Can the foregut nematode Haemonchus contortus and medicinal plants influence the fecal microbial community of the experimentally infected lambs?

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.

The Effect of Age, Gender and Body Mass Index on Tear Film Neuromediators and Corneal Nerves.

Purpose: To evaluate the effect of age, gender and body mass index (BMI) on the levels of tear film neuromediators and corneal nerve parameters in healthy individuals.Methods: Twenty-six healthy subjects were screened for any neurological deficits. The concentration of substance P and calcitonin gene-related peptide (CGRP) in tears was measured by enzyme-linked immunosorbent assay. Corneal nerve fibers were imaged using confocal microscopy and assessed by automated image analysis software. Associations between the clinical variables were analyzed using Pearson or Spearman correlation. Univariate general linear regression was performed to examine the independent relationship between age, BMI and gender of the subjects with concentrations of substance P, CGRP and corneal nerve fiber parameters.Results: Fifteen (58%) of the study participants were male. The mean age of the study cohort was 36 ± 12 years (range, 21-59) with an average BMI of 25 ± 4 kg/m2. The median [IQR] concentrations of substance P and CGRP was 715 [372-1463] pg/mL and 38 [15-74] ng/mL respectively. Moderate but significant positive correlations were found between the concentration of substance P and corneal nerve fiber density (r = 0.467, P = .016), nerve fiber length (r = 0.528, P = .006) and nerve fractal dimension (rs = 0.614, P = .002). There was a significant age-dependent reduction in the concentration of substance P (-6% pg/mL per year, P = .001) and CGRP (-8% ng/ml per year, P < .001). Corneal nerve fiber density (-0.171 no./mm2 per year, P = .029) and nerve fractal dimension (-0.001 per year, P = .021) showed reductions with advancing age. Gender and BMI did not influence any of the measurements.Conclusions: The concentrations of substance P and CGRP in tears, as well as corneal nerve fiber density and nerve fractal dimension, are significantly reduced with advancing age. Age should be considered when evaluating patients for diagnosis and follow-up of corneal neuropathy or ocular surface disorders.

A Comparative Study on the Diagnostic Utility of Corneal Confocal Microscopy and Tear Neuromediator Levels in Diabetic Peripheral Neuropathy.

AIMS: To determine the utility of corneal confocal microscopy and tear neuromediator analysis in the diagnosis of diabetic peripheral neuropathy (DPN) as a result of type 1 and type 2 diabetes. METHODS: Seventy individuals with either type 1 diabetes or type 2 diabetes (T1D/T2D) underwent corneal confocal microscopy to assess the corneal nerve morphology. The concentration of substance P and calcitonin gene-related peptide (CGRP) in tears was measured by enzyme-linked immunosorbent assay. Motor excitability studies were conducted on the median nerve to assess axonal ion channel function. Based on total neuropathy score (TNS), participants were stratified into DPN (DPN+ve; TNS ≥ 2; T1D, n = 19; T2D, n = 16) and without DPN (DPN-ve; TNS ≤ 1; T1D, n = 19; T2D, n = 16). Areas under the receiver operating characteristic curves (AUCs) were calculated to obtain specificity and sensitivity of the measures to diagnose DPN. RESULTS: In T1D, the concentration of substance P and confocal microscopy measures were significantly reduced (P < .010) in DPN+ve. Also, for the nerve excitability measures, mean peak response, percentage of threshold electrotonus at peak and after 90-100 ms, superexcitability and subexcitability were significantly reduced (P < .050) in DPN+ve. In T2D, except for inferior whorl length (P = .190), all other corneal confocal microscopy measures were significantly reduced (P < .010) in DPN+ve, but there was no difference in substance P concentration. For the diagnosis of DPN in T1D, the AUC for inferior whorl length (0.910), mean peak response (0.800) and concentration of substance P (0.770) were high and in T2D, the AUC for corneal nerve fiber length (0.809) and nerve fractal dimension (0.777) were high. CONCLUSION: Corneal confocal microscopy parameters provide a better diagnostic ability to detect DPN in T1D and T2D than nerve excitability measures or concentrations of tear neuromediators. The concentration of substance P could also be useful in diagnosing DPN but for T1D only.

Tear film substance P: A potential biomarker for diabetic peripheral neuropathy.

OBJECTIVE: To explore the changes that occur in the concentrations of substance P (SP) and calcitonin gene-related peptide (CGRP) in tears as a result of corneal denervation and its association with diabetic peripheral neuropathy (DPN). METHODS: Sixty-three individuals with type 1 diabetes/type 2 diabetes (T1D/T2D) and 34 age-matched healthy controls underwent a detailed assessment of neuropathy using the Total Neuropathy Score (TNS). The concentration of SP and CGRP in tears was measured by enzyme-linked immunosorbent assay. The corneal sub-basal nerve plexus was imaged using corneal confocal microscopy. Corneal nerve fibre length, fibre density, branch density, total branch density, nerve fractal dimension and inferior whorl length were quantified. RESULTS: In T1D, the median [IQR] concentration of SP in tears was significantly reduced in those with DPN, (130 [61-692]pg/mL) compared to both control subjects (763 [405-1555]pg/mL, P < 0.01) and in those without DPN (914 [339-1832]pg/mL, P = 0.01); the concentration of CGRP was not changed. In T2D, there was no difference in neuropeptides between participants with diabetes and controls, regardless of neuropathic status. In T1D and T2D, corneal nerve parameters were significantly different between those with DPN or without DPN and controls. A significant correlation was noted between the concentration of tear film SP and TNS in T1D (r = -0.49; P < 0.001) and corneal nerve fibre density (r = 0.45; P < 0.001). The concentration of tear film CGRP was correlated significantly with the reduction of corneal nerve fibre density (r = 0.41; P = 0.01) in T1D. CONCLUSION: Tear film SP may provide a potential non-invasive biomarker for assessing neuropathy in T1D.

Thymol in the intestinal tract of broiler chickens after sustained administration of thyme essential oil in feed.

Plant compounds occurring in phytogenic feed additives are involved in different pharmacological activities in the animal organism. Since the digestive tract acts as a first line of defence against foreign compounds, it is necessary to outline its response to dietary supplementation with bioactive plant components. Little information is available on the bioactivity of thymol as the main bioactive compound of Thymus vulgaris L. essential oil (TEO). The main objective of the present study was to provide a detailed view of the concentrations of thymol in plasma and the content of individual intestinal segments (duodenum, jejunum, ileum, caecum and colon) of broiler chickens after 4 weeks of dietary supplementation with different TEO concentrations. 32 one-day old Ross 308 hybrid broilers were randomly divided into four dietary treatment groups (0.00%, 0.01%, 0.05%, 0.1% w/w of TEO in the diet). Thymol concentrations in the duodenal chyme presented around 7% on average from the thymol amount administered in the feed. A significantly increased thymol amount was observed after 0.1% TEO addition to the diet compared with 0.01% TEO enrichment in the duodenal wall and gut content of jejunum, ileum, caecum and colon (p < 0.05). Thymol levels in the colon were significantly higher than in the ileum and about 1.7 times higher on average than those in the caecum. Significant coefficient of correlation was observed between thymol concentrations in plasma and feed, gut content of all intestinal segments as well as duodenal wall. Our results point to intensive thymol absorption in the initial sections of the digestive tract. In the current study, the role of intestine in biotransformation of thymol was observed, and it would be desirable to investigate whether thymol itself or thymol metabolites are responsible for beneficial effects in intestine.

Ovicidal and larvicidal activity of extracts from medicinal-plants against Haemonchus contortus.

The use of medicinal plants (MP) containing bioactive compounds is an alternative strategy to control of parasitic nematode of small ruminants Haemonchus contortus at various stages of their life cycle. The aims of this study were to determine the in vitro anthelmintic activity of both aqueous and methanolic extracts from 13 medicinal plants typical for Central Europe, and to determine quantity of selected plant secondary metabolites (PSMs) in the methanolic extracts. In vitro egg hatch test and larval development tests were conducted to determine the possible anthelmintic effects of methanolic and aqueous extracts of the roots of Althaea officinalis L., Petasites hybridus L. and Inula helenium L.; flowers of Malva sylvestris L. and Chamomilla recutita L.; leaves of Plantago lanceolata L. and Rosmarinus officinalis L.; seeds of Foeniculum vulgare Mill. and stems of Solidago virgaurea L., Fumaria officinalis L., Hyssopus officinalis L., Melisa officinalis L. and Artemisia absinthium L. on eggs and larvae of H. contortus. Ultra-performance liquid chromatography and tandem mass spectroscopy was used for quantifying six PSMs: gallic acid (GA), rutin (RU), diosmin (DI), hesperidin (HE), quercetin (QU) and kaempferol (KA). RU content of the most effective methanolic extracts was in the order: M. sylvestris (9.33 mg/g DM) > A. absinthium (6.10 mg/g DM) > C. recutita (0.42 mg/g DM). The highest concentration of QU (44.8 mg/g DM) and KA (6.59 mg/g DM) were detected in stems of F. officinalis comparing to the other evaluated plants. The most significant (p < 0.05) anthelmintic effects exhibited methanolic extracts of A. absinthium in both in vitro tests (i.e., egg hatch test and larval development test). Additionally, only two methanolic extracts of C. recutita and M. sylvestris were comparable to activity of A. absinthium using the larval development test. Wider spectrum of aqueous extracts exhibited stronger ovicidal activity in comparison to methanolic extracts. The similar trend was observed in evaluating of larvicidal activity of aqueous and methanolic plant extracts.

Modulation of ruminal and intestinal fermentation by medicinal plants and zinc from different sources.

Two experiments were conducted on sheep to determine the effect of dietary supplementation with zinc and a medicinal plant mixture on haematological parameters and microbial activity in the rumen and large intestine. In Experiment 1, 24 male lambs were randomly divided into four groups: One group was fed an unsupplemented basal diet (control), and three groups were fed a diet supplemented with 70 mg Zn/kg diet in the form of Zn sulphate (ZnSO4 ), a Zn-chelate of glycine hydrate (Zn-Gly) or a Zn-proteinate (Zn-Pro), for five months. The ruminal content was collected separately from each lamb, and batch cultures of ruminal fluid were incubated in vitro with mixture of medicinal plants (Mix) with different roughage:concentrate ratios (800:200 and 400:600, w/w). Bioactive compounds in Mix were quantified by UPLC/MS/MS. In Experiment 2, four sheep were fed a diet consisting of meadow hay and barley grain (400:600, w/w), with Zn-Gly (70 mg Zn/kg diet), Mix (10% replacement of meadow hay) or Zn-Gly and Mix (Zn-Gly-Mix) as supplements in a Latin square design. Mix decreased total gas (p < 0.001) and methane (p < 0.01) production in vitro. In Experiment 1, caecal isobutyrate and isovalerate concentrations varied among the dietary treatments (p < 0.01). The isovalerate concentration of the zinc-supplemented groups in the distal colon was higher (p < 0.001) compared with the control. In Experiment 2, the molar proportion of isobutyrate was the highest in the faeces of the sheep fed the diet with Zn-Gly-Mix (p < 0.01). The plasma zinc concentration was higher in the groups fed a diet supplemented with zinc (p < 0.001). The haematological profile and antioxidant status did not differ between the dietary groups (p > 0.05). The diets containing medicinal plants and organic zinc thus helped to modulate the characteristics of fermentation in ruminants.

Identification and quantification of thymol metabolites in plasma, liver and duodenal wall of broiler chickens using UHPLC-ESI-QTOF-MS.

In the present study, we aimed to develop a method for thymol sulfate and thymol glucuronide determination in plasma, liver and duodenal wall of broiler chickens after feeding with a Thymus vulgaris essential oil at the different concentrations (0.01, 0.05 and 0.1% w/w). UHPLC coupled with accurate-mass QTOF-MS was used for identification and quantification of thymol metabolites. Novel Waters Oasis Prime HLB solid-phase extraction cartridges were applied to sample clean-up with extraction recoveries ranged from 85 to 92%. The presence of thymol glucuronide was confirmed by MS software according to molecular formula, score, mass error and double bond equivalent. In terms of validation, calibration curves of thymol sulfate were constructed in matrix samples with linearity from 3.91 to 250.0 ng/mL and correlation coefficients were within the range of 0.9979-0.9995. Limits of detection were 0.97, 0.29 and 0.63 ng/mL and limits of quantification were 3.23, 0.97 and 2.09 ng/mL for plasma, liver and duodenal wall, respectively. Intra-day and inter-day precision expressed as relative standard deviation were <4.35%. To highlight, thymol metabolites were directly detected for the first time in liver and duodenal wall and this method was shown to be successfully applicable for investigation of thymol metabolism in chickens after thyme essential oil ingestion.

Effect of Thyme Essential Oil Supplementation on Thymol Content in Blood Plasma, Liver, Kidney and Muscle in Broiler Chickens.

The absorption and metabolism of phytogenic feed additives in poultry is studied related to the metabolism and deposition of their main compounds in tissues intended for food production. Fifty-six non-sexed Ross 308 broilers were allocated to seven dietary treatments and fed a diet containing graded levels of thyme (Thymus vulgaris L.) essential oil (EO) (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1%, w/w). Thymol concentration was measured in plasma, liver, kidney and breast muscle tissue using solid phase micro-extraction followed by gas chromatography/mass spectrometry. We found the highest concentrations of thymol in kidney and plasma, and the lowest in breast muscle and liver. Thymol content in plasma and kidney significantly increased when 0.05 and 0.1%, w/w, EO and in liver and breast muscle only when 0.1%, w/w, EO was added to the diet (p<0.05). Our results indicate intensive metabolism of thymol in liver and its accumulation in kidney tissue. We confirm low deposition of thymol in the muscle tissue. It is necessary to.-keep in mind the selection of a sufficient concentration of EO in the feed additive for animals without the risk of thymol residues in edible tissues.

Low-dimensional compounds containing bioactive ligands. V: Synthesis and characterization of novel anticancer Pd(II) ionic compounds with quinolin-8-ol halogen derivatives.

Three novel palladium(II) complexes, NH2(CH3)2[PdCl2(CQ)] (1) (CQ=5-chloro-7-iodo-quinolin-8-ol), NH2(CH3)2[PdCl2(dClQ)] (2) (dClQ=5,7-dichloro-quinolin-8-ol) and NH2(CH3)2[PdCl2(dBrQ)] (3) (dBrQ=5,7-dibromo-quinolin-8-ol) have been prepared and characterized. Their structures contain square-planar [PdCl2(XQ)](-) complex anions in which deprotonated XQ ligands are coordinated to the Pd atoms via the pyridine nitrogen and the phenolato oxygen atoms, other two cis-positions are occupied by two chlorido ligands. Negative charges of these anions are balanced by uncoordinated dimethylammonium cations. Coordination of the XQ ligands to Pd(II) atom was confirmed by the differences in the stretching ν(OH) and ν(CN) vibrations in the IR spectra of ligands and prepared complexes while bands of aliphatic CH and NH stretching vibrations observed in the spectra of 1-3 confirm the presence of dimethylammonium cations in the complexes. The binding of complexes 1-3 to calf thymus DNA was investigated using UV-visible and fluorescence emission spectrophotometry. The fluorescence spectral results indicate that the complexes can bind to DNA through an intercalative mode. The Stern-Volmer quenching constants obtained from the linear quenching plot are in the 1.04 × 10(4) to 4.35 × 10(4) M(-1) range. The complexes exhibit significant anticancer activity tested on A2780 cells and cisplatin resistant cell line A2780/CP.

Regioselectivity and tautomerism of novel five-membered ring nitrogen heterocycles formed via cyclocondensation of acylthiosemicarbazides.

A series of 1-acyl-4-phenyl/(acridin-9-yl)thiosemicarbazides 3, including four new compounds, were prepared in order to study substituent effects on cyclization reactions with oxalyl chloride (producing imidazolidine-4,5-diones 4), dimethyl acetylenedicarboxylate (to give thiazolidin-4-ones 7 and 8) and autocondensation under alkaline conditions (to yield 1,2,4-triazoles 9). A positional isomer, 10 of compound 3f was also prepared. Altogether, twenty new compounds characterized and identified by IR, UV,1H, 13C and 2D NMR and quantum chemical calculations are described. The tautomerism of the products and regioselectivity of the reactions were evaluated. Compounds 3f-h,3h.2HCl, 7b,d and 10 were screened for cytotoxic activity against the L1210 leukemia cell line and all compounds, except for 3f, exhibited promising inhibitions of cell growth.