**3. Results**

#### *3.1. Preparation of Nervilia fordii Ethanol Extract*

The extraction parameters for *Nervilia fordii* ethanol extract was first optimized using single factor experiments by investigating the effects of variables such as the ethanol concentration, temperature and solid-to-solvent ratio on the antioxidant activity and yields of *Nervilia fordii* ethanol extract. Regarding the ethanol concentration, from Figure 2a, it can be seen that no significant difference (*p* < 0.05) in DPPH scavenging rate between the two ethanol concentrations (60% and 70%) was observed. For the extraction temperature, it was found that the increase of temperature would cause an enhanced DPPH scavenging activity (Figure 2(b1)), which may be ascribed to the accelerated molecular movement and decreased solvent viscidity. There was no statistical significance (*p* > 0.05) among the extraction temperatures with the value of 70 ◦C vs. 80 ◦C. In addition, higher temperature could cause the degradation of the flavonoids and phenolic compounds, as exhibited in the yields (Figure 2(b2)), a similar phenomenon was reported by Altemimi et al. [21]. Figure 2(c1) shows that DPPH activity was significantly increased (*p* < 0.05) with the increase of the solid-to-solvent ratio from 1:5 to 1:15, and no significant difference (*p* < 0.05) was observed between 1:10 and 1:15. This finding agreed with Prasad et al. who found that the permeation of compounds into the solvent can be enhanced under the higher solid-liquid ratio [22]. However, too high of a liquid-solid ratio can restrain the cavitation effect (Figure 2(c2)). Hence, from an economic point of view, the optimized process for *Nervilia fordii* ethanol extract was: ethanol concentration 60%, extraction temperature 70 ◦C and solid-to-solvent ratio 1:10.

After that, Box-behnken design (BBD) and response surface methodology (RSM) were then adopted for the optimization of extraction parameter. The levels for response surface design are shown in Table S1. The ANOVA results (Table S2) revealed that the model was remarkably significant (*p* < 0.0001), and the R-squared value obtained was 0.9968, indicating the model was very consistent with the experiment results. The fitted equation was: DPPH scavenging rate (%) = 38.95 − 0.75\*A + 1.53\*B + 1.69\*C + 0.31\*B\*C + 0.28\*A\*C − 0.25\*A\*B − 0.43\*A<sup>2</sup> − 0.81\*B<sup>2</sup> − 1.04\*C<sup>2</sup> (A—Ethanol concentration; B—Temperature; C—Solid to liquid ratio). Besides that, two-dimensional contour plots and three-dimensional response surface for the correlation between any two variables are displayed in Figure S1. The optimal condition for extraction of *Nervilia fordii* ethanol extract was obtained as follows: ethanol concentration 55.55%, extraction temperature 75 ◦C and solid–liquid rate 14.2:1. Regarding the practical situation, the optimal condition was adjusted to an ethanol concentration of 56%, extraction temperature of 75 ◦C and solid–liquid rate of 14:1. Under which, the DPPH scavenging rate of *Nervilia fordii* ethanol extract obtained was 41.5%, which is closer to the theoretical prediction value of 40.9%, suggesting that the model was desirable.

**Figure 2.** Effects of extraction condition on the extract's DPPH radical scavenging activity and yield. ((**a1**–**c1**) indicated the effects of ethanol concentration, temperature and solid-liquid rate on the DPPH radical scavenging rates of *Nervilia fordii* ethanol extract, respectively; (**a2**–**c2**) indicated the effects of ethanol concentration, temperature and solid-liquid rate on the yields of *Nervilia fordii* ethanol extract, respectively).

#### *3.2. Antioxidant Capacities of Different Solvent Extracts*

Then, different solvents (petroleum ether, ethyl acetate and n-butyl alcohol) were used to further extract the above obtained *Nervilia fordii* ethanol extract and their antioxidant activities were evaluated. From Figure 3a, it can be seen that the activity increased with the increase of extract concentration, and the scavenging abilities on DPPH radicals were in the order of ethyl acetate extract of *Nervilia fordii* (NFE) > n-butyl alcohol extract of *Nervilia fordii* (NFB) > petroleum ether extract of *Nervilia fordii* (NFP), which reached 94.5%, 60.5%, 33% and 18.4% at 3 mg/mL, respectively. However, a better scavenging rate was exhibited by Vc (96.3% at 0.1 mg/mL). As summarized in Table 1, the EC50 values of DPPH radical-quenching activity for NFE, NFB and NFP were 0.66, 2.43 and 4.25 mg/mL, respectively. Lipid peroxidation (LPO) is also considered to be another type of free radical oxidation, which is related to cellular damage. Similar to the tendency of DPPH scavenging activity of extracts, the LPO inhibition potency of different extracts were also concentration dependent and NFE possessed the highest inhibitory effect. Figure 3b shows that the inhibition rate rose from 24.9% to 77.4% for Vc and from 12.8% to 88.7% for NFE with the concentration increasing from 100 μg/mL to 500 μg/mL, respectively. In particular, a higher inhibition rate was achieved for NFE than that of Vc when the antioxidant

concentration >300 μg/mL. From the IC50 values of LPO suppression activity in Table 1, NFE (0.307 mg/mL) has considerable activity in comparison to Vc (0.310 mg/mL), while NFB was 0.347 mg/mL and NFP was 0.436 mg/mL. These results clearly indicated that all of the solvent extracts had a noticeable effect on the inhibition of LPO, especially for the NFE, suggesting its potential in inhibiting the oxidation of fatty food or the application for functional foods.

**Figure 3.** The DPPH radical scavenging ability (**a**) and the LPO suppression ratio (**b**) of different extracts and Vc.

#### *3.3. Total Phenol and Flavonoid Contents*

Being the main chain-breaking antioxidants that contributed to the antioxidant activity of extracts, the total phenolic and flavonoid contents in different extracts were examined using gallic acid equivalents (GAE) and rutin equivalents (RE), respectively. As presented in Table 1, the NFE had significantly higher contents of total phenols (86.67 ± 2.5 mg GAE/g extract) and total flavonoids (334.56 ± 4.7 mg RE/g extract), followed by NFB and NFP. It can be concluded that the polar solvent extracts (n-butanol and ethyl acetate) exhibited a higher content than nonpolar solvent extracts (petroleum ether) did, which may contribute to the stronger antioxidant activity of NFE and NFB than that of NFP. Taken the polarities of used solvents into consideration, it has been revealed that flavonoids from *Nervilia fordii* were more extractable by solvents with high polarity.

**Table 1.** Extraction yields, and total phenols, total flavonoid contents in different extracts.


Values (mean ± SD, *n* = 3) in the same column followed by a different letter are significantly different (*p* < 0.05). a GAE, Gallic acid equivalents. b RE, Rutin equivalents. c EC50 means the effective concentration of sample that can decrease 50% of DPPH radical scavenging rate. d IC50 means the effective concentration of sample that can inhibit 50% of lipid peroxidation.

#### *3.4. Encapsulation of NFE into PVA/PVP Electrospun Nanofibers*

As shown in Figure 4, the different NFE content-loaded PVA/PVP fibers did not show different morphology compared to the neat PVA/PVP fibers. However, the diameters of nanofibers significantly increased with the addition of NFE, which could be due to the solution characteristics as shown in Table 2. Although the conductivity decreased with the increase of NFE into the PVA/PVP solution, no significant decrease was observed. The viscosity of the solution increased from 1535 Pa·S to 1693 Pa·S because of the molecule entanglements, which was favorable for the formation of thick fibers [23]. A similar phenomenon was observed in tomato peel extract-loaded gelatin fibers [24]. In addition to fiber diameter, the DPPH radical scavenging rate also enhanced by increasing NFE concentration, suggesting the potential for antioxidant packaging (Table 2). When the NFE concentration was 4.0 mg/mL, the scavenging rate reached 78.4%, and there was no significant difference (*p* > 0.05) compared to that of 8 mg/mL. Besides that, the encapsulation efficiency values for 2 mg/mL, 4 mg/mL and 8 mg/mL NFE were 96.57 ± 1.46%, 94.32 ± 1.78%, and 91.42 ± 2.45%, respectively. These results suggested that almost no loss of NFE occurred during the electrospinning and more than 90% of NFE could be encapsulated into PVA/PVP fibers, indicating the efficient nano-encapsulation of NFE by electrospinning.

**Figure 4.** SEM images of different NFE content-loaded PVA/PVP fibers and its diameter distribution.


**Table 2.** Characteristics of electrospun solutions and the obtained fibers.

Note: different letters in the same column indicates the statistically different (*p* < 0.05).

#### *3.5. Characterization of Electrospun Nanofibers*

The interactions and compatibility among different components were examined by FTIR spectroscopy. Figure 5 shows that NFE exhibited peaks of O–H stretching (3330 cm<sup>−</sup>1), C=O stretching (1705 cm<sup>−</sup>1), and C–O–C) stretching (1242 and 1079 cm<sup>−</sup>1). Compared to neat PVP and PVP fibers, almost no changes in the spectra of PVP/NFE and PVA/NFE were observed, suggesting that NFE could be compatibly entrapped into the PVP and PVA fibers. When NFE was added to the PVA/PVP matrices, the infrared spectra of NFE-loaded PVA/PVP fiber mat exhibited the characteristic peaks of both polymers, while the presence of NFE peaks almost disappeared, indicating the encapsulation of NFE into the fiber mat due to the formation of various kinds of intra/inter-molecular hydrogen bonds. In particular, the intensity and width of the peak broadening over 3380 cm<sup>−</sup><sup>1</sup> was in the following order of PVA/PVP/NFE > PVA/PVP, which indicated that a stronger hydrogen bond was formed in PVA/PVP/NFE.

**Figure 5.** FTIR of different samples.

A crystallinity analysis by XRD pattern was carried out to confirm the inter-molecular interaction and biocompatibility among different components. As shown in Figure 6, pure NFE exhibited patterns of a crystalline state with diffraction peaks at 19.67◦, 22.75◦ and 24.63◦; however, these characteristic peaks for NFE were absent in the diffractograms of PVA/NFE, PVP/NFE and PVA/PVP/NFE. It can be suggested that NFE was converted into an amorphous state, which was ascribed to the electrospinning process and the interaction with amorphous PVP polymer. The rapid evaporation of solvent caused the quick transformation of polymer solution into solid form without enough time for crystallization [25]. Besides that, it can be seen that the first peak of PVP/NFE at 10.82◦ significantly diminished in PVP/PVA/NFE, and the intensity of the PVA/NFE peak at 19.47◦ was weakened and shifted to the higher 2θ position in PVP/PVA/NFE. These findings corresponded with the FTIR study where the intermolecular interaction existed among different components, particularly as hydrogen bonds.

**Figure 6.** X-ray diffractograms of different samples.

TGA was used to evaluate the thermal stability of the obtained fiber mat. From Figure 7, the weight loss around 60~180 ◦C was attributed to the evaporation of free, freezing bound and chemical bound water for all curves. Free water is usually related to the absorption water and the freezing bound water is weakly interacted with the polymeric chain, while the chemical bound water is regarded as the water molecules bound to the polymeric chains via hydrogen bound. Thus, the mass loss in the first region of the thermograms related to different samples can be ascribed as evaporation of different types of water. For the PVA/NFE curve, another two main weight loss regions were displayed. The first region around 280~330 ◦C was due to the degradation of the side chain in the PVA matrix, like the C–O bond, and the second stage (400~460 ◦C) appeared as the cleavage of the C–C backbone in polymers, leading to so-called carbonization. In the case of PVP/NFE film, the region around 400~460 ◦C was attributed to the degradation of PVP. In particular, the higher degradation temperature is a result of increased number of hydroxyl groups among components. These findings are consistent with the previous results of FTIR and XRD analysis. Taking all results into consideration, it can be concluded that the obtained PVA/PVP/NFE fiber mat possesses adequate thermal stability and can be safely used for packaging application.

**Figure 7.** TGA curves of different samples.

#### *3.6. Effectiveness of the Active Films against Lipid Oxidation of Fish Oil*

Oxidation is one of the major causes of oil deterioration. For example, fish oil, a nutritive functional food, is highly susceptible to oxidation due to the presence of polyunsaturated fatty acids, thus limiting its application. Hydroperoxides are the primary products formed in the first stage of oxidation, which can be reflected by peroxide values (PV), an indicator that represents the extent of early phase lipid oxidation [26]. For an edible food product, the PV levels should be less than 30 meq peroxide/kg oil [27]. Herein, the effect of different antioxidant films on the oxidation extent of fish oil was determined by measuring the PV at 25 ◦C for 60 days. Unpacked fish oil was used as blank. As depicted in Figure 8, the initial raw fish oil presented a PV value of 3.03 meq peroxide/kg oil. It was increased markedly for non-packed fish oil after day 10, reaching a maximum level of 378 meq peroxide/kg oil at day 50, and then declined to 343 meq peroxide/kg oil at day 60 (*p* > 0.05), which may be due to the degradation of peroxides as secondary oxidation began [28]. A similar trend was also noted for fish oil packed with PVP/PVA film, although there was a slight suppression in the PV values compared with the control, which could be related to the excellent gas barrier property of PVA-based films [29]. On the contrary, the different forms of antioxidants resulted in different extents of inhibitory effects on oxidation, as

the PV values of samples packed with PVP/PVA/NFE were always lower than that of the oil treated with free NFE on all sampling days. For instance, the PV value (30 meq peroxide/kg oil) occurred on the 10th, 30th and 40th day with respect to the unpacked oil and oil packed with PVA/PVP film and free NFE, respectively. The results indicated that the incorporation of natural antioxidants into an electrospun nanoscale delivery system was able to maintain its activity and was more effective than that of the non-encapsulated antioxidant due to the improved solubility. A similar trend was found in the edible guar gum-based nanofibrous mat for the encapsulation of tannic acid to inhibit the oxidation of flaxseed oil [19].

**Figure 8.** X-ray diffractograms of different samples. Different lowercase letters indicated a significant difference (*p* < 0.05) over different incubation time that treated with the same film. Different capital letters indicated a significant difference (*p* < 0.05) among different packaging film at a specific incubation time.
