Evaluation of antioxidant activity of nano‐ and microencapsulated rosemary (Rosmarinus officinalis L.) leaves extract in cress (Lepidium sativum) and basil (Ocimum basilicum) seed gums for enhancing oxidative stability of sunflower oil

Abstract There has been interest in the use of plant extract as a natural preservative agent for improving the oxidative stability of vegetable oils. However, plant extracts have low stability against heat and environmental stress. In this study, the antioxidant potential of nano‐ and microencapsulated Rosmarinus officinalis L. extract (RE) obtained using the ultrasonication method was measured. The total phenolic and flavonoid content of the extract was 174.4 ± 25.9 mg gallic acid/g extract and 78.30 ± 3.2 mg rutin/g extract, respectively. Antioxidant activity of 50, 100, 200, and 400 ppm of RE was measured by DPPH free radical scavenging methods, ferric reduction assay, and β‐carotene/linoleic acid assay, and then compared to the 100 ppm of TBHQ as a common synthetic antioxidant. The results showed that the antioxidant activity increased with increasing the concentration of the extract in all evaluating methods. The antioxidant activity of 200 ppm of the free and encapsulated extract in cress (Lepidium sativum) and basil (Ocimum basilicum) seed gums at different ratios (1:0, 1:1, and 0:1) was compared to sunflower oil without antioxidants, and oil‐containing TBHQ which was stored at 60°C for 24 days. The oxidation indexes of oil samples include peroxide value, thiobarbituric acid value, and p‐anisidine value measured at 4‐day intervals. A lower oil oxidation was observed in oil‐containing nanoencapsulated extract followed by microencapsulated extract, free extract, and TBHQ. Since producing nanoencapsulated RE requires a higher time and speed of homogenization and due to no statistically significant difference between the antioxidant properties of nanocapsules and microcapsules in oil, the use of microcapsules of RE in basil seed gum to increase the shelf life of sunflower oil is recommended.


| INTRODUC TI ON
Sunflower (Helianthus annuus) is one of the significant oilseeds in the world. Due to high oil yield, and lack of anti-nutritional factors, the area under cultivation has increased (Yazdan-Bakhsh et al., 2021).
Sunflower oil (SFO) contains a high amount of linoleic acid, essential fatty acids, and vitamin E (Franco et al., 2018). It may suffer rancidity when exposed to high temperature, and the presence of oxygen which could lead to the loss of quality (Jia et al., 2021). Oil oxidation has harmful effects on human health due to the formation of rancid odors, unpleasant flavors, and discoloration .
However, an artificial antioxidant such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) could inhibit oil oxidation and prolong oil shelf life (Farahmandfar et al., 2017;Jia et al., 2021). Because of the world demand for more natural antioxidants, many efforts have been devoted to finding new natural components for minimizing or replacing synthetic additives.
Rosemary (Rosmarinus officinalis L.) is a commonly used foodflavoring spice plant (Moczkowska et al., 2020). It is famous as a natural antioxidant due to its strong antioxidant capacity (Yang et al., 2016). Several studies have reported the antioxidant activities of rosemary extract (Saini et al., 2020;Wang et al., 2018). It has been reported that RE could be effective in retarding oil oxidation (Chammem et al., 2015;Guo et al., 2016;Moczkowska et al., 2020;Wang et al., 2018;Yang et al., 2016). Direct use of plant extracts is not applicable because of interaction with food components, unpleasant color, odor, taste, and potential decomposition reactions during processing, and storage (Hosseinialhashemi et al., 2021).
Nanoencapsulation technology providing final product functionality includes controlling the release rate, protecting food ingredients, and novel food delivery systems . By using nanotechnology, extracts can be encapsulated in the form of double or multilayer nanoemulsions. Nanoemulsions are one of the most current colloidal systems for food applications. The selection of wall material as an encapsulant to fabricate nanoparticles has to be carefully made with full considerations (Razavi et al., 2020).  (Mirzabe et al., 2021).
Although the antioxidant activity of RE was investigated in several kinds of literature, there is no study on the properties of nanoencapsulated RE with BSG and CSG wall materials and its antioxidant activity on sunflower oil. Therefore, the aims of this study were to (1) determine the total phenolic content of RE, (2) investigate the antioxidant activity of RE with different methods, (3) encapsulate the RE in cress (Lepidium sativum) and basil (Ocimum basilicum) native seed gums, (4) evaluate the properties of nanoencapsulated RE, and (5) compare the antioxidant ability of TBHQ, free, and encapsulated extract of rosemary in sunflower oil during storage.  (Hammi et al., 2015).
The total phenolic content of RE was determined by means of Folin-Ciocalteu reagent . Briefly, 50 µl of RE was added to the Folin-Ciocalteu phenol reagent (125 µl), which was diluted with distilled water in a 1:4 ratio. After 5 min, 250 µl of sodium carbonate (25%) solution was added. The solution was mixed thoroughly and then allowed to stand for 30 min at a 40°C water bath (Memmert, Schwabach, Germany). The absorbance was measured at 760 nm vs. the prepared blank which is containing all chemicals and reagents except the extract .

| Antioxidant activity of extract
To evaluate the antioxidant activity and optimum concentration of RE, different assessment methods were used. The DPPH radical scavenging activity and ferric reduction power were measured according to the method described by Rashidaie Abandansarie (2019).
The method described by Dias et al. (2015) was used to measure the β-carotene/linoleic acid system (Dias et al.,

| Preparation of nano-and microcapsule of RE
The 0.05% w/v solution of BSG and CSG as coating materials at different ratios (1:0, 1:1, and 0:1) was prepared by dispersing dried powder in 40°C deionized water and after cooling mixed overnight to enhance hydration. To prepare a stable emulsion, RE (10 ml) combined with tween 80 emulsifier (40 ml) and sunflower oil (50 ml) under magnetic stirring for 15 min. Then, the nano-and microemulsions were created by mixing the solution in an Ultra-Turrax homogenizer (IKA T25D, Germany) at 10,000 rpm for 5 min and 15,000 rpm for 10 min, respectively. After that, emulsions were encapsulated with 0.05% of coating materials at 1:5 ratios to form water-in-oil in water emulsion (Mohammadi et al., 2016;Najafi et al., 2011;. Nano-and microemulsions were dried using a freeze dryer (L101, Liotop, São Carlos, Brazil) at 0.017 mPa and −57°C for 48 hr (Chranioti & Tzia, 2013).

| Particle size, zeta potential, PDI, and encapsulation efficiency of capsules
The Z-average diameter, polydispersity index (PDI), and the zeta potential of the particles were measured using the Zetasizer (nano ZS90 equipment, Malvern, UK) based on the laser light scattering method. Samples were diluted with deionized water 10 times and placed in the tube (Joye et al., 2015). The concentration of phenolic compounds in the hexane phase was quantified, and encapsulation efficiency (EE) was calculated by Equation 1 (Kenari et al., 2020):

| Scanning electron microscopy (SEM)
The scanning electron microscope (S4800, Hitachi, Japan) was used to study the morphology of particles. Samples coated with a thin layer of gold and transferred to a vacuum evaporator. A beam of high-velocity electrons with an accelerated voltage of 26 kV was applied to the samples, and the scanned images were obtained (Chatterjee & Bhattacharjee, 2013).

| Release of phenolic compounds
The particles were poured into the McCartney bottles and stored at 60 ± 2°C for 24 days, and the release properties was performed according to Esmaeilzadeh Kenari et al., (2020) at 4-day intervals.
Thus, 5 g of particles was mixed with 5 g of phosphate buffer (pH 7) and centrifuged (Centurion K 2 S series, UK) at 1500 g at room temperature for 90 min. Then, the lower phase was collected carefully, and total phenolic content was determined by means of Folin-Ciocalteu reagent. The amount of phenolic compounds released was determined using the following formula: Release rate (%) = 100 − (R 2 × 100/R 1 ), where R 2 is the percentage of encapsulated compounds (total phenolics) in the outer aqueous phase, and R 1 is the percentage of compounds in the inner phase .

| Oil tests
The oil samples include (1)

| Statistical analysis
Statistical analysis was performed using the SPSS software Version 20.0 (Inc., Chicago, IL). The results of extract and oil samples are presented as mean ± SD of three replications. Comparison of the means was performed by a post-hoc Duncan's test at 95% confidence level and 5% significance level using the one-way ANOVA test.

| Antioxidant activity, total phenolic (TPC), and flavonoid content (TFC) of extract
The TPC and TFC of RE obtained using ultrasound were 174.25 ± 4.9 mg gallic acid/g and 78.30 ± 3.2 mg rutin/g, respectively. The previous study by Wang et al. (2018) investigated the effect of extraction time of 15, 30, 60, 120, and 150 min on the total phenolic content of RE obtained with ethanol 80%. Their results reported the amount of TPC in the range of 10.30-160.70 mg GA/g extract, which is lower than the amount reported in the current study (Wang et al., 2018). Similarly Saini et al. (2020) reported TPC and TFC for rosemary leave extract to be 136.66 ± 7.41 mg gallic acid/g and 37.13 ± 6.04 mg rutin/g, respectively (Saini et al., 2020). The TPC of rosemary extract reported by other researcher were 32.42 mg GA/gDM (Moczkowska et al., 2020), 112.0 mg GAE/g (Chammem et al., 2015), and 162.0 mg GAE/g extract (Erkan et al., 2008). The TFC of rosemary extract was reported 24 mg CE/g extract by Wang et al. (2018) (Wang et al., 2018). The reason for the difference may be related to the growth conditions, harvest season, plant variety, genetics, extraction conditions, polarity, and type of solvent (Erkan et al., 2008;Yazdan-Bakhsh et al., 2021).
Antioxidant activity of RE was evaluated by the DPPH radical scavenging activity, β-carotene/linoleic acid assay, and ferric reduction power. As can be seen in Figures 1, 2,

| Characteristics of nanocapsules and encapsulation efficiency
Determination of particle size, PDI, zeta potential, and encapsulation efficiency of particles are very important factors which affect the stability of colloidal system and properties of final product. The results of particle size, PDI, zeta potential, and encapsulation efficiency of encapsulated RE are shown in Table 1 A higher PDI was observed in particle prepared with basil seed gum.
Also, nanocapsules exhibited higher PDI. All particles had the PDI index ≤0.300, which shows monotone distribution. An increase in particle diameter caused a decrease in PDI. These results are in agreement with a study reported by Chaari et al. (2018). They reported lower PDI in microemulsions of carotenoids in comparison with nanoemulsions and also slight decrease in PDI of microemulsions by increasing the particle diameter (Chaari et al., 2018). A study stated that the encapsulated extract in particle with shahi (Lepidium sativum) seed gum wall had the largest particle size than that of en-  . Higher viscosity of wall material caused an increase in encapsulation efficiency of particles . Therefore, the particles prepared by basil seed gum showed a higher encapsulation efficiency of extract.

| Release rate
Determination of phenolic compounds release rate of particles during storage time is very important. The controlled release of phenolic compounds during incubation period affects the antioxidant activity of particles and shelf life of products. The results of phenolic compounds release from different particles during incubation period are shown in Table 2. As can be seen, in all particles, phenolic compounds were released over time and statistical differences (p < .05) were observed. The size of particles is critical factor which affects the release rate of phenolic compounds and small particles exhibited higher amount of phenolic content. The samples of NBSG and MBSG due to lower rate of release at the end of incubation period were selected for addition in sunflower oil.

| Peroxide value
Peroxide value (POV) determines the concentration of peroxides in the early stages of lipid oxidation. A lower POV implies a higher oxidative stability (Ganji & Sayyed-Alangi, 2017;Yang et al., 2016). The changes in POV of different oil samples during incubation period are shown in Figure 5. It can be seen that in all sunflower oil samples, the peroxide value has increased over time (Asadi & Farahmandfar, 2020;Chammem et al., 2015;Farahmandfar & Ramezanizadeh, 2018) and the significant statistical difference was observed at different incubation periods except days 0 and 4 of oil samples containing antioxidant. The control sample had the highest POV. Similarly, Yang et al. (2016) reported that during 24-day storage, POVs of the soybean oil samples with and without added antioxidants increased sharply and oil with rosemary extract showed lower oxidation (Yang et al., 2016). The drop in POV of CNTL sample at the end of storage period was due to the unstable primary oxidation products that are sensitive to decomposition. These products form the carbonyl compounds (Yang et al., 2016). In all incubation periods, the blank sunflower oil had significantly (p < .05) higher POVs than oil containing antioxidants. The lower POV of oil with plant extract than oil with synthetic antioxidant during frying process or storage also reported by other researchers (Agregán et al., 2017;Farahmandfar et al., 2015;Yazdan-Bakhsh et al., 2021). As can be seen in Figure 5, encapsulated rosemary exhibited higher antioxidant activity than synthetic antioxidant which is in line with the findings in a recent study by other researchers for Mentha piperita (Royshanpour et al., 2021) extract and nanoencapsulated Ferula persica extract (Estakhr et al., 2020).

| Thiobarbituric acid value
The

TA B L E 2
The release of phenolic compounds from encapsulated rosemary extract during storage (%) that TBARS gradually increased in sunflower oil during storage period and increased acceleration after the 4th day. They also reported maximum TBARS for control sample (Chen et al., 2014), which is in line with the results of current study. Previous studies by other researchers also showed that encapsulated extract of Heracleum lasiopetalum (Yazdan-Bakhsh et al., 2021) and Fumaria parviflora  can retard oil oxidation in terms of TBARS and extend the shelf life of sunflower oil.

| P-anisidine value
P-anisidine analysis is suitable method to evaluate the secondary lipid oxidation. As oil oxidation continues, the formed peroxides were no longer assayed. The color changes which occurred due to aldehydecarbonyl bound generation during secondary lipid oxidation were measured as a P-anisidine value (Guo et al., 2016). The results of changes in the P-anisidine value (PAnV) of different oil samples during incubation period are illustrated in Figure 7. In this study, as expected, there was a statistically significant (p < .05) increase in PAnV throughout the incubation period, regardless of samples treated with antioxidant. CNTL oil showed the highest PAnV, which is in line with the result reported by Moczkowska et al., (2020). A higher PAnV indicates that higher rancid oil is produced. Guo et al. (2016) revealed that the highest inhibitory effect on the generation of secondary oxidation products was observed in the palm oil containing natural antioxidant rosemary extract (Guo et al., 2016). Dias et al. (2015) studied the antioxidant activity of rosemary extract in soybean oil under accelerated heating, compared with synthetic antioxidant TBHQ. The results in this study were similar to theirs, and RE was more effective than TBHQ in decreasing the PAnV of oil (Dias et al., 2015). In a study by specified that during incubation, flaxseed oil with an addition of RE had the lowest POV and PAnV which is related to phenolic compounds specially carnosol and rosmarinic acid (Chen et al., 2014;Wang et al., 2018). Unsurprisingly, oil without antioxidant was the most easily oxidized sample. The samples of POV, TBARS, and PAnV with RE incorporated were significantly lower than that of oil with added TBHQ synthetic antioxidant, indicating that RE was more effective in stabilizing oil against oxidative deterioration compared to synthetic antioxidant. The antioxidant activity of TBHQ is because of its pure phenolic compound. However, RE is a sort of crude extract and it contained a large amount of the mixture of phenolic compounds with synergy effect (Guo et al., 2016). The antioxidant activity of phenols is due to their scavenging ability on free radicals, chelate metals, and donate electron or hydrogen atoms (Chen et al., 2014).