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. 2026 Oct 12. Online ahead of print. doi: 10.1039/d6ra06507d

Green-synthesized ZnO and ZnO–MgO nanoparticles from Abelmoschus esculentus extract for multifunctional sago starch active packaging films

Aneseh Ebrahimi a, Masoumeh Hosseinzadeh a,✉, Saba Najafi b, Mohammad Javad Mohammadi a
PMCID: PMC13655793  PMID: 42865771

Abstract

This study developed multifunctional sago starch active packaging films incorporating Abelmoschus esculentus extract (AE), green-synthesized ZnO nanoparticles (ZnONPs), and ZnO–MgO nanoparticles (ZnO-MgONPs). XRD confirmed the successful formation of crystalline nanoparticles, with average crystallite sizes of 22.4 ± 2.1 nm for ZnONPs and 18.7 ± 1.8 nm for ZnO-MgONPs. FESEM/TEM showed quasi-spherical nanoparticles with sizes of 25–35 nm and 20–30 nm, respectively. Compared with the AE-only film, the addition of ZnONPs and ZnO-MgONPs significantly improved film performance: tensile strength increased from 1.43 MPa to 2.12 MPa and 2.79 MPa, respectively, while elongation at break decreased from 60.39% to 46.12% and 32.33%. AE increased film moisture content from 6.98% to 8.78%, whereas nanoparticle incorporation improved barrier and structural properties. FTIR, XRD, FESEM, TGA, and UV-vis analyses confirmed good compatibility among the components, enhanced thermal stability, and stronger UV-shielding behavior in nanocomposite films, especially in the ZnO–MgO-containing system. The incorporation of AE enhanced the DPPH radical-scavenging activity of the films, indicating the contribution of AE-derived bioactive compounds to their antioxidant functionality. The nanocomposite films retained appreciable antioxidant activity while exhibiting improved mechanical, barrier, and antimicrobial properties. The films also exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus, indicating their potential for active food packaging applications. Overall, the AE/ZnO-MgONPs film showed the best balance of mechanical strength, thermal stability, barrier performance, and antimicrobial function, making it a promising eco-friendly packaging material.

This study developed multifunctional sago starch active packaging films incorporating Abelmoschus esculentus extract (AE), green-synthesized ZnO nanoparticles (ZnONPs), and ZnO–MgO nanoparticles (ZnO-MgONPs).graphic file with name d6ra06507d-ga.webp

1. Introduction

Consumers increasingly value both the quality and visual appeal of food. However, food quality can deteriorate rapidly during handling, transport, and storage due to microbial growth and chemical reactions, particularly lipid oxidation. For this reason, preventing microbiological and chemical spoilage remains a major concern for the food industry. In response, innovative packaging systems have been developed to preserve food quality and extend shelf life. Among them, active packaging has emerged as a promising strategy because it can incorporate functional components into the packaging matrix to provide antibacterial and antioxidant properties that are not present in conventional packaging.1–3

However, the development of active packaging materials requires a careful balance between biodegradability, mechanical strength, moisture resistance, optical protection, and antimicrobial activity. Therefore, the incorporation of natural bioactive compounds and functional nanomaterials into biodegradable polymer matrices has received increasing attention as a strategy for developing multifunctional packaging films.4

Polysaccharides are attractive renewable resources for producing edible coatings and films due to their biocompatibility, abundance, and film-forming capacity. Naturally derived polysaccharides are generally non-toxic and can be tailored to control gas diffusion, particularly for oxygen and carbon dioxide, making them useful barriers in food-packaging applications. Their polysaccharide backbone and hydrogen-bonding network confer effective oxygen barrier properties and facilitate formation of cohesive coatings. Consequently, polysaccharide-based films are widely used to extend the shelf life and preserve the quality of fruits and vegetables.5,6 Starch is a naturally occurring biopolymer that has attracted considerable interest for packaging applications due to its low cost, renewability, and wide availability. However, its inherent brittleness and hydrophilic nature significantly limit its direct use as a packaging material, particularly where moisture resistance and barrier performance are critical. Although starch-based films have been proposed as alternatives to conventional plastics, their poor resistance to water remains a major limitation. Among various starch sources, sago starch is a promising candidate because its properties are closer to those required for conventional food packaging materials.7,8 Starch properties can be improved by blending it with other materials, including chemically or physically modified compounds, plasticizers, plant extracts, and nanoparticles. However, the addition of plasticizers often reduces mechanical performance, which can limit film strength and durability. Among the commonly used nanofillers, zinc oxide has attracted considerable attention in food and pharmaceutical applications because of its strong antibacterial and antifungal activity.9–11

Despite these advantages, starch-based films generally exhibit poor water resistance, high moisture sensitivity, limited mechanical strength, and inadequate stability under humid conditions. These limitations restrict their direct application in food packaging. Consequently, the incorporation of plant extracts and inorganic nanoparticles has been explored as a strategy to modify the starch network and improve the functional performance of starch-based films. However, improvement in one property may adversely affect another. For example, hydrophilic plant extracts may increase water absorption, whereas rigid nanoparticles may enhance tensile strength but reduce film flexibility. Therefore, the development of an optimized multifunctional film requires the simultaneous evaluation of structural, mechanical, barrier, optical, thermal, morphological, and antimicrobial properties.

Various inorganic nanomaterials, including nanoclay, ZnO, TiO2, MgO, and silver-based compounds,12 have been incorporated into polymer matrices to improve their functional performance. Among these materials, zinc oxide nanoparticles (ZnONPs) have attracted considerable attention due to their antimicrobial and UV-protective properties as well as improved mechanical properties.13,14 Therefore, ZnO-based nanomaterials have been investigated as functional additives for active food packaging films.15 In addition, ZnO structures containing MgO and Mg have been investigated as functional materials because the incorporation of Mg may alter the crystal structure, surface properties, thermal behavior, and dispersion of ZnO-based materials.16–18 Green synthesis using plant extracts is a promising approach to prepare such nanomaterials because plant-derived compounds may act as reducing, capping, and stabilizing agents during the formation of nanoparticles. The use of a plant-based synthesis route may also reduce the dependence on conventional chemical stabilizers and support the development of materials based on renewable resources. Appropriate references should be added to support these statements.19–22

Plant-mediated synthesis has recently received considerable attention as an alternative to conventional nanoparticle preparation because phytochemicals can act as reducing, capping, and stabilizing agents. Recent reports on green-synthesized ZnO and MgO-based nanomaterials have confirmed their antimicrobial potential and suggested that biogenic synthesis may improve their compatibility with biopolymer matrices. Nevertheless, the translation of green-synthesized mixed-metal oxides, particularly ZnO–MgO systems, into starch-based active packaging films remains insufficiently investigated.23

Abelmoschus esculentus (AE), commonly known as okra, is a rich source of polysaccharides and bioactive compounds. Okra extracts have been associated with antioxidant, antimicrobial, moisturizing, and film-forming properties, making them attractive for functional food-packaging materials.24,25 The polysaccharides and phytochemicals in AE may participate in hydrogen bonding with starch chains and contribute to the stabilization and dispersion of metal-oxide nanomaterials. Accordingly, AE can serve a dual role: as a bioactive additive that enhances the antioxidant and antimicrobial functionality of the starch matrix, and as a green synthesis agent for nanoparticle preparation. In this context, ZnO and ZnO–MgO nanomaterials may complement these effects by acting as reinforcing, UV-shielding, and barrier-modifying fillers. Despite growing interest in starch-based nanocomposite films, the combination of AE with green-synthesized ZnO and ZnO–MgO nanomaterials in sago starch matrices has not been reported. Recent studies have explored related systems: Syarifa et al.26 investigated sago starch films with commercially sourced ZnO particles but without plant extracts or green synthesis. Mohammadi et al.16 studied sago starch films with flaxseed extract and ZnO/ZnO–MgO nanoparticles, although the nanoparticles were not prepared via a plant-mediated route. Erami et al.27 incorporated ZnO nanoparticles into sago starch films with phycocyanin, reporting a tensile strength of 2.18 MPa. Torche et al.14 developed cassava starch films with green-synthesized ZnO (∼63.7 nm) from date palm pit extract, but observed no antibacterial activity against E. coli. Kumar et al.15 compared chemically and green-synthesized ZnO in corn starch films using neem extract, demonstrating that green-synthesized nanoparticles exhibited stronger antimicrobial effects. However, none of these studies simultaneously employed a single plant extract as both the green synthesis agent for nanoparticles and the bioactive additive in the film matrix. Furthermore, the comparative effect of ZnO versus ZnO–MgO nanoparticles on AE-containing sago starch films, particularly with respect to the balance among mechanical, barrier, thermal, optical, and antimicrobial properties, has not been established.

The novelty of the present study therefore lies in three main aspects. First, okra extract is used simultaneously as a green reducing/capping agent for the synthesis of both ZnO and ZnO–MgO nanoparticles and as a bioactive film additive a dual functionality not previously reported for sago starch-based systems. Second, the bimetallic ZnO–MgO system, which is rarely investigated in starch matrices, is compared with ZnO alone to evaluate the effect of MgO incorporation on nanoparticle size, crystallinity, and film performance. Third, a systematic comparison of neat sago starch, AE-containing films at multiple concentrations, and AE/ZnO and AE/ZnO-MgO nanocomposite films is conducted using complementary characterization techniques (XRD, FTIR, FESEM/EDX, TEM, TGA, UV-vis, mechanical testing, WVTR, and antimicrobial assays) to determine whether the formulation produces balanced improvements across multiple properties rather than enhancing a single attribute. Accordingly, this study aimed to develop multifunctional sago starch-based films containing AE, green-synthesized ZnO, and ZnO–MgO nanomaterials and to identify the formulation showing the most favorable combination of tensile strength, moisture resistance, UV protection, thermal stability, and antibacterial activity against E. coli and S. aureus.

2. Material and methods

2.1. Reagents and chemicals

Sago starch (purity ≥95%, food grade) was obtained from local agricultural suppliers in Bandar Abbas, Iran. Glycerol (≥99.5%, analytical grade) and sorbitol (≥99%, analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc nitrate hexahydrate, Zn (NO3)2·6H2O, magnesium nitrate hexahydrate, Mg (NO3)2·6H2O, sodium hydroxide (NaOH), and all other reagents were of analytical grade and used as received without further purification. Deionized water (resistivity ≥ 18.2 MΩ cm) was used throughout the experiments. The bacterial strains Escherichia coli (PTCC 1769) and Staphylococcus aureus (PTCC 1431) were obtained from the Persian Type Culture Collection (PTCC, Tehran, Iran). Fresh okra (Abelmoschus esculentus) was purchased from a local market, washed, air-dried, and then dried prior to use.

2.2. Instruments

Surface morphology of the films by (FESEM) according to a previous report,28 the fracture surface of films after tensile testing was observed using field emission scanning electron microscopy (FESEM). The samples were placed on the FESEM sample stub, coated with carbon and gold to reduce static charge, and observed using a model TE-SCAN with 20 kV and 8 mA. Fourier transform infrared spectroscopy (FT-IR) X-ray diffraction analysis was performed according to a previous report.29 Samples and control films were analyzed using a PHILIPS PW 1800 at 25 °C, 40 kV and 30 mA for X-ray diffraction testing. The samples were scanned from 2θ = 3° to 40°. Thermogravimetric analysis TGA and DTG according to a previous report, thermogravimetric analysis was carried out using a TGA and DTG analyzer (TGA/SDTQ600 TA USA) following the method described.30 The specimens were approximately 5 mg and heated to 650 °C at a rate of 10 °C min−1 under a nitrogen atmosphere. UV-visible transmission spectra the UV-visible spectra of the starch films (2 × 2 cm) were recorded from 200 nm to 1100 nm using a UV-7315 (Jenway, England) spectrophotometer. An empty cell containing air was used as the reference.

2.3. Sample collection and preparation

Abelmoschus esculentus (AE) were collected in Amol, Mazandaran province, Iran, in March 2025 and were deposited at the Laboratory of Phytochemistry and Plant Resources at Azad University of Qaemshahr, Iran.

2.4. Preparation of Abelmoschus esculentus extract

20 g of dried AE were properly crushed, added into 100 mL distilled (DI) water, and stirred at 90 °C for 2 h. Subsequently, the mixture was filtered (Whatman No 1 filter paper) three times to obtain the okra extract, and the extract was stored at 4 °C. The chemical composition of AE was analyzed using a gas chromatograph (GCMASS) (Hewlett-Packard 6890N) equipped with a column HP-5MS (30 m × 250 µm × 0.25 µm) coupled to a mass spectrometer (MS) (Hewlett-Packard 5973N). The individual compounds were identified and confirmed with authentic samples and available library data of the GCMSSS system (WILEY 2001 data software).

2.5. Green synthesis of nanocomposites

2.5.1. Green synthesis of ZnONPs from AE

To fabricate ZnONPs, 50.0 mL7.43 g of Zn (NO3)2·6H2O (0.50 M) salt was dissolved in 50 mL of distilled water and stirred at 25 °C for 30 min. Afterward, 30 mL of AE extract was added dropwise to the salt solution. Once the final solution was stirred at 70 °C for 6 h to form a green gel, which was calcined at 700 °C for 2 h to achieve ZnONPs.31

2.5.2. Green synthesis of ZnO-MgONPs from AE

The prepared extract was used directly in the green synthesis of ZnO-MgONPs. For the synthesis of ZnO-MgONPs, using Zn (NO3)2·6H2O and Mg (NO3)2·6H2O as sources of metallic salts we prepared two different solutions: one containing 50.0 mL of Zn (NO3)2·6H2O solution (0.50 M) and another containing 50.0 mL of Mg (NO3)2·6H2O solution (0.50 M). The ratio of Zn to Mg in these solutions was 1 : 1. We then incrementally combined the prepared solutions and agitated them for 40 minutes. Subsequently, a volume of 30.0 mL of aqueous AE extract was introduced into the solution and agitated at a temperature of 80 °C for a duration of 8 hours. The (M (OH)2) gel was dried at 100 °C for 8 hours and then calcinated at 700 °C for 2 hours to produce the white-coloured ZnO–MgO nanocomposite (Fig. 1 and Table 1) illustrates the biosynthetic process of the ZnO–MgO nanocomposite.16

Fig. 1. A schematic of the film-forming process.

Fig. 1

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Table 1. Composition of films formulations.
Designation of films Sago (g) Nanoparticles (g) AE (w/w) Glycerol/sorbitol (mL)
AE0 4 — — 1 : 3
AE1 4 — 2.0 1 : 3
AE2 4 — 4.0 1 : 3
AE3 4 — 6.0 1 : 3
AE/ZnONPs 4 0.01 6.0 1 : 3
AE/ZnO-MgONPs 4 0.01 6.0 1 : 3
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2.6. Experimental design

The experimental design was established to investigate the effects of AE concentration and nanoparticle incorporation on the performance of sago starch-based films. The control formulation (AE0) contained sago starch without AE or nanoparticles. AE-containing films were prepared by incorporating 2, 4, and 6% AE, designated as AE1, AE2, and AE3, respectively. The 6% AE formulation was subsequently used as the base matrix for incorporating 0.01 g of ZnONPs or ZnO-MgONPs, resulting in the AE/ZnONPs and AE/ZnO-MgONPs formulations, respectively. The amount of sago starch, plasticizer ratio, water volume, casting conditions, and drying conditions were kept constant among the formulations, unless otherwise stated. The composition of all formulations is summarized in Table 1. The films were compared in terms of their structural, physicochemical, mechanical, optical, thermal, barrier, morphological, and antimicrobial properties.

2.7. Preparation of films

2.7.1. Preparation of sago/AE films

4 gr sago starch solution was prepared by adding sago starch to distilled water 100 mL, and heating it to 90 °C for starch gelatinization. The mixture was stirred continuously for 45 minutes to ensure homogeneity. A mixture of sorbitol and glycerol (3 : 1) was added for plasticization. Different concentrations (2.0% (w/w), 4.0% and 6.0% w/w) of AE extract were added to the mixture to prepare films AE1, AE2 and AE3, respectively. Films without AE extract were labeled as AE 0%. Each suspension was cast in a Petri dish and dried in the oven at 40 °C for 24 hours. The thickness of each film was measured to the nearest 0.001 mm.32

2.7.2. Preparation of sago/AE/ZnONPs films

A concentration of 0.01 g of ZnONPs was dispersed in 100 mL of distilled water and stirred using a magnetic stirrer hotplate at room temperature (28 °C) for 60 minutes. The ZnONPs solution was then used to prepare aqueous 4% (w/v) sago starch dispersions. A mixture of glycerol and sorbitol (1 : 3) at 40% (w/w) of the total sago starch was added to the dispersion. The sago starch nanocomposites were heated to 90 ± 2 °C and held for 45 minutes to accomplish starch gelatinization and create a homogenized dispersion. The dispersion was then cooled to around 45 °C, and a concentration of 1 mL of AE extract (6.0% w/w) was mixed for 60 minutes at the same temperature to achieve homogenized dispersions AE/ZnONPs. This suspension was cast in a Petri dish (140 mm diameter), and the films were dried in the oven at 40 °C for 20 hours. After drying, the films were peeled off and kept at 23 ± 2 °C and 50 ± 5% relative humidity (RH) until tested. The thickness of each film was measured to nearest 0.001 mm at five random positions on the films, and the average thickness was determined.27

2.7.3. Preparation of sago/AE/ZnO-MgONPs films

A concentration of 0.01 g of ZnO-MgONPs was dispersed in 100 mL of distilled water and stirred using a magnetic stirrer hotplate at room temperature (28 °C) for 60 minutes. The ZnO-MgoNPs solutions were then used to prepare aqueous 4% (w/v) sago starch dispersions. A mixture of glycerol and sorbitol (1 : 3) at 40% (w/w) of the total sago starch was added to the dispersion. The sago starch nanocomposites were heated to 90 ± 2 °C and held for 45 minutes to accomplish starch gelatinization and achieve a homogenized dispersion. The dispersion was then cooled to around 45 °C, and a concentration 6.0% of 1 mL AE extract was mixed for 60 minutes at the same temperature to achieve homogenized dispersions (AE/ZnO-MgONPs). This suspension was cast in a Petri dish (140 mm) and the films were dried in the oven at 40 °C for 20 hours. After drying the films were peeled off and kept at 23 ± 2 °C and 50 ± 5% relative humidity (RH) until tested. The thickness of each film was measured to the nearest 0.001 mm. The average thickness was determined at five random positions on the films (Fig. 1).9,33

2.8. Phytochemical analyses

2.8.1. Thickness analysis

The thickness of films was measured at five random points using a dial thickness digital caliper (Neoteck Co., Canada).

2.8.2. Mechanical properties

The mechanical properties of the films were estimated based on ASTM Standard Method (D882–18) and were determined using a universal testing machine (STM, SANTAM, IRAN). With a slight modification, the mechanical properties were then measured by cutting, the films into strips (1 × 10 cm) and conditioning them at 50% RH for approximately 48 hours. The force and distance were measured during the extension of the strips at a speed of 60 mm min−1 until they broke. Initial grip separation and mechanical crosshead speed were set at 30 mm and 1 mm s−1, respectively. Tensile strength (TS) and elongation at break (EAB) were calculated. At least five replicates were carried out for each sample.34

2.8.3. Moisture content

The films were cut into small pieces (2 × 2 cm), placed on glass Petri dishes, and the weights of the films were then determined by drying them in an oven at 105 °C for 24 hours until a constant weight was reached (dry sample weight). The moisture content of the films was determined according to the method.35 Three replications of each film treatment were recorded before and after the oven for calculating the moisture content.

2.8.3. 1

where M1 and M2 are the initial weight of the film and the weight of the film after drying.

2.8.4. Water solubility

The film's water solubility was determined according to.36 Pieces of film (2 × 2 cm) were cut from each film and stored in a desiccator with silica gel (0% RH) for 1 day. Samples were weighed to the nearest 0.001 g and placed into beakers with 50 m of deionized water. The mixture was shaken continuously using a shaker (JENWAY Japan) for 24 hours at room temperature (approximately 25 °C). The remaining pieces of film after soaking were filtered through filter paper (Whatman no.1), followed by oven drying at 105 °C to a constant weight. Samples were measured in three replicates and the percentage of total soluble matter (% solubility) was calculated as follows:

2.8.4. 2

2.8.5. Swelling test

The films swelling ratio was determined according to.37 The swelling ratio of the film samples was determined on 2 × 2 cm square dried at 60 °C for 6 hours in an oven, and then accurately weighed. Films were immersed in deionized water for 24 hours. The obtained wet samples were then tapped with filter paper. The swelling ratio was determined according to the following eqn (3)

2.8.5. 3

All measurements were carried out in three replicates.

2.8.6. Water vapor transmission rate (WVTR) test

The water vapor transmission rate (WVTR) of the films was determined according to ASTM E96-00 test method.38 With a diameter of 5.0 cm were placed at room temperature and 30% relative humidity (RH). Circular glass tubes containing 15 mL of distilled water were used; the tube head was mounted by the sample film. The films were then placed into desiccators with silica gel at 25 °C. The films were weighed periodically at time intervals of 3 hours for 24 hours. Three film specimens were tested for each type of film. The WVTR of the sample was calculated according to eqn (4):

2.8.6. 4

where, G is the weight loss during time t, G/t is the slope of the straight line, and A is the cross-sectional area of the glass tube. Through this test, reliable values of water vapor transfer through permeable (or semi-permeable) films can be obtained.

2.9. Antimicrobial activity evaluation of films

The film sample was sectioned into disks with a diameter of 5 mm and thereafter positioned on Mueller–Hinton agar plates. The plate was previously inoculated with 0.1 mL of a solution containing about 105–106 CFU mL−1 of Escherichia coli and Staphylococcus aureus bacteria. We subsequently incubated the plate at 37 °C for 24 hours. After the incubation period, we measured the width of the film disk's inhibitory zone and estimated its area.

We examined the antibacterial efficacy of sago, sago/AE, AEZnONPs and AE/ZnO-MgONPs blend films against E. coli and S. aureus using qualitative and quantitative analyses, respectively. We subjectively assessed the antibacterial efficacy of films using a disc diffusion experiment, after which the films slices were positioned on the medium. We assessed the bacterial growth and determined the diameters of the inhibition zones for these samples after a 24-hours incubation period. We conducted the tests three times for each film. We assessed the inhibition rates of films against E. coli and S. aureus using the optical density (O.D.) technique. We submerged 0.5 g films in 30 mL of nutrient agar culture medium containing E. coli and S. aureus, using blank nutrient medium as the control. We incubated the samples in thermostatic shakers for 12 hours, maintaining a shaking velocity of 50 rpm at 37 °C. We then measured the absorbance of the culture fluid at 600 nm using a UV-visible spectrophotometer. The formula for calculating the inhibition ratio (IR) is as follows: IR = (Acon).

2.10. Antioxidant activity evaluation of films

In the present study, 0.3 g of film sample was immersed in 5 mL methanol and mixed solution containing the film sample was stirred at 40 °C for 60 mim, and then centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant was taken for following antioxidant capacity determination. The efficacy of the films to scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals was determined using spectrophotometry method on basis of bleaching of the bluish-red or purple color of DPPH solution as a reagent, according to39 with minor changes and then a 2.8 mL of film extract solution were mixed with 0.2 mL of 1 Mm methanolic solutions of DPPH. The absorbance at 517 nm was measured after the solution had been allowed to stand in the dark at ambient temperature for 30 min. Against the corresponding blank solution using a spectrophotometer UV-7315 (Jenway, England). The percentage of DPPH radical-scavenging activity was calculated using following equation:

2.10. 5

2.11. Statistical analysis

Experimental data were reported as mean ± SD for triplicate measurement. SPSS 25.0 software was used for statistical analysis, with significance evaluated by One-way ANOVA and Duncan's multiple range test (p < 0.05). All figures were created using by ORIGINPRO 2022 software (Stat-Ease Inc., Minneapolis, USA).

3. Results and discussion

3.1. Chemical composition of Abelmoschus esculentus extract

The compositional analysis of ethanol extract GC-MS was also identified (Fig. 2 and Table 2) 19 various compounds. The major components were linoleic acid ethyl ester (35.53%), 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z) (12.17%), gamma-Sitosterol (9.98%), (3methyl,24R)-ergost-5-en-3-ol (3.33%). Other researchers have also shown to be the main component in Abelmoschus esculentus extract.40

Fig. 2. GC-MS chromatograms of Abelmoschus esculentus extract.

Fig. 2

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Table 2. Chemical composition of Abelmoschus esculentus extract.

No. T R min Compound Peak area % Molecular formula MW
1 8.16 Butyrolactone 0.48 C4H6O2 86.09
2 12.72 Benzeneacetaldehyde 0.44 C8H8O 120.15
3 16.22 2,3-Dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one 1.53 C6H8O4 144.12
4 16.82 2,4-Difluoroaniline 1.16 C6H5F2N 129.11
5 19.04 5-Hydroxymethylfurfural 0.84 C6H6O3 126.11
6 19.46 5-Hydroxymethylfurfural 0.84 C6H6O3 126.11
7 26.08 Cytidine 1.19 C9H13N3O5 243.22
8 36.04 Hexadecanoic acid 18.1 C16H32O2 256.42
9 36.23 Hexadecanoic acid, ethyl ester 4.80 C18H36O2 284.5
10 38.57 Phytol 0.64 C20H40O3 296.5
11 39.83 Linoleic acid ethyl ester 35.53 C20H36O2 308.5
12 40.02 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- 12.17 C20H34O2 306.48
13 46.75 Methyl 8,11,14-heptadecatrienoate 0.33 C18H30O2 278.4
14 46.91 Palmitin 0.70 C19H38O4 330.5
15 48.78 Linoelaidic acid 2.24 C18H32O2 280.4
16 50.21 Octacosane, 2-methy- 0.30 C29H60 408.8
17 54.38 (3methyl,24R)-ergost-5-en-3-ol 3.33 C28H48O 400.7
18 54.88 Stigmasterol 2.93 C29H48O 412.7
19 55.90 Gamma-sitosterol 9.68 C29H50O 414.7
20 58.44 Stigmast-4-en-3-one 1.44 C29H48O 412.7
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3.2. Proposed mechanism of nanocomposites formation

3.2.1. Structural properties XRD

The patterns of XRD of green-synthesized ZnO and ZnO–MgO nanoparticles (Fig. 3) showed typical peaks of hexagonal wurtzite structure of ZnO. The presence of high crystallinity with preferential orientation in plane (101) was confirmed by the presence of prominent diffraction peaks at 2-degree values of 31.8 (100), 34.4 (002), 36.3 (101), 47.5 (102), 56.6 (110), 62.9 (103) and 67.9 (112). The ZnO MgO nanocomposite also had more peaks of 37.8 o (111), 43.3 o (200), 64.9 o (220), 74.5 o (311), and 78.3 o (222), which corresponded to the face-centered cubic (FCC) structure of MgO. The crystallite size determined by the Debye–Scherrer equation was 22.4 ± 2.1 nm in ZnO and 18.7 ± 1.8 nm in ZnO 18.7 ± 1.8 nm ZnO 18.7 ± 1.8 nm ZnO ZnO 18.7 ± 1.8 nm ZnO 18.7 ± 1.8 nm ZnO. The crystallite size was calculated as 22.4 + 2.1 nm. There were no peaks of impurities, which indicates purity of the phases. The narrowed peak is an indication of a reduced crystallite size caused by the Mg2+ doping effects accompanied by the slight peak broadening witnessed in ZnO–MgO, which is in line with recent reports on bio-synthesized bimetallic nanocomposites.41,42

Fig. 3. XRD pattern of the nanocomposite.

Fig. 3

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3.2.2. Structural properties FTIR spectra

Fig. 4 affirmed that the okra extract was present with capping agents to the nanoparticles. Pure ZnO exhibited the typical values of 3425 cm−1 (O–H stretching), 1635 cm−1 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching), 1384 cm−1 (C–H bending) and 465 cm−1 (Zn–O vibration). Another peak of 574 cm−1 (Mg–O vibration) and 443 cm−1 (Zn–O–Mg stretching) was observed in the ZnO–Mg nanocomposite, which confirmed the effective incorporation of Mg. The presence of polyphenolic O–H stretching of flavonoids and tannins in okra extract as capping agents was determined by peaks of 3369 cm−1 and 3157 cm−1. C–O–C vibration of polysaccharides was found to be 1084 cm−1, and carbonyl group (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) of quercetin and other phenols were detected at 1990 and 1912 cm−1. The functional groups ensure stable agglomeration and prevent colloidal stability as has been reported in the recent studies on green synthesis.by.43

Fig. 4. FTIR analysis of the nanocomposite.

Fig. 4

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3.2.3. Structural properties FESEM, EDX and TEM images

Fig. 5a indicated the existence of quasi-spherical morphology, as well as the average particle size of 25–35 nm of ZnO and 20–30 nm of ZnO–MgO nanoparticles. The mixed metal oxides and phytochemical capping of ZnO resulted in a synergistic stabilization of the ZnO which contributed to a more homogeneous distribution and a reduction in the aggregation of the nanocomposite compared to the pure ZnO.

Fig. 5. FESEM (a), EDX (b), and TEM (c) of the nanocomposite.

Fig. 5

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The surface roughness analysis showed that the R 2 nm of ZnO and ZnO MgO are 2.8 nm and 1.9 nm, respectively, which showed that ZnO and ZnO MgO had smoother morphology in the bimetallic system. Elemental composition of ZnO: ZnO (Zn: 48.2 wt% O: 31.6 wt%); ZnO–MgO (Zn: 35.4 wt% Mg: 12.7 wt% O: 38.1 wt%) was verified by energy-dispersive X-ray (EDX): no impurities were detected.

3.3. Phytochemical analyses

3.3.1. Thickness and mechanical properties of films

The addition of AE at (2.0%, 4.0%, and 6.0% w/v), followed by the addition of either AE (6.0%) ZnONPs (0.1%), and AE (6.0%) ZnO-MgONPs increased the thickness of the films in comparison with neat sago film. It was reported that the thickness of AE films was approximately 0.31–0.36 mm with AE and approximately 0.38 mm with AE + ZnO or AE + ZnO–MgO. This growth is due to further crosslinking and intermolecular interactions of the sago starch chains, AE components, as well as the nanoparticles, resulting in a denser and more heterogeneous network structure and, hence, a greater amount of solid per unit area.44

The mechanical characteristics of the sago-based films are the competition between the okra extract plasticization (AE) and ZnO and ZnO–MgO nanoparticles reinforcement (Fig. 6). This balance is transparent in tensile strength (TS) and elongation at break (EAB). In the inclusion of 6% (w/v) AE in the absence of nanoparticles (AE 6.0 film) the TS reduced to around 1.43 MPa and EAB rose to around 60.39%. It means that AE components are the plasticizers, which insert into the chains of starch, weaken intermolecular hydrogen bonds, and enhance free volume and movement of chains. Consequently, the movie gets softer and stretchier, although less flexible to the applied stress (reduced TS). Conversely, addition of ZnO or ZnO–MgO nanoparticles to the AE-containing films resulted in a significant increase in TS and reduction in EAB. In the case of the AE/ZnONPs film, TS increased to approximately 2.12 MPa and EAB decreased to approximately 46.12% with TS also going up to approximately 2.79 MPa and EAB decreasing to approximately 32.33% in the case of AE/ZnO-MgONPs film. The behavior is common with nanocomposites systems: rigid, well dispersed nanoparticles have high interfacial interaction (e.g. hydrogen bonding) with hydroxyl groups of starch and functional groups of AE to form a smaller and more interconnected network. The nanoparticles become stress bearing points, which enhances the load conveying within them as well as the resistance to deformation thus the film is able to withstand more stress before failure (higher TS) although it stretches less (lower EAB). Moreover, the existence of ZnO and ZnO–MgO decreases the accessibility of free hydroxyl to bind water that is likely to reduce moisture content and water-plasticization in the matrix. Less plasticization of water also leads to increased stiffness and strength and a decrease in the extensibility. All these effects explain the reason why nanocomposite films exhibit better TS and lower EAB than the AE-only film.45

Fig. 6. The TS and EAB of films with different components.

Fig. 6

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Results also suggest that incorporation of ZnONPs and ZNO-MgONPs significantly decreases the moisture content of sago starch films (Fig. 7). The moisture content of these films correlates with the elongation of films caused by ZnONPs and ZNO-MgONPs at breakage. Water functions as a plasticizer inside the bio composite network. Drying films can decrease their flexibility and enhance the strength of the films produced. The homogeneous distribution of nanoparticles throughout the matrix is responsible for the enhanced tensile strength of nanocomposite films. Nanoscale particles enhance the surface characteristics and bolster the strength of polymers. The results of previous studies similarly revealed that adding AE, ZnONPs and ZNO-MgONPs to the sago starch matrix significantly improved its mechanical properties.46

Fig. 7. The moisture, solubility, swelling and water vapor of films with different components.

Fig. 7

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The reinforcement effect of ZnONPs and ZnO-MgONPs can be explained by the combined influence of nanoparticle-polymer interfacial interactions and the resulting modification of the starch-AE network. FTIR spectra of the nanocomposite films showed changes in the O–H stretching region and broadening of the C–O–C region, suggesting increased hydrogen-bonding interactions between hydroxyl-containing groups of starch and AE and the hydroxylated surfaces of the metal-oxide nanoparticles. These physical interactions may reduce polymer-chain mobility and enable the nanoparticles to act as stress-transfer sites within the matrix.

In addition, FESEM observations showed no obvious large-scale particle aggregates on the examined film surfaces, while the XRD results confirmed the presence of ZnO and MgO-related crystalline phases in the starch matrix. The improved thermal stability observed by TGA is also consistent with a more constrained polymer network and enhanced resistance of the nanocomposite films to thermal degradation. Therefore, the higher tensile strength, particularly for the AE/ZnO-MgONPs film, is plausibly related to stronger matrix-filler interactions, reduced free volume, and a more tortuous and compact structure. However, because elemental mapping and quantitative dispersion analysis were not performed, the uniform distribution of nanoparticles throughout the film matrix cannot be conclusively confirmed.

3.3.2. Moisture content, water solubility, swelling test and water vapor transmission rate (WVTR)

The packing material must efficiently control the moisture levels inside the box. Film moisture content, water solubility, and water vapor transfer rate are critical parameters in the food packaging industry, particularly for packaged products (Fig. 7). Provides a summary of the findings for moisture content, water solubility, and water vapour transfer rate (WVTR). The moisture content exhibited a notable rise from 6.98% to 8.78% in direct correlation with the increase in AE concentration (p < 0.05) but decreased with sago film. The lowest moisture and solubility percentage was for the film sample containing a combination of AE (6%) ZnO-MgONPs decreased them of starch films due to the incorporation of different levels of nanoparticles can be attributed to the formation of strong hydrogen bonds between the starch network and the nanoparticle less. The addition of ZnO nanoparticles to the polymer matrix reduces the number of hydroxyl groups available to water molecules, resulting in a decrease in the hydrophilicity of starch films.34

During study, the effect of AE concentration increases up to 8.78% decreased the end result moisture content of the films; the effect of the introduction of ZnO and more so ZnO–MgO was more effective at decreasing the final moisture content of the films compared to the AE-only films. AE has polar compounds that bind water and serve as additional plasticizing species, and therefore the more AE the more equilibrium moisture. Conversely, the presence of ZnO and ZnO–MgO with hydroxyl groups of starch (and AE) causes a reduction in the number of free –OH sites to bind water and improves the density of the network and the non-hygroscopicity of the film; which is consistent with recent literature of modified sago starch films where the higher the hydrophobicity and the intensified internal interconnection, the lower the moisture content in the film. Reduced moisture typically relates with increased stiffness and improved barrier properties, which coincides with increased TS and reduced EAB of your nanocomposite films.47

The water solubility indicates the water resistance and indirectly, the possibility of the biodegradability of the films. In yours, AE alone has the propensity to enhance the aqueous solubility since it contains hydrophilic groups and disrupts the formation of starch to starch hydrogen bonds, whereas the mixture of AE 6% and ZnO–MgO offers the lowest aqueous solubility amongst the formulations. The cause of this reduction has been blamed on the low hydrogen bonding and potential coordination interactions of starch between the starch chains and the surfaces of nanoparticle that reduces the mobility and leaching of polymer segments into water. The same thing has been documented with heat-moisture-treated and octenyl-succinylated sago starch films, with a greater proportion of hydrophobic substitution resulting in lower water solubility than native starch films. Recent works in sago based bionanocomposite films with plant extracts and metal oxide nanoparticles also indicate that proper nanoparticle loading can greatly decrease the solubility and still provide acceptable mechanical properties.48

Swelling index is the ratio of the quantity of water that is absorbed by the film but not entirely dissolved. In your results, the control AE0 film showed extremely high swelling rates after 24 h (approximately 122.2%), which is a high hydrophobic, and loosely cross-linked matrix that is able to absorb a lot of water. Movies with more AE were even more swollen as the amount of hydrophilic groups favoring water absorption and network growth were abundant. The swelling ratio however reduced a lot when ZnO and ZnO–MgO were added (AE 6%/ZnONPs or AE/ZnO-MgONPs) which you had rightly explained as due to the more hydrophobic nature and filler effect of the nanoparticles: they fill free volume, enhance inter chain interactions and form a more compact structure, which limits penetration of water, and chain relaxation. Similar behavior has been observed with sago starch films that have undergone chemical modification or nano filler reinforcement, in which dual modification or addition of a nanoparticle decreases swelling power and water uptake as compared to unmodified starch systems.49

Food packaging needs WVTR because it speaks of how the film can control the movement of moisture between food and the surrounding. In study, an increase of AE content increased WVTR, which also indicated the greater hydrophilicity and more open network formed by AE, which enabled water vapor diffusion. The Sago/AE/ZnONPs and in particular the SagoAE/ZnO-MgONPs films had low WVTR than the AE films which you described in terms of tortuosity: the presence of dispersed nanoparticles forms a more intricate and lengthy diffusion pathway of water molecules and hence low permeation. This understanding is congruent with recent research on sago-starch and other starch-based films in which hydrophobic modification (octenyl succinylation) or addition of metal-oxide nanoparticles (TiO2, ZnO) reduced water vapor permeability and enhanced moisture-proof functionality by many folds. It has also been reported that sago composite film made of starch and gelatin will exhibit WVTR values in a similar range, and would exhibit lower WVTR with a more closely packed and well-organized network.50

Although the present results indicate improved moisture-related barrier performance, water-vapor transmission alone is not sufficient to fully establish packaging suitability. Future studies should determine oxygen permeability and carbon dioxide permeability, particularly for foods susceptible to oxidative deterioration or requiring modified-atmosphere conditions. In addition, grease resistance and long-term storage stability under different temperature and relative-humidity conditions should be evaluated. These analyses, together with real food-storage experiments, will provide a more comprehensive assessment of the practical performance of the developed films.

3.3.3. Effects of AE, AE/ZnO-MgONPs on surface morphology of sago starch

FESEM imaging of the sago starch film surfaces shows (Fig. 8) a clear morphological modification on introduction of okra extract (AE) and AE/ZnO-MgONPs nanoparticles, which gives an idea of their compatibility, dispersion, and the organization into microstructure.

Fig. 8. FESEM micrographs of the surface of the sago control film (A and B), AE (6.0%) ZnO-MgONPs (C–F) and cross-section.

Fig. 8

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Smooth baseline morphology sago film control sago film (Smooth Baseline Morphology). The face of pure sago control film is smooth and uniform without any cracks or pores or irregularities. Such a homogenous microstructure does show good entanglement of chains and high hydrogen bonding of the gelatinized sago starch network, which is characteristic of plasticized starch films in which glycerol/sorbitol (3 : 1 ratio) is an effective bridging polymer chain to prevent phase separation. This fluid morphology is associated with the mechanical and barrier baseline values of your research. Sago/AE 6.0 film (heterogeneous, roughened surface). Adding of 6% (w/v) AE forms a heterostructure with higher surface roughness, that is more visible cavities, and droplets that are entrapped by the starch network. Phase incompatibility is additionally caused by the okra extract, which is rich in polysaccharides, polyphenols, and low-molecular-weight compounds (as confirmed by your GC-MS analysis that reveals the presence of linoleic acid derivatives and phytosterols): hydrophilic AE components partially break the starch–starch hydrogen bonds forming micro-domains of extract aggregation. These regions are presented in the form of surface droplets/cavities, which enhance the overall roughness, but film integrity (no macroscopic cracks). Such a coarser morphology is responsible for the plasticizing behavior observed in mechanical testing (reduced TS, increased EAB) and increased water-related properties (greater moisture, swelling, WVTR), because the heterogeneous structure is more structure sites to contact water and has fewer network sites. Microstructure: sago-AE 6.0-ZnO-MgONPs film (redefined microstructure). The most notable change is in AE/ZnO-MgONPs films whereby, the surface experiences increased roughness further as a result of nanoparticle induced texturing and retained droplet morphology of AE. Key observations include: no massive aggregation of nanoparticles, indicating good dispersion due to the presence of AE phytochemicals acting as steric stabilizers (which is in line with the ability of okra mucilage to stabilize nanoparticles), above all. Improved micro-cavities and surface protrusions, presumably due to nanoparticles incorporated at the starch-AE interface to form a more complicated 3D topography. Cross-sectional images verify that nanoparticles are not surface-bound but that they are present throughout the thickness of the matrix. This morphology is conducive to the nanocomposite reinforcement mechanism: ZnO–MgO particles (20–30 nm, quasi-spherical per your FESEM/TEM) are well dispersed multifunctional fillers that: connect starch-AE domains by interaction through surface hydroxyl. Seal micro-pores caused by AE plasticization. Enhance surface tortuosity, which results in enhanced mechanical strength (greater TS), decreased water interactions, and low WVTR. Similar results were reported in earlier studies, when the incorporation of natural compounds in different edible films.

3.3.4. FT-IR spectra analysis

Fourier transform infrared (FT-IR) spectroscopy (4000–650 cm−1 range) reveals molecular-level interactions between sago starch, AE, ZnO nanoparticles, and ZnO–MgO nanoparticles (Fig. 9), confirming hydrogen bonding, structural compatibility, and absence of chemical degradation in the nanocomposite films. Characteristic bands in all films all formulations exhibited core sago starch spectral features: broad O–H stretching (3093–3563 cm−1): hydrogen-bonded hydroxyl groups from starch amylose/amylopectin and residual water/glycerol plasticizers. C–H stretching (2984 cm−1) and bending (1334 cm−1): aliphatic hydrocarbon chains from starch and glycerol/sorbitol. C–O stretching (1639 cm−1): residual bound water and glycosidic linkages (C–O–C) in starch backbone. These baseline peaks confirm successful starch gelatinization and film formation across all samples. AE incorporation effects (sago/AE) AE addition introduced subtle peak broadening and intensity changes without new characteristic bands: O–H region shift/intensification: AE polysaccharides, polyphenols (linoleic acid derivatives, phytosterols per your GC-MS), and low-MW compounds compete for hydrogen bonding sites with starch, weakening starch–starch interactions. No new peaks in fingerprint region (1200–650 cm−1): indicates physical dispersion/partial miscibility rather than chemical reaction between AE and starch. This supports AE's plasticizing role observed in mechanical testing (↓TS, ↑EAB) and correlates with increased surface heterogeneity (FESEM). Nanoparticle Effects (AE-ZnONPs and AE-ZnO-MgONPs Films). The most significant changes appeared in nanocomposite spectra: O–H band shift to lower wavenumbers (∼50–100 cm−1 shift): direct evidence of new hydrogen bonds between: nanoparticle surface hydroxyls/O2− sites and starch hydroxyls AE functional groups (phenolics, polysaccharides) and metal oxide surfaces. Peak broadening in 1000–1200 cm−1 (C–O–C, glycosidic): increased network complexity from nanoparticle-starch-AE matrix interactions. No new covalent peaks (no Zn–O–C ∼550–600 cm−1): confirms physical compatibility without harsh chemical modification or starch degradation. Enhanced intensity in fingerprint t region: reflects matrix reinforcement and uniform nanoparticle dispersion (corroborated by your FESEM showing no aggregation). Key interpretation: matrix-reinforcement bonding the observed O–H shift and broadening without peak disappearance or new covalent signatures indicates: strong physical interactions (hydrogen bonding, van der Waals) between components. Excellent compatibility: nanoparticles bridge plasticized starch-AE domains, explaining enhanced TS (2.79 MPa), reduced EAB (32.33%), and improved barrier properties AE stabilization role: Okra phytochemicals likely cap nanoparticle surfaces (as in green ZnO synthesis), preventing aggregation and promoting uniform matrix integration.51

Fig. 9. FT-IR spectra of sago film (control), sago/AE, sago/AE/ZnONPs and sago/AEZnO-MgONP5.

Fig. 9

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3.3.5. X-ray diffraction

According to (Fig. 10) shows the X-ray diffraction patterns sago, AE, AE/ZNPs and AE/ZnO-MgONPs loaded sago starch films. All samples show three maxima main peaks about 17.24°, 19.59.0° and 22.39° indicate that semi crystalline type of sago starch is intermediate to that of Type A and Type B starches after added AE/ZnONPs the peak, observed at 2θ values of 31.3°, 33.9°, 35.5°, 47.2°, 56.2°, 62.4° and 67.5°, correspond to the reflections at (100), (002), (101), (102), (110), (103), (112) planes respectively. Almost similar value have also been reported by52 and for AE/ZnO-MgONPs pick about 32.29° and 39.19° observed magnesium in the structure. The above XRD pattern also consists of some additional sharp peaks showing presence of semi crystalline starch matrix.

Fig. 10. XRD patterns of sago film, AE, AE/ZnONPs and AE/ZnO-MgONPs.

Fig. 10

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3.3.6. Thermogravimetric analysis

The food package's resistance to external conditions depended on its thermal stability. Thermogravimetric analysis was used to measure the thermal stability and weight loss of the sago starch films under heating at a regulated pace. The TGA profile normally includes details on the film's composition and incineration resistance. The sago films in our investigation displayed nearly the same thermal profile, and as temperature rose, so did the rate of moisture/water evaporation and the degree of degradation. Four distinct stages of weight loss were seen in the TGA patterns during heating, as illustrated in (Fig. 11a). The first phase was visible below 200 °C and was caused by the evaporation of moisture and other solvents. The second phase was visible between 160 and 224 °C and was caused by the evaporation and decomposition of glycerol. The third phase was visible at 300–360 °C and films loss maximum weight was caused by the decomposition of polymers. The final stage occurred at temperatures between 360–600 °C, during which the construction decomposed due to crosslinking with ZnONPs and ZnO-MgONPs. However, in the third and final stage, the films exhibited improved thermal stability, with more weight remaining as AE concentrations increased. By adding AE, the interaction between ZnONPs and ZnO-MgONPs was promoted, resulting in a more stable structure with enhanced thermal stability.53 This result is also consistent with DTG curve (Fig. 11b). The maximum decomposition rates of the sago starch film containing AE, the ZnONPs-containing film, and the ZnO-MgONPs-containing film were −1.09, −0.98 and −0.89 mg °C−1, respectively.54

Fig. 11. Curves of TGA (a) and DTG (b) for sago, AE, AE/ZnONPs and AE/ZnO-MgONPs.

Fig. 11

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3.3.7. Ultraviolet-visible spectroscopy (UV-vis)

Fig. 12 displays the light absorbance of sago films enhanced with varying amounts of AE, ZNPs, and ZnO-MgONPs content. The UV absorbance varied between 0% and 3%, whereas the visible absorbance ranged from 0.5% to 2.5% for the control film, 0.8% to 0.5% for sago films integrated with AE, 0.8% to 1.5% for AE/ZnONPs film 0.8% to 2.0% and ZnO-MgONPs. For 1.0% to 2.3%. The lowest amount of UV light absorbance was related to the control sample, and the highest amount was observed in films containing ZnO-MgONPs. Similar results were reported in a study by55 these researchers demonstrated that edible films containing ZnONPs and ZnO-MgONPs nanoparticles had a higher percentage of UV light absorbance compared to the control film.

Fig. 12. UV-vis spectrums of sago, sago/AE, AE/ZnONPs and AE/ZnO-MgONPs.

Fig. 12

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3.3.8. Antimicrobial activity evaluation

The antibacterial activity of the sago, sago/AE, AE/ZnONPs, and AE/ZnO-MgONPs films was assessed against Escherichia coli and Staphylococcus aureus by measuring the inhibition zones and inhibition rate. As illustrated in (Fig. 13), the films produced distinct inhibition zones, with diameters of 7.0 ± 0.1 mm, 5.2 ± 0.1 mm, and 4.8 ± 0.2 mm against E. coli, and 4.7 ± 0.2 mm, 5.4 ± 0.1 mm, and 4.9 ± 0.2 mm against S. aureus, respectively.56–58 The antibacterial activity of the nanocomposite films is likely associated with the combined action of AE-derived bioactive compounds and ZnO-based nanoparticles. A plausible mechanism for ZnO-containing systems involves the gradual release of Zn2+ ions, which may interact with negatively charged bacterial cell-envelope components, disturb membrane permeability, and interfere with essential cellular functions. In addition, ZnO nanoparticles may generate reactive oxygen species (ROS), including superoxide radicals and hydrogen peroxide, which can induce oxidative damage to membrane lipids, proteins, and nucleic acids. Direct contact between nanoparticles and bacterial cells may further contribute to membrane disruption and leakage of intracellular constituents. The comparatively stronger antibacterial activity of the AE/ZnONPs film may therefore be related to the availability of ZnO active sites and its smaller particle size, whereas the activity of the ZnO–MgO-containing film may be influenced by the altered surface chemistry and interaction of the mixed-metal oxide with the starch-AE matrix. Moreover, phytochemicals present in AE may contribute to antimicrobial activity through membrane perturbation and interference with microbial metabolism. Nevertheless, because Zn2+ release, ROS generation, membrane integrity, and intracellular leakage were not directly measured in this study, these mechanisms should be considered proposed rather than experimentally confirmed.9,59,60 Despite the promising antibacterial performance, the food-contact safety of the developed nanocomposite films cannot be established without migration assessment. In particular, the potential release of Zn and Mg species from the starch matrix should be evaluated in relevant food simulants representing aqueous, acidic, alcoholic, and fatty food conditions under appropriate time–temperature scenarios. Total migrated Zn and Mg may be quantified by ICP-OES or ICP-MS, while particle-sensitive techniques, such as single-particle ICP-MS, could help distinguish dissolved ionic species from possible nanoparticle migration. Previous assessments of ZnO-containing food-contact materials indicate that migration may predominantly involve soluble zinc species rather than intact nanoparticles; nevertheless, migration behavior is strongly dependent on the polymer matrix, nanoparticle characteristics, food type, contact conditions, and aging of the material. Therefore, comprehensive migration and toxicological assessment will be essential before the films can be considered for practical food-contact use and regulatory evaluation.

Fig. 13. Photograph of the inhibition zones of the sago, sago/AE, AE/ZnONPs, and AE/ZnO-MgONPs blend films against (a) E. coli and (b) S. aureus, and the bacteriostatic rates of the films against (c) E. coli and (d) S. aureus.

Fig. 13

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3.3.9. Antioxidant activity evaluation

The antioxidant activity of the sago starch films increased significantly with increasing Abelmoschus esculentus (AE) extract concentration (p < 0.05) Fig. 14. The control film (AE0) exhibited the lowest DPPH radical-scavenging activity, whereas films containing AE showed higher antioxidant capacity, with the 6% AE formulation exhibiting the greatest activity. This improvement can be attributed to bioactive constituents of the AE extract, including phenolic compounds and other reducing components, which can donate hydrogen atoms or electrons to neutralize DPPH radicals. Incorporation of ZnONPs and ZnO-MgONPs into the film containing 6% AE maintained or further enhanced DPPH radical-scavenging activity. This behavior may be related to favorable interactions between the nanoparticles and bioactive compounds in the AE extract, which could improve the stability or availability of antioxidant constituents within the starch matrix. Nevertheless, the antioxidant activity is expected to originate mainly from AE-derived phytochemicals rather than from the metal-oxide nanoparticles themselves. Although the AE-containing film showed considerable antioxidant activity, incorporation of ZnONPs or ZnO-MgONPs resulted in a relative reduction in DPPH radical-scavenging activity. This decrease may be attributed to interactions between the nanoparticle surfaces and phenolic or other antioxidant compounds in the extract, which may reduce the accessibility of active groups to DPPH radicals. Despite this reduction, the nanocomposite films retained antioxidant functionality due to the presence of AE-derived bioactive compounds.

Fig. 14. DPPH scavenging activity of statistically significant difference (p < 0.05).

Fig. 14

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4. Conclusions

This study demonstrated that the incorporation of Abelmoschus esculentus extract (AE), green-synthesized ZnO nanoparticles, and ZnO–MgO nanoparticles modified the structural and functional properties of sago starch films. Increasing the AE concentration increased film thickness and water-vapor transmission rate (WVTR), while decreasing tensile strength. These results suggest that AE produced a more hydrophilic and less compact starch network, probably because its constituents exerted a plasticizing effect, reduced starch–starch intermolecular interactions, and increased polymer-chain mobility. The incorporation of ZnONPs and ZnO-MgONPs improved the tensile strength, thermal stability, and UV-shielding performance of the AE-containing films. These improvements are likely related to physical interactions between the nanoparticles and the starch-AE matrix, which may promote a more compact network and restrict polymer-chain mobility. Nanoparticle incorporation also altered the optical and moisture-related properties of the films. Overall, the AE/ZnO-MgONPs formulation showed the most favorable balance of mechanical strength, moisture resistance, thermal stability, UV protection, and antibacterial activity among the evaluated formulations.

The antibacterial activity of the nanocomposite films may result from the combined effects of AE-derived bioactive compounds and ZnO-based nanoparticles. However, Zn2+ release, reactive oxygen species generation, bacterial membrane disruption, and intracellular leakage were not directly assessed in the present study. Therefore, these mechanisms should be regarded as plausible explanations rather than experimentally confirmed mechanisms. Future studies should quantify Zn2+ release and ROS generation and evaluate bacterial membrane integrity to clarify the antimicrobial mechanism.

Despite the promising laboratory-scale results, several limitations should be acknowledged. The present study did not evaluate the migration of Zn and Mg species into food simulants, cytotoxicity or broader toxicological safety, real-food storage performance, sensory properties, long-term film stability, or industrial-scale production feasibility. In addition, although the AE-mediated synthesis route uses an aqueous plant extract and avoids additional synthetic capping agents, the reproducibility of nanoparticle production may be affected by natural variations in extract composition. Standardization of plant material, extract preparation, precursor-to-extract ratio, reaction conditions, and calcination parameters will therefore be necessary during scale-up.

Moreover, the energy demand associated with drying and calcination at 700 °C should be considered when assessing the economic and environmental feasibility of large-scale production. Consequently, the developed sago starch nanocomposite films should be considered promising candidate materials for further investigation in sustainable active food-packaging applications, rather than materials ready for commercial use. Comprehensive migration and cytotoxicity assessments, food-storage trials, long-term stability studies, reproducibility testing, and pilot-scale economic and life-cycle analyses are required before practical commercialization can be considered.

In addition, the incorporation of Abelmoschus esculentus extract imparted antioxidant functionality to the sago starch films, as demonstrated by their DPPH radical-scavenging activity. This effect is mainly attributed to the bioactive constituents of the plant extract, which can donate electrons or hydrogen atoms to neutralize free radicals. The incorporation of ZnONPs and ZnO-MgONPs enabled the development of multifunctional films that combined antioxidant activity with improved mechanical, barrier, UV-shielding, thermal, and antibacterial properties. Although nanoparticle incorporation may have reduced the accessibility of some antioxidant compounds to DPPH radicals, the nanocomposite films retained measurable antioxidant activity while providing substantial improvements in mechanical, barrier, thermal, UV-shielding, and antibacterial properties.

Conflicts of interest

The authors declare that they have no known competing financial interests.

Acknowledgments

The authors gratefully acknowledge Islamic Azad University, Qaemshahr Branch, Qaemshahr, Iran, for providing laboratory facilities and institutional support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All experimental data, including numerical results and graphs used to support the conclusions of this work, are presented within the figures and tables of the main article.

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Associated Data

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Data Availability Statement

All experimental data, including numerical results and graphs used to support the conclusions of this work, are presented within the figures and tables of the main article.

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