Evaluation of antioxidant and antibacterial interactions between resveratrol and eugenol in carboxymethyl cellulose biodegradable film

Abstract The aim of present study was to compare the in vitro antioxidant and antibacterial properties of carboxymethyl cellulose (CMC) films containing resveratrol (RES) and eugenol (EUG), alone and in combination, and to calculate the dose interactions between them. At first, the total phenolic content of CMC films was evaluated. Then, their antioxidant and antibacterial effects of films were determined using DPPH, reducing power, disk diffusion, and broth dilution methods. Finally, concentrations of RES and EUG which showed better results in the CMC films were added in combination forms to calculate their antioxidant and antibacterial interactions. The results showed that addition of RES and/or EUG to CMC films increased the total phenolic content, free radicals scavenging activity, reducing power, and antibacterial activities of the films (p ≤ .05). Gram‐positive bacteria were more susceptible than Gram‐negatives. In addition, the combined use of RES and EUG in CMC films had synergistic antioxidant and antagonistic antibacterial effects. The best results belonged to the film containing RES (8 µg/ml) + EUG (8 mg/ml) (p ≤ .05). Considering the results of the present research, we can utilize CMC biodegradable film containing RES and EUG as a natural active packaging in food industry.

RES and EUG which showed better results in the CMC films were added in combination forms to calculate their antioxidant and antibacterial interactions. The results showed that addition of RES and/or EUG to CMC films increased the total phenolic content, free radicals scavenging activity, reducing power, and antibacterial activities of the films (p ≤ .05). Gram-positive bacteria were more susceptible than Gramnegatives. In addition, the combined use of RES and EUG in CMC films had synergistic antioxidant and antagonistic antibacterial effects. The best results belonged to the film containing RES (8 µg/ml) + EUG (8 mg/ml) (p ≤ .05). Considering the results of the present research, we can utilize CMC biodegradable film containing RES and EUG as a natural active packaging in food industry.
Food packaging is one of the most important processes for maintaining the microbial and chemical quality and safety of products (Han, 2005). Nowadays, synthetic polymers are widely used in food packaging industry due to their desirable characteristics such as stability, flexibility, and ease of production (Yadav et al., 2018). However, synthetic polymers can cause problems such as adverse environmental effects, the release of toxic gases, and contribution to global warming. In addition, the transfer of chemical compounds such as monomers and solvent residues, to food is another problem of synthetic polymers. Environmental and consumer health concerns have led to an increase in the replacement of biodegradable polymers with synthetic polymers (Mignon et al., 2019). Biodegradable polymers such as edible films can be considered as a suitable barrier against moisture and gases (oxygen and carbon dioxide) as well as good carriers for antimicrobial and antioxidant compounds as active packaging (Hasheminya et al., 2019). Carboxymethyl cellulose (CMC) is one of the most widely used polymers in the food packaging industry due to its hydrophilic properties, optimal film ductility, biological compatibility, gas transfer inhibition, and internal stability of the network structure (Roy & Rhim, 2020). This biodegradable polysaccharide is non-toxic and shows good functional characteristics in the formation of active packaging (Raeisi et al., 2012). Previous studies have shown that CMC film and coating are good carriers for several bioactive agents such as natural antimicrobial and antioxidant compounds (Dashipoor et al., 2014;Dashipour et al., 2015;Panahirad et al., 2021;Raeisi et al., 2015). According to Panahirad et al. (2021), CMC-active coatings could reserve active ingredients in their structure and gradually release them to the surface of fruits and vegetables to improve the performance of the active ingredients. As a result, the quality and shelf life of the product will be promoted (Panahirad et al., 2021).
The use of antimicrobial and antioxidant compounds in food products is a good solution to maintain the safety of consumers. Despite the widespread use of synthetic antimicrobial and antioxidant compounds in food industry, the adverse effects of these compounds on human health are considered as a serious public health concern.
In this respect, comprehensive researches have been performed to replace them with natural compounds (Davidson et al., 2014;Shahidi & Ambigaipalan, 2015).
Resveratrol (RES) is a stilbene polyphenolic compound which exists in grapes, peanuts, red wine, and mulberries. Several studies have been conducted and reported antimicrobial, antioxidant, and potential health benefits of resveratrol (Arcanjo et al., 2019;Ma et al., 2018;Oh & Shahidi, 2018).
Eugenol (EUG) is a natural and aromatic compound that is generally extracted from cloves, bay laurel, and cinnamon bark. This phenolic compound has been interested as an additive applied in the food, agricultural, pharmaceutical, and cosmetic industries, due to its low-cost, and desirable antimicrobial and antioxidant activities (Zheng et al., 2019).
Previously, several studies have been performed on the antimicrobial and antioxidant potential of biodegradable films containing resveratrol and eugenol alone (Busolo & Lagaron, 2015;Ma et al., 2018;Pastor et al., 2013;Suppakul, 2016;Zheng et al., 2019). But according to best of our knowledge, no comparative studies have been conducted on the antimicrobial and antioxidant potential of resveratrol and eugenol in biodegradable films yet. Therefore, the objective of the present study was to compare the antibacterial and antioxidant properties of CMC biodegradable films containing RES and EUG, alone and in combination, and to calculate the dose interactions between them using an in vitro model.

| Preparing CMC films
In order to preparation of CMC films containing RES and EUG, one gram of CMC powder and 0.01 g calcium chloride were dissolved in 100 ml of sterile distilled water. The film solution was then stirred using a magnetic stirrer at 70°C and 1200 rpm for 45 min to completely dissolve the CMC and to obtain a uniform solution. Then, 0.5 ml of glycerol (50% v/w based on CMC weight) was added to the suspension as a plasticizer and was mixed at 70°C for 10 min.

Concentrations of RES and EUG which showed better antibacterial
and antioxidant effects were also incorporated into CMC film in combination form to evaluate the dose interactions between them.
Then, film-forming solutions were homogenized with ultra-turrax homogenizer (IKA T10 basic) at 13,500 rpm for 3 min at room temperature. CMC solution without addition of RES and EUG was considered as the control group. Then, 25 ml of the film-forming solutions was poured on Teflon plates (ϕ = 10 cm) and placed in the oven to dry for 30 h at 37°C. The dried films were then separated from the plates and stored in a desiccator at 25°C and 53% relative humidity until testing. Prior to antibacterial testing, the films were exposed to ultraviolet light for 2 min under the laminar hood in order to eliminate possible contaminants (Dashipour et al., 2015).

| Total phenolic content of CMC films containing RES and EUG
Folin-Ciocalteu method was used to measure the phenolic compounds of CMC films containing different concentrations of RES and EUG. For this purpose, 25 mg of each film was placed in 3 ml of distilled water and it was homogenized for 5 min. Then, 0.1 ml of the film extract was mixed with 7 ml of distilled water and 0.5 ml of Folin-Ciocalteu reagent and kept at room temperature for 8 min. Consequently, 1.5 ml of sodium carbonate (2% w/v) and distilled water were added to obtain the final volume of 10 ml. Afterward, the obtained mixture was kept in darkness at the room temperature for 2 h and the absorbance was measured at 765 nm by a spectrophotometer (HACH, DR 5000). The calibration curve was drawn using gallic acid at specific concentrations, and the results were determined using the following equation: where T is total phenolic content (mg GAE/g film), C is the gallic acid concentration obtained from the standard curve (mg/ml), V is the volume of film extract (ml), and M is the weight of dried film (g). The experiment was carried out in triplicate (Moradi et al., 2011).

| Evaluation of antioxidant activities of CMC films containing RES and EUG
2.4.1 | DPPH free radical scavenging activity 25 mg of each film sample was dissolved in 3 ml of distilled water, and then, 2.8 ml of each film extract was added with 0.2 ml of 1 mM DPPH methanolic solutions. The absorbance was measured at 517 nm by a spectrophotometer after incubation for 30 min at room temperature. Films which contain 1 mg/ml BHT and film without any antioxidant compound were considered as positive and negative controls, respectively. The percentage of inhibition was measured by the following equation: where Abs DPPH is the absorption of DPPH methanolic solution and Abs sample is the absorption of CMC films (Hashemi et al., 2019).

| Ferric (Fe 3+ ) reducing power
At first, 10 mg of each film was added to the test tubes containing 1.25 ml of potassium phosphate buffer (pH 6.6, 0.2 M) and 1.25 ml of potassium ferricyanide (1%), and it was kept at 50°C for 20 min. Afterward, 0.5 ml of trichloroacetic acid (10%) was added to the tubes and centrifuged at 3000 rpm for 10 min.
Finally, 1.25 ml of the supernatant was transferred to the tubes containing 1.25 ml of potassium phosphate buffer and 0.25 ml of ferric chloride. After 10 min incubation, the absorbance of test tubes was measured at 700 nm. The higher adsorption rate of the final mixture, the greater the reducing power of the sample (Jridi et al., 2019).

| Calculating the antioxidant interactions between RES and EUG in CMC films
The antioxidant interactions between RES and EUG in CMC films which were determined by DPPH and reducing power tests were calculated using the combination index (C.I). For this purpose, the following formulas were used: where DPPH ab and R.P ab are the obtained results from the combined use of RES and EUG in CMC films, and DPPH a , DPPH b , R.P a , and R.P b are the values obtained from the use of RES and EUG alone in CMC films. C.I values equal, smaller or greater than 1, indicate an additive, antagonistic, or synergistic effects, respectively (Hashemi et al., 2019).

| Preparation of studied bacterial strains
Antibacterial effects of CMC films were studied against two grampositive bacterial strains including S. aureus and L. monocytogenes, and two gram-negative bacterial strains including E. coli and S. enteritidis. First of all, stock bacteria (vials containing 20% w/v glycerol in BHI broth and kept at −20°C) were inoculated in 15 ml BHI broth and incubated at 37°C for 24 h at 150 rpm (repeated at least twice). Afterward, inoculums were centrifuged three times at 6000 rpm for 5 min to be separated from BHI broth. During the last 2 centrifugation steps, the supernatant was eliminated and physiological saline was added. In order to prepare and count bacterial inoculations, optical density (OD) method was used.
Various dilutions were prepared from bacterial cultures, and their absorbance was read at 600 nm using a spectrophotometer to adjust to 0.5 McFarland standard turbidity (~1-2 × 10 8 CFU/ml).
Afterward, bacterial suspensions were diluted to achieve the desired concentration for each test. In order to confirm the results, bacterial counting was carried out in BHI agar at 37°C for 24 h (Abdollahzadeh et al., 2014).

| Disk diffusion method
In this test, 100 µl of each studied bacterial suspension (~1-2 × 10 6 CFU/ml) was cultured in BHI agar medium. Consequently, disks with a diameter of 6 mm were prepared from CMC films containing different concentrations of RES and EUG (alone and in combination) and placed on the culture medium. Disks which contain chloramphenicol (30 μg/disk) and CMC films without any antimicrobials were used as positive and negative controls, respectively. Then, the plates were incubated at 37°C for 24 h and antimicrobial activity of CMC films was assessed by measuring the inhibition zone diameter by a caliper (Moghimi et al., 2017). Accordingly, the antibacterial activities of films were classified as follows (Lv et al., 2011): 12 mm ≥ inhibition zone: Weak antibacterial activity; 20 mm > inhibition zone > 12 mm: Moderate antibacterial activity; 20 mm ≤ Inhibition zone: Strong antimicrobial activity.

Calculating the antibacterial interactions between RES and EUG in CMC films using disk diffusion method
To evaluate the antibacterial interactions between RES and EUG in CMC films, at first the inhibition zone of CMC films containing RES was statistically added to the inhibition zone of CMC films containing EUG and recorded as "expected inhibition zone". Then, the inhibition zone of CMC films containing a combination of RES and EUG at similar concentrations was compared with them. If the expected inhibition zones are equal, smaller or greater than the inhibition zones of CMC films containing a combination of RES and EUG, indicate an additive, synergistic or antagonistic effects, respectively (Moussaoui & Alaoui, 2016).

| Broth dilution method
In this method, 25 mg of each CMC films was transferred to test tubes containing 10 ml of bacterial suspension (BHI broth containing ~1-2 × 10 5 CFU/ml bacteria). Afterward, the tubes were placed in shaking incubator at 37°C for 24 h. A test tube without film and a test tube without bacteria were used as positive and negative controls, respectively. Then, 0.1 ml of each tube was used to prepare serial dilutions that were spread on nutrient agar plates. The plates were incubated at 37°C for 24 h, and the colonies were counted and calculated using a dilution factor (Sugumar et al., 2015). In order to perform the next calculations, positive control was counted immediately after preparing the suspension and 24 h after incubation.

| Calculating the bacterial reduction index
In order to calculate the bacterial reduction index of CMC films con-

Calculating the percentage of bacterial growth inhibition
The percentage of bacterial growth inhibition CMC films containing various concentrations of RES and EUG was calculated using the following formula: where P.I is the percentage of bacterial growth inhibition; CON is the mean of bacterial counts of the control sample after 24 h (24-h control); Treatment is the mean of bacterial counts of the treated samples after 24 h.

| Statistical analysis
All tests were performed in triplicate. Statistical analysis of the data was performed using one-way ANOVA with SPSS software (Version 18.0 for Windows; SPSS Inc.). Tukey test was also used to determine the significant difference between the means (α = .05). Pearson test was also used to determine the correlation between the data.

| Total phenolic content
Phenolic compounds, including terpenes and flavonoids, are responsible for the strong antimicrobial/antioxidant activities of some plant preservatives (Aziz & Karboune, 2018). The total phenolic compounds in the CMC films containing different concentrations of RES and/or EUG using Folin-Ciocalteu method was expressed as mg GAE/g film which was calculated by using an equation that was obtained from the standard graph (R² = 0.9992): Abs 765 = 0.0011 mg gallic acid + 0.0075.
Based on the results (Figure 1), the amount of total phenolic compounds in CMC films containing different concentrations of RES and EUG was in the range of 7.88-15.15 and 9.09-16.36 mg GAE/g film, respectively. In this regard, Talón et al. (2019) have reported 3.24 mg GAE/g film total phenolic content in corn starch film containing 1.59% EUG (Talón et al., 2019). Also, according to the study conducted by Adhikari et al. (2018), the results showed that there was a positive correlation between the total phenolic content and the amount of RES in seeds and sprouts of Korean peanuts (Adhikari et al., 2018). In another study, the total phenolic content of sodium alginate film containing 0.25-4 μg/ml RES was 6.82-11.56 mg GAE/g film which was in accordance with the results of present study (Hashemi et al., 2019). Moreover, the results of present study showed that there was a significant increase in total phenolic con-

| DPPH free radical scavenging activity
DPPH method is a comprehensive test for measuring the free radical scavenging ability of edible films containing natural antioxidants with plant origin. This method is very suitable for evaluating the free radical scavenging power due to its simplicity, speed, and independence from sample polarity (Salarbashi et al., 2014). The results of antioxidant activity of CMC films containing RES or EUG measured using DPPH free radical scavenging method are shown in Table 1.
As can be seen, the films prepared without adding RES and EUG (control group) did not show any antioxidant activity and the antioxi- which was in accordance with the results of the present study. In the aforementioned study, the antioxidant capacity of the films has been attributed to the reaction between the hydroxyl groups of EUG and free radicals and the formation of stabilized phenoxyl radicals. Film containing EUG is able to act as a strong donor of electrons or hydrogen atoms and subsequently turn purple color of DPPH free radicals to yellow (Zheng et al., 2019). In a study conducted by Busolo and Lagaron (2015), it was reported that polyethylene film enriched with 1000 ppm RES had antioxidant activity of about 80% based on the DPPH method (Busolo & Lagaron, 2015). In another study, chitosan and methylcellulose films containing RES showed favorable antioxidant activity and it was recommended that these films could be used as a packaging material for foods which are sensitive to autoxidation (Pastor et al., 2013). In the study by Hashemi et al. (2019), the DPPH free radical scavenging activity of sodium alginate film containing 4 µg/ml RES was 72.66%, which verifies the results of present study (Hashemi et al., 2019 Also, the reducing power of all films was significantly increased by increasing the concentrations of RES or EUG to CMC films (p ≤ .05).
The highest reducing power among films containing RES was related to the concentration of 8 μg/ml and among the films which contained EUG was related to the concentration of 8 mg/ml (p ≤ .05).

TA B L E 3 Antioxidant interactions between RES and EUG in CMC films (Mean ± SD)
it has been declared that RES has a greater reducing power than BHT. The final outcome of reducing reactions is to terminate the radical chain reactions (Gülçin, 2010).

| Antioxidant interactions between RES and EUG in CMC films
The RES and EUG concentrations which showed better antioxidant activities in CMC films based on DPPH and reducing power tests were incorporated into films in combination forms to evaluate their antioxidant interactions. For this purpose, concentrations of 4 and 8 µg/ml of RES as well as 4 and 8 mg/ml of EUG were added to films in combination forms. studied the antioxidant activity of RES in combination with gallic acid, caffeic acid, and quercetin using the DPPH method and found that this phenolic mixture had an antagonistic effect in inhibiting DPPH free radicals, while the combination of RES and catechin indicated a synergistic effect (Skroza et al., 2015). In another study, the antioxidant interactions of RES and Zataria multiflora Boiss essential oil in sodium alginate film were evaluated and the synergistic effect was shown between them (C.I > 1) (Hashemi et al., 2019).

| Correlation between total phenolic content and antioxidant capacity
Phenolic compounds as antioxidant agents are able to trap free radicals, donate electrons or hydrogen atoms, and inactivate them

| Disk diffusion method
The results of antibacterial activities of CMC films containing RES and EUG using disk diffusion method are presented in Tables 4 and   5 the outside, and disruption of the bacterial enzyme system (Sandri et al., 2007). The higher susceptibility of gram-positive bacteria to RES and EUG compared with gram-negatives has been proven by another studies (Ma et al., 2013(Ma et al., , 2018. In the study by Zheng   of gram-negative bacteria may be able to extrude RES from the cell (Ma et al., 2018).

| Broth dilution method
The results of antibacterial effects of CMC films containing RES using broth dilution method are shown in Table 6 The results of antibacterial activities for CMC films containing EUG using broth dilution method can be observed in Table 7  Values followed by different letters within the same rows are significantly different according to the Tukey's test (p ≤ .05).

| Antibacterial interactions between RES and EUG in CMC films
The results of antibacterial interactions between RES and EUG in CMC films using disk diffusion method are presented in Table 8. In general, the inhibitory zones of combined treatments were higher than single treatments and films containing 8 mg/ml EUG along with different concentrations of RES demonstrated strong antimicrobial activity against gram-positive bacteria (Inhibition zone ≥ 20 mm).
However, these results demonstrate the antagonistic effects of the combined use of RES and EUG compared with their alone use in the films. In other words, the "expected inhibition zones" were smaller than the inhibition zones of CMC films containing a combination of RES and EUG. Also, the inhibitory effects of the films containing RES (8 µg/ml) + EUG (8 mg/ml) and RES (4 µg/ml) + EUG (8 mg/ml) against all studied bacteria were significantly more than other combined treatments (p ≤ .05).

The results of antibacterial interactions between RES and EUG
in CMC films using broth dilution method are presented in Table 9.
In general, the antibacterial effects of combined treatments were higher than single use of RES or EUG in CMC films. Comparing the re-   antimicrobials (Santiesteban-López et al., 2007). The possible disruption of one or more of these mechanisms increases the possibility of antagonistic effects.

| CON CLUS ION
The use of RES and EUG to the edible CMC films increased the total phenolic content and increased their antioxidant and antibacterial activities against gram-positive bacteria (L. monocytogenes and S. aureus) and gram-negative bacteria (S. enteritidis and E. coli).
Antioxidant and antimicrobial properties of the CMC films increased with increasing concentration of phenolic compounds. Gram-positive bacteria were more sensitive than gram-negatives, and the highest antibacterial activity was observed against L. monocytogenes. The combined use of RES and EUG in CMC films produced the synergistic antioxidant effects. However, despite the stronger antibacterial effects of combined use of RES and EUG than single use of each of them in the film, the combined use of them produced the antagonistic antibacterial effects. The best results were related to the CMC film containing RES (8 µg/ml) + EUG (8 mg/ml). Therefore, based on the findings of the present study, the edible CMC films containing RES and EUG can be used for the food packaging industry.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in "figshare" at https://figsh are.com/artic les/datas et/CMC_films_ conta ining_RES_and_EUG_xlsx/15062280.