**2. Methodology and materials**

#### **2.1 Ozonization of emu oil**

The experiments were carried out at pressure and room temperature in a semi-continuous type reactor. A constant temperature of ±2°C below the melting temperature of 16°C was maintained.

The ozone is fed continuously and is bubbled to the oil contained in a jacketed reactor, water exert as cooling fluid through a recirculation bath. The reactor is designed in the lower part with a porous ceramic plate (diffuser), an output at the top for monitoring the remain ozone leaving the reactor and has a step valve used to obtain samples at 2, 5, 7 and 10% of ozone.

Ozone is generated from oxygen (acquired from INFRA) with a purity of 99.5%, by means of an ozone generator "AZCO". The refined grade A Willow Springs emu oil was used as raw material, which was applied the extraction technology as described by Marquez [14]. The ozone/oxygen mixture from the generator is introduced to the reactor from the bottom and is evenly distributed in the aqueous solution by means of the diffuser. In the upper part of the reactor there is an output that is connected to a gas phase ozone analyzer BMT-930 connected to a computer that receives the data to be processed in "MATLAB", and generates a graph of the concentration of ozone at the exit of the reactor against time (ozonogram) [7, 15]. The working conditions were: oil weight: 9 g, initial ozone concentration: 30 ± 0.5 mg/L, ozone flow: 0.5 L/min.

#### **2.2 Peroxide value (PV)**

This method is based on the determination in the test solution of the amount of peroxides contained by means of a titration based on the ISO 3960: 2017 standard.

The peroxide index indicates the milliequivalents of oxygen in the form of peroxide per kilogram of fat or oil.

A mass of 5.0 ± 0.05 g of sample is determined inside the flask, 30 cm3 of acetic acid-chloroform solution is added and stirred until the sample is completely dissolved.

**53**

*Effect of the Ozonization Degree of Emu Oil over Healing: An Emerging Oxidation Treatment*

repeat the determination using 0.01 N sodium thiosulfate solution.

contained in a kilogram of fat or oil by means of the following equation:

determinations are made in duplicate at least [16].

*PV* = (*A* − *A*1) × *N* × (

of the sample in grams.

**2.3 31P NMR**

With a Mohr pipette, 0.5 ml of saturated potassium iodide solution is added; it is stirred and allowed to stand for 1 min, after which 30 ml of water are added. It is slowly and carefully titrated with 0.1 N sodium thiosulfate solution; shake vigorously after each addition, until it has a slightly yellow coloration; then add 0.5 ml of starch indicator solution and titration is continued without stirring until the blue color disappears. If the 0.1 N solution of sodium thiosulfate is less than 0.5 ml,

Perform a blank test in the same conditions in which the sample test was carried out. In addition, the milliliters of 0.1 N thiosulfate solution used in the titration should be noted in each case and should not exceed 0.1 ml of thiosulfate. The

The peroxide value is calculated by expressing the milliequivalents of peroxide

where PV = peroxide value; A = milliliters of sodium thiosulfate solution spent in the titration of the sample; A1 = ml of sodium thiosulfate solution spent in the titration of the blank; N = normality of the sodium thiosulfate solution; M = mass

31P NMR analysis was performed in triplicate and was based on the method of Lehnhardt. A detergent solution was prepared containing: sodium cholate (10% w/w), EDTA (1% w/w) and phosphonomethylglycine (PMG) as an internal standard for quantification (0.3 g/l); pH was adjusted to 7.1 using sodium hydroxide. The detergent solution was an aqueous solution containing 20% D2O for deuterium field-frequency lock capability. Sample was mixed with detergent solution (750 μl) by vortexing, then dispersed by ultrasonication with occasional shaking at 60°C for up to 10 min. The amount of sample used depended on its phospholipid content (lecithin standard 15 mg, cream polar lipid 15 mg, BPC60 powder 50 mg, BPC60 lipid 15 mg, lipid-depleted BPC60 residue powder 70 mg, PC700 20 mg, beta serum powder 60 mg, liquid beta serum 200 μl in 500 μl detergent). When required, pH adjustment was made with aqueous NaOH after the sample was fully dispersed in the detergent. The solution was then transferred to a 5 mm NMR tube for analysis. The mixture was then transferred to a 5 mm NMR tube and the 31P-NMR spectra were recorded. The NMR spectral data were acquired in a Bruker 400 MHz system using a delay of 25 s between 90° pulses and a line widening of 4.0 Hz. A minimum of 200 transients were acquired for each sample at room temperature [17]. The 31P-NMR had a standard deviation of ±1.20%. A minimum of 200 transients were acquired for each sample at room temperature [17]. The chemical species at approximately 132.2 ppm had two TMDP groups and, therefore, two phosphorus atoms bound to it, doubling the NMR signal. For this reason, to obtain the moles of water, the peak area at approximately 132.2 ppm was

halved and then it was added to the peak area at approximately 15.9 ppm.

Male NIH mice of 60 days of age, weighing between 25 and 30 g, were maintained in separate cages in a room under controlled conditions with temperature

**2.4 Intensive wound healing activity in vivo**

*2.4.1 Experimental species*

\_\_\_\_\_ 1000

*<sup>M</sup>* ) (1)

*DOI: http://dx.doi.org/10.5772/intechopen.83383*

*Effect of the Ozonization Degree of Emu Oil over Healing: An Emerging Oxidation Treatment DOI: http://dx.doi.org/10.5772/intechopen.83383*

With a Mohr pipette, 0.5 ml of saturated potassium iodide solution is added; it is stirred and allowed to stand for 1 min, after which 30 ml of water are added. It is slowly and carefully titrated with 0.1 N sodium thiosulfate solution; shake vigorously after each addition, until it has a slightly yellow coloration; then add 0.5 ml of starch indicator solution and titration is continued without stirring until the blue color disappears. If the 0.1 N solution of sodium thiosulfate is less than 0.5 ml, repeat the determination using 0.01 N sodium thiosulfate solution.

Perform a blank test in the same conditions in which the sample test was carried out. In addition, the milliliters of 0.1 N thiosulfate solution used in the titration should be noted in each case and should not exceed 0.1 ml of thiosulfate. The determinations are made in duplicate at least [16].

The peroxide value is calculated by expressing the milliequivalents of peroxide contained in a kilogram of fat or oil by means of the following equation:

$$PV = \begin{pmatrix} A \ -A\_1 \end{pmatrix} \times N \times \left(\frac{1000}{M}\right) \tag{1}$$

where PV = peroxide value; A = milliliters of sodium thiosulfate solution spent in the titration of the sample; A1 = ml of sodium thiosulfate solution spent in the titration of the blank; N = normality of the sodium thiosulfate solution; M = mass of the sample in grams.
