**3.1. Dietary formulation, husbandry and sample collection**

**2.4. Chemical structure and properties of carvacrol, cinnamaldehyde and capsaicin**

breast cancer, prostate, lung and mouth cancer cells [28].

106 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

H8

antifungal and anticorrosion properties [29].

**3. Poultry experiments (methodology)**

conditions were used in this chapter.

Cinnamaldehyde (C<sup>9</sup>

Capsaicinoids (C18H27NO<sup>3</sup>

Carvacrol (C10H14O) is a chemical (see **Figure 1**) found in several plants including: wild bergamot, thyme and pepperwort, but it is most abundant in oregano (*Origanum vulgare*) oil [27]. Carvacrol gives oregano a slightly spicy flavour, is colourless, and has a distinct warm odour. Overall, carvacrol has a promising antibacterial, antiviral, antioxidant, and antifungal impact. Carvacrol also demonstrated significant anti-cancer effects when tested against

of several tree species from the genus Cinnamomum. Cinnamaldehyde, the principal component of the essential oil of cinnamon bark, gives the cinnamish odour responsible for the characteristic taste and odour of cinnamon spice. Cinnamaldehyde has strong antimicrobial,

and fungi (see **Figure 1**). They have no flavour or odour, but act directly on the pain receptors in the mouth and throat. Capsaicin, the most common capsaicinoid, is an irritant for most mammals, including humans, and produces a sensation of burning in any tissue with which it comes into contact [30]. However, birds are not sensitive to the capsaicin [31] and can benefit from the nutritional value of the chilli peppers. Capsicum oleoresin (active ingredient capsa-

The experiments described in this chapter followed internationally recognised guidelines for work with poultry. All birds were cared for according to laws and regulations detailed in UK guidelines. Data from four experiments performed under similar environmental and dietary

icin) is found in pepper fruits and has antifungal and antibacterial activity [32].

**Figure 1.** Chemical structure (2-D) of (left to right) carvacrol, cinnamaldehyde and capsaicin.

O) is a chemical (see **Figure 1**) that naturally occurs in the inner bark

) are produced by peppers as a protection against certain mammals

The four experiments employed the same dietary formulations (**Table 1**). Birds were fed one of four diets. There were two control diets based on either wheat (WC) or maize (MC) which were formulated to be iso-energetic (12.13 MJ/kg AME) and iso-nitrogenic (215 g/kg CP).


ME = metabolisable energy.

1 The Vitamin and mineral premix contained vitamins and trace elements to meet the requirements specified by the National research Council (1994). The premix provided (units/kg diet): retinol, 12,000 IU; cholecalciferol, 5000 IU; α-tocopherol, 34 mg; menadione, 3 mg; thiamine, 2 mg; riboflavin, 7 mg; pyridoxine, 5 mg; cobalamin, 15 μg; nicotinic acid, 50 mg; pantothenic acid, 15 mg; folic acid, 1 mg; biotin, 200 μg; 80 mg iron as iron sulphate (30%); 10 mg copper as a copper sulphate (25%); 100 mg manganese as manganous oxide (62%); 80 mg zinc as zinc oxide (72%); 1 mg iodine as calcium iodate (52%); 0.2 mg selenium as sodium selenite (4.5%); 0.5 mg molybdenum as sodium molybdate (40%).

**Table 1.** Composition of the control diets.

The other two diets were the control diets supplemented with a standardised combination of PE (XTRACT 6930; Pancosma S.A., Geneva, Switzerland) including 5% carvacrol, 3% cinnamaldehyde and 2% capsicum oleoresin (100 grams per tonne, respectively, i.e. WC + PE; MC + PE). The PE was added in powder form to the diets and all diets were fed as mash. The diets did not contain any coccidiostat or antimicrobial growth promoters, prophylactic or other similar additives.

A comparative slaughter technique [35] was used to determine the energy retained in the

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Where: FI is the dry matter (kg) consumed for the experimental period. REc is the total energy

The fasting heat production (FHP MJ/bird) was estimated to be 0.450 MJ/d per kg of metabolic body weight (BW)0.70 per day, which correspond to the asymptotic heat production at zero

Concentration of antioxidants in liver was determined by high-performance liquid chromatography (HPLC) [37, 38]. In brief: approximately 300 mg of liver samples were mixed in 0.7 ml 5% sodium chloride solution, then 1 ml ethanol was added and samples homogenised. During homogenisation, 2 ml hexane was added. Then samples were centrifuged and the hexane phase, containing the vitamin E and coenzyme Q10 were collected. Extraction with hexane was performed twice, and the combined phase was evaporated under nitrogen and

Vitamin E (α-, γ- and ϭ-tocopherols) was determined as previously described [39] using an HPLC system (Shimadzu Liquid Chromatograph, LC-10 AD, Japan Spectroscopic Co Ltd., with a Jasco Intelligent Spectrofluorometer 821-FP) fitted with a Spherisorb, type S30DS2, 3 mm C18 reverse phase HPLC column, 15 cm × 4.6 mm (Phase Separations Limited, UK). Chromatography was performed using a mobile phase of methanol–water (97:3, v/v) at a flow rate of 1.05 ml/min. Fluorescence detection of tocopherols and tocotrienols involved excitation and emission wavelengths of 295 and 330 nm, respectively. Standard solutions of tocopherols in methanol were used for instrument calibration and tocol was used as an internal standard.

Coenzyme Q10 was analysed in the same extract by injecting 50 ml into the same HPLC system, but using a Vidac 201TP54 column (5 μm, 25 cm × 4.6 mm) and mobile phase ethanol– methanol–2-propanol (70:15:15, v/v) and flow rate of 1.5 ml/min with a diode array detection

The analyses of relative expression of genes of interest (GOI) in the caecal tonsils were per-

Approximately 30mg of macro-dissected caecal tonsil tissue per sample (previously submerged in RNAlater® (Sigma-Aldrich, USA) and stored frozen at -80°C) was homogenised in 500 uL

at 275 nm [40]. Coenzyme Q10 (Sigma, Poole, UK) standard was used for calibration.

carcase of birds. In brief, dietary NE was calculated using the following equation:

NE (MJ/kg) = (REc + FHP)/FI.

**3.4. Determination of hepatic antioxidant concentration**

re-dissolved in a mixture of dichloromethane–methanol (1:1, v/v).

retained in the carcass (see [50]).

activity (as proposed by [36]).

**3.5. Gene expression analysis**

formed by qStandard (Middlesex, UK).

*3.5.1. Total RNA extraction and reverse transcription*

Day old male Ross 308 broiler chickens were purchased from a commercial hatchery and reared in floor pens littered with wood shavings. The temperatures were kept at 32°C during the first 2 days on birds arrival and were gradually reduced to 20°C by 21 days of age. A standard lighting programme following breeder's recommendations (Aviagen Ltd., Edinburgh, UK) for broilers was used. Access to the feed and the water was *ad libitum*. At 17 days of age, two birds from each pen were transferred to a pen with wire mesh floor and excreta samples were collected for four consecutive days from each pen, immediately dried at 60°C and then milled for further analyses. The birds were weighed on a per-pen basis and the average bird feed intake (FI), weight gain (WG) and gain: feed ratio (GF) were determined.

In the experiments where dietary net energy (NE) was determined, at the end of the study, all chickens were killed by cervical dislocation and the carcass of the birds, including intestine, blood and feather, from each pen were frozen and then minced, thoroughly mixed and sampled, and used for following analysis and calculations.

In the experiments where the hepatic antioxidant content was determined, at 21 days of age, one bird from each pen was randomly selected, stunned/killed and the liver was collected and stored at –20°C prior to analysis of antioxidant contents.

In the experiments where the gene expression was measured, at 21 days of age one bird from each pen was randomly selected, stunned/killed and the left caecal tonsil was collected and stored in RNAlater® (Sigma-Aldrich, USA) at −70°C prior to analysis of the relative expression of selected genes.
