and powerful. Therefore, NEO should be further studied to be applied in swine health.

Development and biological evaluation of pNIPAM-based nanogels as vaccine carriers.

"Smart" nanogels are an attractive tool for the development of new strategies of immunization in veterinary medicine. Here, we reported the synthesis and physicochemical characterization of thermoresponsive nanogels based on poly(N-isopropylacrylamide) (pNIPAM) and theirin vitro, ex vivoand in vivo (mice model) performance. Smart nanogels of ca. 250 nm, with a transition temperature of 32 °C were obtained by precipitation polymerization. Assays to evaluatepNIPAM nanogels cytotoxicity were performed in different cell lines showing high biocompatibility (>70 %). The efficient internalization of the system was studied by confocal microscopy as well as flow cytometry. The ability to protect and deliver antigens was analyzed using the outer membrane lipoprotein A (OmlA), an important virulence factor ofActinobacillus pleuropneumoniae(App)cause of porcine pleuropneumonia. This lipoprotein was synthesized by recombinant technology and its technique was also described. The biodistribution ofpNIPAM nanogels administered intranasally was performedinvivo and ex vivo through Pearl Imaging System, which showed that nanogels were kept mostly in the lungs during the evaluated time. Besides, the efficacy of the proposal nanogel-based vaccine was studiedin vivoby measuring the antibody titers of BALB/c mice inoculated with OmlA encapsulated intopNIPAM nanogels compared to OmlA plus aluminum hydroxide adjuvant. The results proved the ability of nanogels to stimulate a humoral immune response. Therefore, we have demonstrated thatpNIPAM nanogels can be used as an efficient platform for vaccine nanocarriers.

Spray-drying-microencapsulated Minthostachys verticillata essential oil and limonene as innovative adjuvant strategy to bovine mastitis vaccines.

Design of innovative adjuvant strategies with an appropriate safety profile is relevant to developed subunit or inactivated microorganism vaccines for bovine mastitis. Minthostachys verticillata essential oil (EO) has demonstrated ability to stimulate the innate immune response and adjuvant effect similar to Al(OH)3. Here we evaluated the adjuvant effect of EO and its metabolite, limonene (L) alone and microencapsulated by spray-drying, using an inactivated Enterococcus faecium strain bovine-mastitis inducer. The gas chromatography-mass spectrometry analysis showed that microencapsulation process did not alter the EO or L chemistry. Microencapsulated EO (McEO) or L (McL) (2.0, 2.5 and 5.0 mg/ml) decreased the viability of bovine mammary gland epithelial cells in a dose-dependent way. Balb/c mice (n = 32) were subcutaneously inoculated (day 0) and revaccinated (day 14 and 28) with saline solution, inactivated bacteria alone or combined with Incomplete Freund's Adjuvant; EO or L (2.5 mg/ml); McEO or McL (5.0 mg/ml); or microcapsule wall material (Mc) alone (2.5 mg/ml). EO, L, McEO and McL stimulated E. faecium-specific IgG (IgG1 or IgG2a) with opsonizing capacity and increased the proportion of CD4+ and CD8+ T cells producers of IFN-γ. Microencapsulation was an effective strategy to increase the adjuvant potential of EO or L. These new adjuvants deserve further study to evaluate their incorporation into vaccines for bovine mastitis.

Mycofumigation of postharvest blueberries with volatile compounds from Trichoderma atroviride IC-11 is a promising tool to control rots caused by Botrytis cinerea.

Botrytis cinerea, the causal agent of the gray mold, is a filamentous fungus that infects blueberries and can cause important production losses in postharvest storage. Considering that the use of synthetic fungicides is not allowed on blueberries in postharvest conditions, alternative and natural strategies are needed to control gray mold. The objective of this work was to evaluate the capability of volatile organic compounds (VOCs) produced by Trichoderma atroviride IC-11 to control B. cinerea growth in blueberries after harvest. These VOCs inhibited almost completely B. cinerea growth in vitro. The most abundant volatile compound was 6-pentyl-α-pyrone (6PP). In vitro assays with pure 6PP confirmed its antifungal activity. The incidence of gray mold was evaluated in blueberries inoculated with B. cinerea and exposed to volatiles of T. atroviride IC-11. Gray mold incidence among those stored in air at 20 °C for 14 days was 100%, while the incidence among the volatile-treated fruit was 17%. Gray mold incidence among those stored in air at 4 °C for 31 days was 82%, while the incidence among the volatile-treated fruit was 11%. T. atroviride IC-11 VOCs inhibited mycelial growth and conidia germination of B. cinerea. The binding of VOCs to the surface of hyphae caused their vacuolation and deterioration. Selective cytotoxicity of 6PP on B. cinerea was observed but not on human intestinal cells at specific concentrations that controlled gray mold. The postharvest mycofumigation of blueberries with T. atroviride IC-11 VOCs is a promising approach to protect these fruits from gray mold.

Minthostachys verticillata Griseb (Epling.) (Lamiaceae) essential oil orally administered modulates gastrointestinal immunological and oxidative parameters in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Minthostachys verticillata (Griseb.) Epling (Lamiaceae) is a plant used in folk medicine for digestive or respiratory disorders. In addition, it is incorporated as condiment, in foods, as beverage flavoring or mate. The ethnopharmacological interest of M. verticillata resides in its essential oil (EO). Part of group has demonstrated the immunomodulatory ability of EO giving this oil a biological potential not known until that moment and conducted studies to evaluate their possible application in diseases of veterinary interest. However, the immunomodulatory effects of EO administered orally have not been fully characterized. AIM OF THE STUDY: This study evaluated the impact of EO oral administration on gastrointestinal and immune health through measurement of immunological and oxidative parameters in mice. MATERIAL AND METHODS: The EO was extracted from the leaves, slender stems and flowers of M. verticillata by hydrodistillation and chemical analyzed by gas chromatography-mass spectrometry (GC-MS). Prior to in vivo study, the cytotoxic effect of EO was determined using the human colon carcinoma Caco-2 cell line. For in vivo study, three groups of male Balb/c mice (n = 3) were orally administered with saline solution (control group) and EO (5 or 10 mg/kg/day) during 10 consecutive days. Subsequently, histological and hematological parameters, cytokines production, oxidative markers and CD4+ and CD8+ T cells were evaluated. RESULTS: The chemical analysis of EO revealed the presence of a high content of monoterpenes, being the main pulegone (76.12%) and menthone (14.28%). The EO oral administration improved mice growth performance and modulated systemic adaptive immune response by increasing in the total leukocyte number. A high percentage of CD4+ T cells were observed whereas the number of CD8+ T cells was not altered. EO did not alter the morpho-physiology of intestine and improved total antioxidant capacity by decreasing MDA concentrations. In addition, EO decreased the IL-6 levels and increased in the IL-4 and IL-10 concentrations. CONCLUSION: Results indicate that M. verticillata EO modulate inflammatory and oxidative parameters constituting a natural alternative which could be applied to improve gastrointestinal and immune functionality in animals.

A natural oil increases specific anti-OVA IgG levels and induces a cellular immune response combined with aluminum hydroxide.

The efficacy of certain vaccines is improved by the use of adjuvants. Nowadays, the development of new, effective, and safe adjuvants that stimulate the innate immune response is researched. In this context, medicinal plants appear as a suitable alternative. Minthostachys verticillata essential oil (EO) has demonstrated the ability to modulate mechanisms of the innate immune response. Thus, the present work aimed to evaluate the EO adjuvant effect on humoral and cellular immunity, coadministered with OVA as antigen. The chemical analysis of EO by gas chromatography-mass spectrometry revealed a predominant pulegone-menthone chemotype. EO (1.25, 2.5, or 5.0 mg/ml) did not alter the viability of murine fibroblasts (3T3 cell line) neither showed signs of toxicity in Balb/c mice inoculated subcutaneously. The serum of mice immunized with OVA + EO showed increased levels of anti-OVA-specific antibodies of IgG1 subclass compared with the mice immunized with OVA alone revealing an adjuvant effect of EO. The delayed type hypersensitivity showed that the combination OVA + Al(OH)3  + EO was the best to induce a cellular immune response that extended until 48 h postinjection of OVA. M. verticillata EO appears as a new, safe, and effective adjuvant, which should continue to be studied for their possible future incorporation into vaccine formulations.

Minthostachys verticillata essential oil activates macrophage phagocytosis and modulates the innate immune response in a murine model of Enterococcus faecium mastitis.

Minthostachys verticillata essential oil (EO) is a natural product that reports immunomodulatory effects on human T cells as well as anti-inflammatory activity. Bovine mastitis is a worldwide disease, mainly caused by bacteria, affecting milk quality and yield, leading to high economic losses. Environmental pathogens, as Enterococcus faecium, are implicated in the disease. Antibiotic therapy is adequate, although it can leave residues in milk, causing problems in human health. The search of immunomodulatory substances for bovine mastitis treatment is a promising alternative strategy. The aim of this study was to characterize the effect of M. verticillata EO on macrophage phagocytosis and evaluate its immunomodulatory and protective effects in mice challenged with E. faecium. The results showed that EO activated macrophage phagocytosis mechanisms inducing reactive oxygen species production. Moreover, EO modulated the innate immune response in mammary glands of female Balb/c mice challenged with E. faecium decreasing the infiltration of polymorphonuclear neutrophils and IL-1ß and TNF-α mRNA expression. In addition, EO increased the expression of IL-10 in the last hours of infection. Treatment with EO did not increase the number of activated CD4+ or CD8+ T cells or the production of specific antibodies. These results suggest that EO play an important role in helping to resolve the infection in the first hours without activating adaptive immunity. In addition, a marked decrease of the bacterial count in the glands of mice treated with EO was observed. A natural product such as M. verticillata EO could have a potential use to control bovine mastitis.

Potent inhibition of Western Equine Encephalitis virus by a fraction rich in flavonoids and phenolic acids obtained from Achyrocline satureioides

ABSTRACT Achyrocline satureioides (Lam.) DC. Asteraceae, ‘marcela del campo', possess several pharmacological properties. Previously we reported antiviral activity of an aqueous extract of A. satureioides against an alphavirus, Western Equine Encephalitis virus. Alphaviruses are highly virulent pathogens which cause encephalitis in humans and equines. There are no effective antiviral to treat its infections. The aim of this study was to evaluate in vitro cytotoxic and antiviral activities against Western Equine Encephalitis virus of five water extract chromatographic fractions from A. satureioides and identify the main compounds of the bioactive fraction. Also, it was to assess in vivo cytogenotoxic ability of the active fraction. Cytotoxicity studies revealed low toxicity of the most of fractions in Vero and in equine peripheral blood mononuclear cells. Antiviral studies showed that the water crude extract – Sephadex LH 20 – fraction 3 MeOH–H2O (Fraction 3) was active against Western Equine Encephalitis virus with Effective Concentration 50% = 5 µg/ml. Selectivity Indices were 126.0 on Vero and 133.6 on peripheral blood mononuclear cells, four times higher than aqueous extract selectivity index. Regarding the mechanism of action we demonstrated that F3 exerted its action in intracellular replication stages. Further, fraction 3 showed important virucidal action. Fraction 3 contains, in order of highest to lowest: chlorogenic acid, luteolin, 5,7,8-trimethoxyflavone, 3-O-methylquercetin and caffeic acid. Fraction 3 did not induce in vivo toxic nor mutagenic effect. Therefore, it is safe its application as antiviral potential. Further studies of antiviral activity in vivo will be developed using a murine model.

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.

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.

Genotoxic evaluation of a methanolic extract of Verbascum thapsus using micronucleus test in mouse bone marrow.

Verbascum thapsus L. is a medicinal plant and has been used to treat numerous pulmonary diseases, asthma, inflammatory disease, spasmodic coughs and migraine headaches. Several studies have demonstrated that different extracts of V. thapsus present antimicrobial activity. Thus, the goal of this study was to evaluate the genotoxic and cytotoxic activities of a methanolic extract of Verbascum thapsus, using micronucleus test in mouse bone marrow. No toxicity in bone marrow was detected in the extract-treated groups. The methanolic extract of V. thapsus at doses of 100, 300 and 500 mg/kg, did not produce a significant increase in the frequency of MNPCE in bone marrow and neither altered the relationship PCE/NCE respect to negative control. These cytogenotoxic findings contribute the preclinical knowledge of methanolic extract of V. thapsus and provide security in its use as herbal medicine.