Thymol, alpha tocopherol, and ascorbyl palmitate supplementation as growth enhancers for broiler chickens.

Consumer concern on the quality of products and animal welfare has greatly increased during the past decades. Dietary synthetic antibiotic products used as growth promoters have been restricted or banned in many countries. Edible plants, essential oils, or their main components were suggested as natural feed supplements to improve growth, products' quality, and welfare-related parameters. Thymol (THY), a main component of oregano essential oil, has been proved as an effective antimicrobial and antioxidant compound. Tocopherol (TOC) evidenced antioxidant activity with potential as a growth promoter and a synergic antioxidant activity between TOC and ascorbyl palmitate (AP) has also been reported. Herein, we evaluated whether broiler diet supplementation with THY, and THY with a formulation mix containing TOC and AP (1:0.5:0.5, respectively) have potential as growth enhancers under commercial conditions. Potential protective effects against foot pad dermatitis and hock burns were also evaluated. Newly hatched male broiler chicks with similar body weight (BW) were randomly assigned to 1 of 7 groups (4 replicates each) as follows: Basal (no feed supplements added), Promotor (Basal + 6.26 µmol flavomycin/kg feed), BHT (Basal + 1.33 mmol of buthylated hidroxytoluene (BHT)/kg feed), Prom-BHT (Basal + 6.26 µmol flavomycin/kg feed + 1.33 mmol of BHT/kg feed), TOC-AP (Basal + 0.67 mmoles of TOC + 0.67 mmoles of AP/kg feed), THY (Basal + 1.33 mmoles of THY/kg feed), and THY-TOC-AP (Basal + 0.67 mmoles of THY + 0.67 mmoles of a mix 1:1 of TOC-AP). Along 7 wk, BW, feed intake, and feed conversion ratio were evaluated. Skin injuries were assessed at 35 d of age. At the end of the study (42 d), compared to Basal group, similarly enhanced final BW were observed in all groups but TOC-AP. No main differences between groups were detected in feed intake, feed conversion ratio, or skin injuries. Findings suggest that THY itself or in combination with TOC-AP may have value as a natural growth enhancer alternative for broilers.

Thymol as natural antioxidant additive for poultry feed: oxidative stability improvement.

Antioxidant are regularly included in poultry feed as protection from deterioration during storage. Recently the interest for the use of natural phytochemicals in animal diets has been increased. Thymol (THY) has been proven to be an effective antioxidant for extending broiler meat quality during storage with similar action to the widely used butylated hydroxytoluene (BHT). This study evaluates whether THY can also have a protective effect on the feed mash by assessing its antioxidant potential and related changes in fatty acid (FA) balance. Feed mash was assigned to 1 of 4 treatments, control (CON, no additive), vehicle (VEH, ethanol 96%), BHT (400 mg BHT /kg feed) and THY (400 mg THY /kg feed). Three replicates of each treatment were taken after 0, 30, and 60 d of storage at room temperature (23 ± 3°C) and relative humidity (40 ± 5%). Peroxide value (PV), titratable acidity (TA) and FA relative composition were determined. As expected, there were no treatment effects on those variables at 0 d of storage. However, higher PV values were detected in the CON and VEH groups after 30 and 60 d of storage in comparison to the THY and BHT treated samples (CON = VEH > THY = BHT). While a slight increase was also observed in TA through storage time, no particular treatment effects were detected. Relative FA composition changed with storage time only in the CON and VEH group which had a decrease in polyunsaturated fatty acids and an increase in saturated FA. No changes were detected in the Thy and BHT treated feeds. The results suggest a similar THY and BHT protective effect on feed mash lipid oxidation. Thus, THY could be considered as a useful natural alternative to help sustain quality of poultry feed.

In vitro activity of natural phenolic compounds against fluconazole-resistant Candida species: a quantitative structure-activity relationship analysis.

AIMS: To evaluate the antifungal activity and to analyse the structure-activity relationship of eleven natural phenolic compounds against four Candida species which are resistant to fluconazole. METHODS AND RESULTS: Four different species of Candida isolates were used: Candida albicans, Candida krusei, Candida tropicalis and Candida dubliniensis. The phenolic compound carvacrol showed the highest anti-Candida bioactivity, followed by thymol and isoeugenol. The obtained minimum inhibitory concentration (MIC) values obtained were used in a quantitative structure-activity relationship (QSAR) analysis where the electronic, steric, thermodynamic and topological descriptors served as dependent variables. According to the descriptors obtained in this QSAR study, the antifungal activity of phenols has a first action specific character which is based on their interaction with plasma or mitochondrial membranes. The second action is based on a steric descriptor-the maximal and minimal projection of the area-which could explain the inability of some phenolic compounds to be biotransformed to quinones methylene by Candida species. CONCLUSIONS: According to the descriptors obtained in this QSAR study, the anti-Candida activity of ortho-substituted phenols is due to more than one action mechanism. The anti-Candida activity of phenolic compounds can be predicted by their molecular properties and structural characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be employed to predict the anti-Candida activity of new phenolic compounds in the search for new alternatives or complementary therapies to combat against candidiasis.

Effects of thymol and isoeugenol feed supplementation on quail adult performance, egg characteristics and hatching success.

1. A study was conducted to evaluate whether feed supplementation with thymol or isoeugenol can alter Japanese quail growth rate and final body weight, the female onset of puberty, hen-day egg production and the physical and chemical characteristics of the egg, as well as its potential to alter hatchability. 2. From 4 to 16 weeks of age, birds from each cage (1 male: 3 females) were assigned to 1 of 3 treatments that differed in the supplement added to the feed: control, thymol or isoeugenol (400 mg/kg). The average ages (d) at first egg lay (FIRST), at 25% egg production (A25% EP), at 50% egg production (A50% EP) and weekly and cumulative hen-day egg production (HDEP) were calculated. In addition, physical and chemical characteristics of the eggs, their fertility and hatchability were also evaluated for each group. 3. Feed supplementation did not significantly affect growth rate, final body weight, egg production parameters, fertility and physical characteristics of egg or most of the fatty acid components of the yolk. 4. The group treated with isoeugenol showed an increase in the percentage of palmitoleic fatty acid compared to the control, with thymol group showing intermediates values. 5. Both thymol and isoeugenol supplemented groups showed increased hatchabilities, by 18.8% and 11.8%, respectively, compared to their control counterparts. 6. The improvement in the hatching success of the eggs from the thymol and isoeugenol supplemented groups without a negative impact on their performance may have important economic implications for future breeding programmes, particularly if these effects generalise from quail to other more commercially important poultry species, such as chickens or turkeys.

Effects of thymol and carvacrol feed supplementation on lipid oxidation in broiler meat.

Poultry meat is particularly prone to oxidative deterioration due to its high concentration of polyunsaturated fatty acids. The present study evaluates the effects of thymol and its isomer carvacrol on lipid oxidation when supplemented to the feed. Supplementation with the antioxidant butylated hydroxytoluene was used as a positive control. Thus, broiler chickens were assigned to 1 of 4 treatments: vehicle (control), 150 mg/kg of butylated hydroxytoluene (positive control), 150 mg/kg of thymol, or 150 mg/kg of carvacrol. Breast and thigh samples were taken at 0, 5, and 10 d of 4 degrees C storage. Lipid oxidation was determined by the analysis of 2-TBA reactive substances (TBARS). Sample storage for 5 to 10 d significantly increased the levels of TBARS. Feed supplementation did not significantly affect breast sample oxidation. However, after 5 and 10 d of storage, increasingly higher values of TBARS were detected in thigh samples of the control group in comparison to the 3 supplemented groups. Interestingly, the same lower values of TBARS were detected between those feed-supplemented groups. Therefore, the application of the natural antioxidants thymol or carvacrol could be useful to improve poultry meat quality.

Inhibitory effect of cyclic terpenes (limonene, menthol, menthone and thymol) on Fusarium verticillioides MRC 826 growth and fumonisin B1 biosynthesis.

The minimum inhibitory concentration (MIC) of cyclic terpenes (limonene, menthol, menthone and thymol) against Fusarium verticillioides MRC 826 was assessed by using the semisolid agar antifungal susceptibility (SAAS) technique. Limonene, menthol, menthone and thymol were evaluated at final concentrations of 25, 50, 75, 150, 200, 250, 500 and 1000 microL/L of culture medium. Limonene and thymol showed the highest inhibitory effects on F. verticillioides development. Thus, the effects of monoterpenes on fumonisin B1 (FB1) biosynthesis were evaluated by using corn grain (Zea mays) as substrate. The monoterpenes were inserted on maize 1 day before inoculation with a conidial suspension of F. verticillioides to give final concentrations of 75 ppm. At this concentration, thymol was the most active inhibitor on FB1 biosynthesis.

Mosquito repellent activity of essential oils of aromatic plants growing in Argentina.

Mosquitoes are important vectors of diseases and nuisance pests. Repellents minimize contact with mosquitoes. Repellents based on essential oils (EO) are being developed as an alternative to DEET (N,N-diethyl-m-methylbenzamide), an effective compound that has disadvantages including toxic reactions, and damage to plastic and synthetic fabric. This work evaluated the repellency against Aedes aegypti of EO from aromatic plants that grow in Argentina: Acantholippia seriphioides, Achyrocline satureioides, Aloysia citriodora, Anemia tomentosa, Baccharis spartioides, Chenopodium ambrosioides, Eucalyptus saligna, Hyptis mutabilis, Minthostachys mollis, Rosmarinus officinalis, Tagetes minuta and Tagetes pusilla. Most EO were effective. Variations depending on geographic origin of the plant were detected. At a 90% EO concentration, A. satureoides and T. pusilla were the least repellent. At concentrations of 12.5% B. spartioides, R. officinalis and A. citriodora showed the longest repellency times. Comparisons of the principal components of each EO suggest that limonene and camphor were the main components responsible for the repellent effects.

Natural terpenes: self-assembly and membrane partitioning.

Monoterpenes (MTs) are highly hydrophobic substances present in essential oils. They cover a wide spectrum of biological effects with a membrane interaction as a common point. Here we studied the surface activity of camphor, cineole, thymol, menthol and geraniol, and their ability to reach and incorporate into model membranes affecting some features of their dynamic organization. All the MTs studied self-aggregated in water with critical micellar concentrations (CMC) between 3 and 8 microM. Their octanol-water and membrane-water partition coefficients were correlated with one another. They all penetrated in monomolecular layers of dipalmitoyl-phosphatildylcholine at the air-water interface, even at surface pressures (pi) above the equilibrium lateral pressure of bilayers; thymol exhibited the highest (61.3 mN/m) and camphor the lowest (37 mN/m) pi(cut-off) value. They affected the self-aggregation of Triton X-100, increasing its CMC from 0.16 mM in the absence of MTs up to 0.68 mM (e.g. for geraniol), and the topology of sPC vesicles, increasing its surface curvature, suggesting their location at the polar head group region of the membrane. The latter was supported by their ability to increase differentially the polarity of the membrane environment sensed by two electrochromic dyes. Dipole moment values (between 1.224 and 2.523 D) and solvation areas (between 80 and 97 A(2)) were calculated from their energy-minimized structures. The relative contribution of each experimental, theoretical and structural property to determine MTs' effects on membrane dynamics were evaluated by a principal component analysis.

Aromatic plants essential oils activity on Fusarium verticillioides Fumonisin B(1) production in corn grain.

The minimum inhibitory concentration (MIC) of Origanum vulgare, Aloysia triphylla, Aloysia polystachya and Mentha piperita essential oils (EOs) against Fusarium verticillioides M 7075 (F. moniliforme, Sheldon) were assessed, using the semisolid agar antifungal susceptibility (SAAS) technique. O. vulgare, A. triphylla, A. polystachya and M. piperita EOs were evaluated at final concentrations of 10, 20, 40, 50, 100, 200, 250, 500, 1000 and 1500 epsilonl per litre (epsilonl/l) of culture medium. A. triphylla and O. vulgare EOs showed the highest inhibitory effects on F. verticillioides mycelial development. This inhibition was observed at 250 and 500 epsilonl/l for EOs coming from Aloysia triphylla and O. vulgare, respectively. Thus, the effects of EOs on FB(1) production were evaluated using corn grain (Zea mays) as substrate. The EOs were inserted on the 5th, 10th, 15th and 20th day of maize postinoculation with a conidia suspension of F. verticillioides. O. vulgare and A. triphylla were applied to give final concentrations of 30 ppm and 45 ppm, respectively. Different effects were observed in the toxicogenicity at the 20th day treatment. The O. vulgare EO decreased the production level of FB(1) (P < 0.01) while A. triphyla EO increased it (P < 0.001) with respect to those obtained in the inoculated maize, not EOs treated. Results obtained in the present work indicate that fumonisin production could be inhibited or stimulated by some constituents of EOs coming from aromatic plants. Further studies should be performed to identify the components of EOs with modulatory activity on the growth and fumonisins production of Fusarium verticillioides.

Air pollutants effect on monoterpenes composition and foliar chemical parameters in Schinus areira L.

Monoterpenes, pigments, lipid peroxidation products, sulfur and water content expressed as dry weight/fresh weight ratio were measured in leaves of Schinus areira L. collected in Mendoza, Argentina. The sampling sites showed different SO(2), NO(2) and PM(10) levels, which allowed setting out zones by mapping. Foliar chemical parameters in S. areira were analysed in relation to these zones. High hydroperoxy conjugated dienes (HPCD) and alpha-phellandrene content in foliar samples coming from areas with high SO(2) levels were measured. Principal component analysis (PCA) showed a positive association among SO(2), HPCD, malondialdehyde, sabinene and beta-pinene. In relation to NO(2) levels high values of chlorophyll (Chl)-b and Chl-b/Chl-a ratio were found. PCA showed for NO(2), an association with Chl-b/Chl-a, phaeophytin-a/Chl-a ratios and alpha-phellandrene. As regard PM(10), no significant differences were found in the chemical parameters indicating foliar damage, whereas the contents of tricyclene, alpha-pinene and beta-pinene showed low values in the area with high levels of this pollutant. From the results, it can be inferred that the more noticeable changes in the foliar chemistry of S. areira were detected in relation to the different SO(2) levels. This response was associated to an increase in the foliar oxidation processes accompanied by modifications in the levels of terpenes. This is important, as SO(2) was the only pollutant whose values in the annual arithmetic mean were over the standard value of 30 ppb.

Composition and antimicrobial activity of the essential oil of Baccharis notosergila.

The essential oil of the aerial parts of Baccharis notosergila was examined by GC and GC-MS. Thirty-one constituents were identified representing 96.4% and alpha-pinene, limonene, beta-caryophyllene, and spathulenol were found to be the major components. Furthermore, the oil was tested against eight gram-positive and -negative bacteria and it was found that they exhibited moderate antibacterial activity.

Chemical constituents of the essential oil of Lippia laxibracteata (Verbenaceae)

The oil from the aerial parts of Lippia laxibracteata Herzog (Verbenaceae) was obtained by hydrodistillation and analyzed by GC and GC/MS. The major component of the oil was thymol (67%) with minor amounts of p-cymene (10%), cis-beta-ocimene (7%) and carvacrol (4.5%).

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.

Essential oils of Achyrocline satureioides, Achyrocline alata and Achyrocline tomentosa.

Essential oils obtained from the inflorescences of ACHYROCLINE SATUREIOIDES (Lam.) DC, ACHYROCLINE ALATA (HBK) DC and ACHYROCLINE TOMENTOSA Rusby, collected from central Argentina, were analyzed by GC and GC/MS. Fifty-two compounds representing 93-98% of the oils were identified. Caryophyllene was the most abundant component in all oils studied (39-48%).

Anxiogenic-like effects of Tagetes minuta L essential oil on T-maze and tonic immobility behaviour in domestic chicks.

In a first experiment, four doses (ranging between 0.04 and 0.45 mg/kg of body weight) of the essential oil from Tagetes minuta L. were subcutaneously injected in two-day-old chicks and a dose-response curve assessed for escape performance in a T-maze test. The 0.1, 0.25 and 0.45 mg/kg doses impaired the first escape performance suggesting an anxiogenic-like effect of the essential oil. After 3 h the same chicks were tested for a second escape performance, without being injected again, and no differences were observed compared to controls, suggesting that the essential oil did not affect retention. Furthermore, the effects of the essential oil were observed in the three sections of the T-maze apparatus. So, the performance was impaired in the isolation chamber section, suggesting the induction of increased anxiogenic behaviour, and also in the mirror section, suggesting that the social reinstatement behaviour was modified by an increased anxiety level. Changes in the principal corridor section were not observed, suggesting that the locomotor activity was not affected by these oil doses. The second escape performance was not affected in any of the T-maze sections, confirming that these doses did not affect learning ability. In a second experiment, a middle dose of the essential oil (0.25 mg/kg) increased the tonic immobility reaction in 15 days old chicks similarly to an anxiogenic dose of FG 7142 (1 mg/kg), while an anxiolytic dose of diazepam (0.08 mg/kg) did not affect this behaviour. Taken together, the present results suggest that the essential oil from Tagetes minuta L. may exert a negative modulation on the GABAergic function without affecting the learning ability.

Essential oils of three Baccharis species.

The essential oils of the aerial parts of three Baccharis species ( B. articulata, B. myrtilloides, and B. rufescens) were examined by GC and GC/MS. The major constituents were for B. articulata: beta-caryophyllene (16.8%), trans-nerolidol (15.6%); for B. myrtilloides: germacrene D (9.5%) and for B. rufescens: limonene (14.4%) and trans-nerolidol (14.5%).

Analysis of the essential oil of Grindelia discoidea.

The essential oil from aerial parts of Grindelia discoidea was analyzed by GC and GC/MS. Forty-six components were identified, representing more than 95% of the oil. The main constituents were ( E,E)-farnesol (> 9.0%) and ( Z,E)-farnesol (> 15.7%).

The essential oil from Tagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain.

The hypothesis that the essential oil from Tagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude members from chick brains. The essential oil from T. minuta L. inhibited [3H]FNTZ specific binding to chick brain members. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (Kd) = 2.47 +/- 0.32 nM, maximal binding (Bmax) = 556 +/- 5 fmoles/mg protein, and Hill coefficient (n) = 1.00 +/- 0.07 in the absence, and Kd = 6.73 +/- 1.4 nM, Bmax = 583 +/- 69 fmoles/mg protein, and n = 1.02 +/- 0.08 in the presence of 29 microgram/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 microgram/mL. The marked increase in [3H]FNTZ nonspecific binding starting at 60 micrograms of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with members. [3H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oil's micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oil's components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oil's CMC suggests that this effect may be due to oil's partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface.