2.8.1. Experimental Condition

Lard oil was used to evaluate the functionality of the rosemary extract as an antioxidant. In other countries, rosemary extracts defined the sum of carnosol and carnosic acid, is permitted at 50 μg/mL in lard oil. Therefore, these two compounds, except rosmarinic acid, were used for the functional evaluation. Two antioxidant components of rosemary extract (carnosic acid and carnosol) were added to lard oil to provide a combined concentration of 50 μg/mL. In addition, to compare the effects of synthetic antioxidants (carnosol and carnosic acid standards) and natural antioxidants, rosemary leaf extract was prepared and analyzed by HPLC to calculate its carnosol and carnosic acid concentrations. This leaf extract was then added to lard oil to provide a combined carnosol and carnosic acid concentration of 50 μg/mL. The mixture of carnosol and carnosic acid standards was also added to lard oil to provide a combined concentration of 50 μg/mL. To provide a positive control, butylated hydroxyanisole (BHA) was added to lard oil at a concentration of 50 μg/mL. The prepared samples were stored in a dry oven at 50 ◦C for 42 days. The peroxide value, acid value, and residual antioxidant level of each sample were evaluated every 7 days.

#### 2.8.2. Measurement of Antioxidant Activity

Antioxidant activity was evaluated using the peroxide value and acid value. The peroxide value and acid value of each sample were analyzed by modifying the Official Methods and Recommended Practices of the American Oil Chemists' Society (AOCS, 1993) Cd 8-53 and Te 1a-64, respectively [18]. The peroxide value was proceeded as follows. First, 2 g of sample was dissolved in 10 mL of acetic acid and chloroform mixture (3:2, *v/v*) in a 100 mL Erlenmeyer flask and 0.4 mL of KI saturated solution was added and kept in a dark place for 10 minutes. After that, 12 mL of distilled water and 0.4 mL of 1% starch solution were added in succession, and the mixture was shaken. The resultant was titrated with 0.01 N Na2S2O3 solution. Acid value was measured by the following method. 5 g of sample is precisely weighed, placed in a stoppered Erlenmeyer flask, and dissolved in 100 mL of a neutral ethanol/ether mixture (1:2). Using phenolphthalein solution as an indicator, titrate with 0.1 N potassium hydroxide ethanolic standard solution until pale red color persists for 30 s. The peroxide value and the acid value were measured every seven days. The peroxide value and the acid value results were respectively expressed as milli-equivalents of hydroperoxides per kg of lipids (meq/kg) and in terms of the number of mg of KOH required to neutralize the free fatty acids contained in 1 g of lipid (mg KOH/g).

#### 2.8.3. Measurement of Residual Antioxidant Level

The residual amount of antioxidant compound was evaluated using HPLC. Sample preparation was carried out as described in Section 2.5. The residual amount of antioxidant in each experimental group was expressed as the rate of change of antioxidant over time. The results of each test group measured on the first day are displayed as 100%, while the later results are displayed as a percentage of the first day's result.
