3.1. Extraction Yield, Total Phenolic Content (TPC) and Antioxidant Activity
The extraction yield, total polyphenols (TPC), and antioxidant activity in extracts of bearberry leaves obtained with 50:50
v/
v ethanol:water are shown in
Table 1. On average, 1.6 ± 0.01 g of extract pulp was recovered from 5 g of bearberry extract after 3 days freeze drying (
p > 0.05).
Pegg
et al. (2005) reported that the total phenolic content of BL extract was 312 mg/g DW for 95% (
v/
v) ethanol extraction [
6]. It was much higher than what we reported for 50% (
v/
v) ethanol extract. However, the total phenolic content value obtained from the BL water infusion was very low with 160.78 ± 2.84 g/kg sample [
24]. The mixtures of alcohol and water was more efficient in extracting phenolic compounds and gave a better yield than water because some phenolic constituents do not dissolve in water. Meanwhile, the antioxidant activity of BL assessed using the TEAC method was 90.42 mmol of TE/g DW. A recent study reported the antioxidant activity of BL in the TEAC assay was 3.19 ± 0.01 mol of TE/kg sample following extraction by an infusion method which is much lower than our value [
24]. The antioxidant activity of bearberry leaf achieved in the TEAC assay indicates the potency of the extract to scavenge the radical cation ABTS
•+ (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) generated in the assay. The use of several methods allows a more general assessment of the antioxidant properties of the plant. The variations of data were influenced by the sample preparation, type of extraction (solvent, temperature,
etc.), selection of end-points, and method of expression of the results. Several studies have determined the antioxidant activity of BL extract using
in vitro analysis. A few studies reported bearberry extract scavenging ability using the DPPH radical and the ability of the extract to reduce ferric(III) ions to ferrous(II) ions using the FRAP (fluorescence recovery after photobleaching) method [
15,
16,
17]. The polyphenol constituents in the extract contribute the most to the antioxidant activity. The infusion of BL showed an abundance of phenolic acid components at trace concentrations including catechin and its derivatives, epigallocatechin gallate, epigallocatechin, and epicatechin. These catechins have a strong antioxidant capacity mainly linked to their radical scavenging activity [
18].
3.2 Analysis of Free Radical Activity Assays
In the present study, methanolic extracts from BL were examined by EPR spectroscopy for their capacity to act as radical scavenger towards the methoxy radical (CH
3O
•) generated by the Fenton reaction. This method was used to evaluate the scavenging ability of plant extracts for the free methoxy radical (CH
3O
•).
Figure 1 shows the decrease in the EPR signal with increasing concentration of BL extract. The free radical scavenging activity of the extracts against methoxy (CH
3O
•) radical was investigated by a competitive method in the presence of DMPO as spin trap and recorded by the spectrum generated by EPR spectroscopy. The CH
3O
• radical generated by the Fenton procedure has a relatively short half-life, which means it must be identified by EPR as the stable nitroxide adduct with DMPO, DMPO–OCH
3 (hyperfine splitting constants, a
N = 13.9 G and a
H = 8.3 G). This stable DMPO–OCH
3 compound can be quantified by the double integration value of the signal from EPR. The extract containing antioxidants at different concentrations may compete with the spin trap DMPO in the scavenging of methoxy radicals. Thus, the effect decreases the amount of radical adducts and, accordingly, decreases the intensity of the EPR signal. The best fit with intensity of EPR signal exhibited a linear function (
Figure 1), given by Equation (1):
where the
x values were in mg/L.
The graph indicates the exponential value of the signal of the spectrum decreased as the amount of bearberry extract increased. Azman
et al. demonstrated the scavenging ability of catechins with methoxy radical using this assay [
18]. These catechins were also found in bearberry extract by Valjkovic
et al. and these compounds contributed to the ability to scavenge methoxy radical in this assay [
24]. Furthermore, BL scavenging ability has been previously reported by Amarowicz
et al. using hydroxyl free radicals (HO
•) measured by EPR [
11].
3.3. Antioxidant Effects in Stored o/w Emulsion
Methods have been developed to understand the effect of natural antioxidants in model foods such as emulsions and active film packaging. Adding natural antioxidants to food not only delays the oxidation process but also enhances the nutritional quality of the food through direct ingestion. In previous work, the effect of bearberry leaf extract in oil-water emulsion has not been described. A model emulsion was used to assess the deterioration of lipids at two stages of oxidation, which were the primary oxidation products (Peroxide Value) and the secondary oxidation products (TBARS). In addition, the change in pH was monitored, since pH tends to fall during oxidation.
The development of primary oxidation products was monitored by the evaluation of hydroperoxide formation (PV) during storage (
Figure 2). Primary degradation of lipids measured by PV occurs due to the reaction between oxygen and unsaturated fatty acids thatform hydroperoxides. The induction time is defined as the time for samples to reach 10 meq hydroperoxides/kg of emulsion. This value can be used as a measure of the stability of emulsions. The limits of oxidation products in fat products (animal, plant and anhydrous) including margarine and fat preparations were set at <10 meq hydroperoxides/kg as a guarantee of the product quality [
20]. When the peroxide value of the sample is greater than 10 meq hydroperoxide/kg, the sample is in a highly oxidised state and starts to become rancid. The PV value of the control emulsion increased rapidly, reaching more than 10 meq hydroperoxide/kg after only 6 days (
p < 0.05). The sample containing BL 1 g/kg reached the end of the induction time after 20 days while the BHT samples reached this state after 36 days of storage. Several studies investigated the effects of adding natural antioxidants to delay the lipid deterioration in food model emulsions. Skowyra
et al. [
16] found that an emulsion containing 48 μg/mL of Tara extract took 13 days to reach more than 10 meq hydroperoxide/kg, and Roedig–Penman and Gordon [
25] reported that an emulsion containing green tea extract required eight days to reach the end of the induction time. Emulsion containing 100 mg/L of rosemary and thyme extract displayed low PV which remained below 10 for 25 days of storage [
25].
Primary oxidation occurs rapidly in the fat phase of the product due to the formation of highly unstable hydroperoxides that break down easily. This process results in the formation of ketones, epoxides, or organic acids which are acidic, and leads to changes in the pH [
16].
Figure 3 shows that the pH value dropped over time, and this change was inversely proportional to the increase of PV. By comparison, emulsion containing BHA showed a significant pH difference from the pH values for BL and the control sample (
p < 0.05). BL samples remained higher than pH 6 for 10 days of storage and the pH declined gradually until 40 days.
A number of researchers suggested a positive effect of pH on oxidation rate which was influenced by natural antioxidants [
16,
17,
26]. Pehlivan
et al. [
27] reported that edible vegetable oils contained heavy metals such as iron up to 0.2 mg/kg oil. The levels of some metal compounds, if high enough, will promote oxidation which affects the pH [
28]. Furthermore, the redox state of metals and the activity, solubility, stability, and chelation capacity of antioxidants are among the parameters that affect the rate of change of pH in oil emulsions [
29].
Analysis of oxidation in the emulsions was extended by measuring a secondary oxidation product in the emulsion using the TBARS method (
Figure 4). Malondialdehyde (MDA) compounds produced are responsible for the alteration of flavor, rancid odor, and the undesirable taste in foods [
30]. The acceptable limits of TBARS value in fat products was set at 1.0 mg malondialdehyde/kg [
31].
Figure 4 shows the secondary oxidation products monitored by TBARS assay. Control sample experienced TBARS value of greater than 4.0 mg malondialdehyde/kg (
p < 0.05) on the 3 weeks storages compare to samples treated with antioxidant with value of TBARS that less than 1 mg malondialdehyde/kg. Over the 25 days of storage, samples with antioxidants had a TBARS value of approximately 1 mg malondialdehyde/kg sample, which may not change the physical and visual properties of the product. Emulsion treated with BHA exhibited the lowest TBARS value throughout the storage period and samples with bearberry had TBARS values of lower than 2 mg malondialdehyde/kg (
p < 0.05).
Many findings demonstrated that the efficiency of natural plant extracts as antioxidants in various concentrations applied possibly depends on the variation in the system tested and initial plant extraction. The active properties of BL was reported several times by Amarowicz
et al. [
11,
32]. Quercetin, one of the main compounds found in the BL extract, showed positive effects against lipid deterioration in oil-in-water emulsion [
9]. The leaves consist of an abundance of phenolic compounds, which may contributed to the antioxidant properties, reducing power, and antiradical properties as described in much of the literature [
7,
11,
32]. The antioxidant activity of phenolic linked with phenolic compounds is related to the hydroxyl group linked to the aromatic ring, which is capable of donating hydrogen atoms and neutralizing free radicals. This mechanism prevents further propagation of lipid oxidation, which can be measured by the TBARS method [
33]. The behavior of BL as a natural antioxidant in a food model was reported in the literature [
12,
34]. Pegg
et al. (2005) [
9] reported 200 ppm of bearberry extract inhibited TBARS formation in cooked pork patties after seven days of storage. Meanwhile, Carpenter
et al. [
12] demonstrated that the addition of BL impeded lipid oxidation in raw pork patties for 9–12 days of refrigerated storage relative to control. To the best of our knowledge, this is the first report of the inhibitory effect of BL using oil-in-water emulsion.