2.1. Materials and reagents
The seeds of C. camphora were collected in the ripening stage from the trees growing at Nanjing Agricultural University (Nanjing, China). Analytical grade standards of protocatechuic acid, gentisic acid, phlorizin, 4-hydroxybenzoic acid, procyanidin B3, citric acid, p-coumaric acid, syringic acid, L-ascorbic acid, ferulic acid, caffeic acid, salicylic acid, quercetin, rutin, catechin and epicatechin were purchased from Henan Putian Tongchuang Biotechnology Co., Ltd. (Henan, China). CMC was obtained from Junsei Chemical Co., Ltd. (Tokyo, Japan), while glycerol was obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylben-zothiazoline-6-sulphonic acid) (ABTS) were obtained from Sigma Chemical Co., Ltd. (St Loius, MO, USA). Gallic acid was purchased from Aladdin Industrial Inc. (Shanghai, China). The sunflower oil was obtained from a local market (Nanjing, China,). All other reagents used were of analytical grade.
2.2. Preparation and purification of CCSE
The extraction experiments were designed as follows: first, seed coats were peeled from fresh fruits of C. camphora and oven-dried for 12 h at 50 ℃. To remove oil from seeds, the crushed C. camphora seeds (25 g) were continuously extracted with 150 mL petroleum ether at 45 ℃ for 6 h in a Soxhlet apparatus. Afterward, the crushed seeds were ground to powder and extracted with 80% aqueous ethanol solution at 45 ℃ for 90 min, with ultrasonication at 300 W (KQ 300DB, 300 W, 0-40 kHz, Kunshan Ultrasonic Instrument, Kunshan, China). After vacuum filtration, the supernatants were obtained and concentrated at 45 ℃ by a vacuum rotary evaporator. The targeted phase was then loaded onto a column (5 × 30 cm) of D101 resin, washed with distilled water at a flow rate of 1.5 mL/min to eliminate strongly polar components. After that, the column was eluted twice with 80% ethanol. The resulting elution was concentrated by a rotary evaporator at 45 ℃ and freeze-dried to obtain purified fractions of CCSE.
2.3. Characterization of phenolic compounds in CCSE
2.3.1. Total phenolic content
The Folin-Ciocalteu method outlined by Islam et al. [33] was adopted to evaluate the total polyphenol content of CCSE. Briefly, 0.5 mL from the sample was added to 1.0 mL from the reagent (Folin-Ciocalteu) was diluted 10 times and left in the dark for 6 min. After that, 2.5 mL from Na2CO3 (75 g/L) was added and the mixture was kept for 30 min at 25 ℃. At 765 nm, the absorbance of the sample was measured, and the amount of total phenolic content was expressed as mg of gallic acid equivalent (GAE) per g of extract. Gallic acid was used as a standard for plotting the calibration curve. All experiments were repeated six times.
2.3.2. LC-ESI-QTOF-MS analysis
Identification and quantification of polyphenols in CCSE were achieved by the LC-ESI-QTOF-MS system (G2-XS QTOF Waters, Manchester, UK). In brief, the extract was prepared at a concentration of 5 mg/mL. After that, the samples were centrifuged at 3,000 rpm (Beckman coulter Avanti J-265XP) for 15 min at 20 ℃. Continuously, 1.5 mL of supernatants were collected in HPLC vials for LC-MS, and 2 µL solution was injected into the UPLC column (2.1 × 100 mm, 1.7 µm particles, ACQUITY UPLC BEH C18 column) with a flow rate of 0.3 mL/min. Buffer A consisted of 0.1% formic acid in the water, and buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was 5% Buffer B for 0.5 min, 5–95% buffer B for 11 min, 95% buffer B for 2 min. Electrospray ionization (ESI) was used as a source in positive modes with a preferred mass spectrum in the m/z range 100-1600. The leucine-enkephalin (m/z 556.2771) was used for recalibration and removing the lock mass. Ionization parameters were the following: the capillary voltage was 2.5 kV, sample cone was 40 V, source temperature was 120 ℃ and desolvation gas temperature was 800 ℃. Data acquisition and processing were performed using Masslynx software version 4.1.
2.3.3. HPLC analysis of CCSE
Aglient 1100 Series HPLC system (USA) with DAD detector and Phenomenex SynergiTM 4 µm RP-Hydro C-18 (0.46 × 250 mm) was used to determine the phenolic composition of CCSE. Two mobile phases were used, formic acid in deionized water 0.1% (v/v) (A) and acetonitrile (B). The flow rate was set at 15-30% from 0 to10 min, 30-50% from 10 to15 min, 50-60% from 15 to 20 min, and 60-70% from 20 to 25 min, and 70-80% from 25 to 30 min. The injection volume and the flow rate were 20 µL and 0.35 mL/min, respectively. The samples were measured at 280 nm and the column temperature was 35 ℃.
2.4. Preparation of films
The films were fabricated by dissolving 6.0 and 3.2 g of CMC and GA, respectively, in distilled water (400 mL). After stirring (800 rpm, 60 ℃, 2 h), the glycerol solution (35% w/w based on CMC and GA used), used as a plasticizer, was added to the mixture and thoroughly homogenized by stirring at 800 rpm at 50 ℃ for 30 min. After that, CCSE was added at the concentration of 0, 0.5, 1.2 and 2% (based on CMC and GA used) to afford groups of CMC/GA (CG, control), CMC/GA with the respective concentrations denoted as (CG-CCSE 0.5, CG-CCSE 1.2, and CG-CCSE 2), respectively. Every group was stirred (40 ℃, 800 rpm, 1 h) to afford a thorough blend. Afterward, 50 mL aliquot from each group was carefully poured into petri-dish plates (150 mm) and oven-dried (45 ℃, 12 h). The films were then peeled off the plates and stored in a desiccator at 30 ℃ until further analysis.
2.5. Characterization and mechanisms of films
2.5.1. Scanning electron microscopy (SEM) analysis
The surface morphology of produced films was analyzed using SEM (SU8010, Hitachi, Japan) at 10 kV. The films were cut (10 × 10 mm) and placed on an aluminum slab using a double-sided sputter covered with gold and carbon tape.
2.5.2. FT-IR spectrophotometry analysis
FT-IR spectrometry (Nicolet iS-50, Thermo, USA) with 4000 to 525 cm −1 frequency range was employed to analyze the prepared film's initial structures.
2.5.3. X-ray diffraction (XRD)
An X-ray diffractometer (D8 Advance, Bruker, USA) was used to identify the crystal structure of the films at 30 kV and 10 mA. Angular range (2 θ = 5 – 80 °) was applied to detect the scattered radiation at 15.6°/min scanning speed.
2.5.4. Differential scanning calorimetry (DSC) analysis
DSC (DSC-60, Shimadzu Corp., Kyoto, Japan) was used to analyze the thermal properties of the films. Briefly, 10 mg of film pieces were sealed in a standard aluminum pan which was heated under a nitrogen atmosphere from 27 to 450 ℃ at a rate of 10 ℃/min
2.5.5. Thickness and mechanical properties analysis
The film thickness was estimated by a digital hand-held micrometer (Mitutoyo Absolute, Tester Sangyo Co. Ltd., Japan). Each sample was measured 10 times randomly at various levels, and the mean was calculated. Additionally, a texture analyzer (TA.XT Plus, Stable Micro Systems Ltd., Surrey, UK) was employed to analyze the mechanical properties (tensile strength percentage (TS) and elongation-at-break (EB) of the films at room temperature. Film strips (10 × 80 mm) were positioned on grips (50 mm) with the tensile power and cross speed processed at 5 kN and 50 mm/min, respectively. The films were examined three times. TS and EB values, per the values of extensibility (mm) and resistance to extension (N), were calculated using the equation.
$$TS \left(MPa\right)=\frac{F}{X\times W}\times 100 \left(1\right)$$
$$EB \left(\%\right)=\frac{\varDelta L}{L}\times 100 \left(2\right)$$
Where F = resistance to extension (N), X = film thickness (mm) and W = the width of the film (mm), ΔL = the increase in the distance at the break (mm) and L = the original length between grips of the film (mm).
2.5.6. Measurements of solubility, swelling ratio and moisture
The film bands (20 × 20 mm) were weighted as wet weight (M0) and dried at 105 ℃ till constant weight to calculate the primary dry mass value (M1). The dried pieces were transferred to 100 ml beakers filled with distilled water (50 mL) covered with plastic wraps and preserved at 25 ℃ for 24 h. Next, the films were dehydrated superficially with filter papers and weight to get (M2). The saturated-hydrate films were dehydrated again to constant weight at 105 ℃ to afford final dry mass (M3). The moisture content (MC), swelling ratio, and solubility were measured using the equation below.
$$Moisture content \left(\%\right)=\frac{{M}_{0}-{M}_{1}}{{M}_{0}}\times 100 \left(3\right)$$
$$Sweling ratio \left(\%\right)=\frac{{M}_{2}-{M}_{0}}{{M}_{0}}\times 100 \left(4\right)$$
$$Film solubility \left(\%\right)=\frac{{M}_{1}-{M}_{3}}{{M}_{1}}\times 100 \left(5\right)$$
Where M0 = initial weight of the film, M1 = initial dry weight of the film, M2 = weight after drenching films in water for 24 h, and M3 = dry weight of the film after drenching films in water for 24 h.
2.5.7. Analysis of water vapor permeability (WVP) of films
The method of Zhang et al. [34] was followed to estimate the WVP values of the films with minor changes. The film samples (5 × 5 cm) and anhydrous CaCl2 were loaded in special glass cups (4 cm) and sealed. After that, the cups were placed in a desiccator at 75% humidity and packed with sodium chloride saturated solution. Thereafter, for 10 h, the weight of the cups was recorded each hour and after 24 h. By using linear regression, the slope of weight difference against time was recorded against time. The WVP (g m−1s−1Pa−1) was estimated as follows:
$$WNP=\frac{Slope\times L}{A\times \varDelta P} \left(6\right)$$
where L = average film thickness (m), A = transfer area (m2), and ΔP = difference in the pressure of partial water vapor.
2.5.8. Film color and opacity
The color of films was measured using a colorimeter (CR-40, Minolta, Camera Co., Japan) with illuminant D65, a 0° viewing angle, and a 0.13 mm diameter viewing area was used to determine the color of prepared films. Before measurement, the Colorimeter was calibrated with a white tile (mod CR-A43). The equations below were used to estimate the total color variation (ΔE), white (WI) and yellow (YI) indexes.
$${{\varDelta E=\left(\right({L}_{i}^{*}-{L}^{*})}^{2}+{ \left({a}_{i}^{*}-{a}^{*}\right)}^{2}+{\left({b}_{i}^{*}-{b}^{*}\right)}^{2})}^{1/2} \left(7\right)$$
$$YI=\frac{142.68\times {b}^{*}}{{L}^{*}} \left(8\right)$$
$$WI=100-{\left({ \left(100-{L}^{*}\right)}^{2}+{a}^{*2}\right)}^{1/2}+{b}^{*2} \left(9\right)$$
UV-722 spectrophotometer (Shanghai Jinghua Science & Technology Instruments Co., Ltd., China) was used to determine the opacity of the samples at 600 nm. The transparency for the sliced bands (80 × 45 mm length) was assessed. The opacity was calculated as equated below.
$$Opacity=A/L \left(10\right)$$
Where, \(A\) = absorbance value at 600 nm and L = film thickness (nm)
2.5.9. Determination of the antioxidant activity of films
The method outlined by Liu et al. [35] was employed to determine the antioxidant activity of the films. Film solution (50 µL) was mixed with 200 µL of working solution from ABTS, the absorbance at 734 nm was measured, and scavenging activity was estimated as shown below.
$$Scavenging activity on ABTS free radicals \left(\%\right)=\left(1-\frac{{A}_{1}-{A}_{2}}{{A}_{0}}\times 100\right) \left(11\right)$$
Where Ao = ABTS+ initial absorbance, A1 = sample absorbance, and A2 = sample with PBS absorbance.
To determine the DPPH radical scavenging activity, 50 µL from the film sample was blended with 200 µL of DPPH methanolic solution (0.2 mM) and put in the dark for 30 min at 30 ℃. Later, the absorbance was measured at 517 nm with ascorbic acid used as the control. The scavenging activity was calculated as follows.
$$Scavenging activity on DPPH free radicals \left(\%\right)=\left(1-\frac{{A}_{1}-{A}_{2}}{{A}_{0}}\times 100\right) \left(12\right)$$
Where \({A}_{0}\) = initial DPPH absorbance, \(A1\) = sample absorbance, and \(A2\) = sample absorbance with water.
2.5.10. Assay of antimicrobial activity of films
The procedure of Khalid et al. [36] was employed to evaluate the antimicrobial activity of the prepared films against Gram-negative (Escherichia (E.) coli CGMCC 1.8721) and two Gram-positive (Staphylococcus (S.) aureus (CGMCC 1.8721) and Bacillus (B.) cereus (CGMCC 1.895)) bacteria. Briefly, each bacterial culture was diluted to afford a final 105 CFU/mL concentration. The cells (0.1 mL) were spread onto a new medium agar plate. Subsequently, the discs of films (12 mm diameter) were prepared, sterilized under UV light, placed carefully in petri-dish plates, and incubated (37 ℃, 24 h). A sliding caliper was used to estimate the area of the inhibitory zones.
2.5.11. Biodegradation test
The composting test described by Tan et al. [37] was adopted to analyze the biodegradation capacity of the prepared films. Briefly, the soil was collected from the experimental area at Nanjing Agricultural University (Nanjing, China). It was then put in a tray made from plastic and buried with the films (2 × 2 cm) at 4 cm depth for 21 days. The soil was treated twice daily with water. Each week, the films were removed and their weight loss calculated using the following equation:
$$Weight loss \left(\%\right)=\frac{{M}_{0}-{M}_{1}}{{M}_{0}}\times 100 \left(13\right)$$
Where M0 = initial film weight and M1 = film weight after 7, 14, or 21 days of biodegradation.
2.6. Experiments of packaging of sunflower oil
2.6.1. Capability of films for oil resistance
The procedure of Wang et al. [38] was adopted to determine the capability of films for oil resistance with minor changes. Briefly, Whatman No. 1 filter paper (6 cm diameter) was dried in the hot-air oven at 50 ℃. The films (4 × 4 cm) were then fitted with ropes above 5 cm of oil-containing glass test tubes and positioned downward for 48 h on the filter paper. Afterward, the weight was recorded, and the oil absorption ratio (OAR) was measured.
$$OAR\left(\%\right)=\left(\frac{m}{{m}_{0}}-1\right) \times 100 \left(14\right)$$
Where \(m\) = filter paper weight after 48 h and \({m}_{0}\) = weight of dried filter paper.
2.6.2. Determination of peroxide value (POV)
Sunflower oil (10 mL) was added into a glass test tube (15 mm × 150 mm) and samples containing sunflower oil were sealed and stored for 28 days at 50 ℃. From each sample, 10 mL was taken on days 1, 7, 14, and 28 to determine the POV by iodometric titration as outlined by the National Standard of the People’s Republic of China GB/ T5009.227-2016 [39] with slight changes. Briefly, sunflower oil (7.0 mL) was dissolved in 30 mL of 2:3 (v/v) acetic acid and trichloromethane before adding 1.0 mL saturated potassium iodide and shaken in the dark for 1 min. Afterward, distilled water (30 mL) was added and shaken for 1 min before adding starch indicator (1%, 0.5 mL) into the mix. The solution was titrated with 0.01 N sodium thiosulfate until the blue color disappeared, and the consumption rate was recorded. POV (mEq/kg) was calculated as shown below:
$$POV (mEq/kg)=\frac{V\times N\times 1000}{W} \left(15\right)$$
Where V = volume of sodium thiosulfate for titration (mL), N = normality of thiosulfate, and W = weight of the lipid (g).