Nigella/honey/garlic/olive oil co-loaded PVA electrospun nanofibers for potential biomedical applications

Abstract

The current work focuses on the formation of nanofibrous mats without the use of toxic solvents and metallic nanoparticles utilizing polyvinyl alcohol (PVA) and a blend of nigella, honey, garlic, and olive oil. Using deionized water (DI) water as a solvent, nanofibrous mats composed of PVA/nigella/honey (PNH) and PVA/garlic/honey/olive oil (PGHO) were developed. Methanol extraction was utilized to extract the therapeutic components of nigella sativa. Antibacterial and moisture management tests (MMT) were employed to examine the antibacterial and absorbance characteristics of the PNH and PGHO nanofibrous. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) tests were employed to analyze the morphological and chemical characteristics. PGHO showed thermal stability up to 245 °C, and PNH withstands until 225 °C. PNH and PGHO both exhibited antibacterial activity against Staphylococcus aureus (S. aureus), with inhibition zones of 36 mm and 35 mm, respectively. The synthesized materials exhibited excellent absorbance properties, thermal stability, cytotoxicity, and the production of thin nanofibers with an average diameter between 150 and 170 nm. The samples were characterized using FTIR spectra, which confirmed the presence of all components in the developed samples. To date, extensive research on electrospinning for biomedical applications has been undertaken using a variety of hazardous solvents and metallic nanoparticles. Briefly, our objective is to develop nanofibrous materials from plant extracts through a process called “green electrospinning” to observe the synergistic effect of multiple biocomponents incorporated nanofibers avoiding toxic solvents and metallic compounds for potential biomedical applications.

Graphical abstract

Introduction

Electrospinning has grown in popularity for the fabrication of polymeric nanocomposite materials due to its ability to produce ultra-fine fibers (Huang et al. 2003), versatile composite material (Huang et al. 2003), biomedical materials (Ali et al. 2020a), and most importantly, its widespread acceptance simplest (Yarin and Zussman, 2004) fiber processing system (Yang et al. 2018). When discussing its applications, health care and biomedical materials would be the most crucial fields as consumers have a preference for hygienic materials (Ding et al. 2019). These environmentally friendly materials may be able to protect themselves from the detrimental impacts of bacterial diseases (Rajendran et al. 2012). Surface tension, viscosity, molecular weight, voltage, heater, the distance between rotary drum to needles, and pumps are all parameters that must be well-managed while producing electrospun fibrous materials (Elsabee et al. 2012; Reneker et al. 2000; Ziyadi et al. 2021a). The surface tension of the solution can be distorted by the high voltage that allows ejecting of electrically charged jets responsible for forming ultra-fine nanofibers that are deposited on rotary drums (Abid et al. 2019; Sadeghianmaryan et al. 2020). Data from several studies suggest that electrospinning has tremendous applications in various fields, for instance, filtration (Frey et al. 2003; Kaur et al. 2019; Liu et al. 2020a, b), protective material (He et al. 2014), electrical and optical applications (Chen et al. 2010), sensors (Laurila et al. 2015; Marzana et al. 2022), tissue engineering (Pangon et al. 2016), drug release (Huang et al. 2003; Jannesari et al. 2011; Panthi et al. 2020), wound dressing (Amalraj et al. 2020; Shahid et al. 2020), and enzyme immobilization (Agarwal et al. 2008 and Panthi et al. 2020).

According to SciFinder statistics, thousands of research have been published on electrospinning to date, but most of them are based on organic solvents for preparing a blending working fluid for a single–fluid electrospinning (Ziyadi et al. 2021b) or several different organic solvents are exploited to prepare various working fluids to load several functional ingredients in the complicated structures such as core–shell (Xu et al. 2022), Janus (Zhang et al. 2022), beads-on-a-string (He et al. 2022), and other multi-compartment structures (Yu et al. 2021). In sharp contrast, very limited studies were conducted on the aqueous solution-based electrospinning, and even limited studies can be found on the encapsulation of multiple plant extracts into the electrospun nanohybrids. Plant extract-based nanofibrous materials, on the other hand, are expected to grow in popularity in the coming years (Zhang et al. 2017). Metallic nanoparticles have been used in the majority of biomedical electrospun nanofibrous studies. Silver, gold, and zinc nanoparticles are among the nanoparticles that have antibacterial and bactericidal capabilities (Jain et al. 2020). These nanoparticles were found to be efficient against multi-resistant and biofilm-forming bacteria, reported by several authors (Atiyeh et al. 2007; Nguyen et al. 2011; Silvestry-Rodriguez et al. 2007).

The use of these metallic nanoparticles, on the other hand, has resulted in life-threatening disorders. Although silver nanoparticles (AgNPs) are commonly used in antibacterial nanofibrous, various research has indicated that they have harmful health impacts. Toxicity to mammalian cells (Braydich-Stolle et al. 2005; Gopinath et al. 2008; Wen et al. 2007), destruction of brain cells (Augustine et al. 2016; Hussain et al. 2005), liver cells (Hussain et al. 2005), and stem cells (Braydich-Stolle et al. 2005) are among them. According to McAuliffe et al. findings, AgNPs impair the male reproductive system due to their high toxicity. AgNPs breach the blood-testes barrier and are deposited in the testes, causing negative effects on sperm cells, according to this study (McAuliffe and Perry 2007), exerting deleterious effects on sperm cells. In their research, Takenaka et al. discovered that AgNPs could harm the cardiovascular system. In rats, they looked at the pulmonary and systemic dispersion of inhaled ultra-fine elemental AgNPs and identified silver. They also found Ag in the blood, kidney, spleen, nose, brain, and heart (Takenaka et al. 2001).

Similarly, gold nanoparticles (AuNPs) and Zinc oxide nanoparticles ZnONPs demonstrated reliable antibacterial properties. In particular, ZnO offers simple fabrication, high surface area, lower cost, and stability (Prabhu et al. 2022). ZnONPs can promote the polarization of electrospun polyvinylidene fluoride nanofibers (PVDF) (Gao et al. 2020; Han et al. 2019). However, the inherent toxicity of these nanoparticles poses a danger to human health. ZnONPs, specifically, are responsible for mitochondrial dysfunction in keratinocytes when they are employed at high concentrations. Lactate dehydrogenase is released, which causes this. ZnONPs also generate carcinogenic changes, as reported by Yang et al. (2009). Chitosan/gold nanofibrous enriched with PVA was developed with a bioactive Punica granatum extract. This nanofibrous showed excellent biocompatibility and antibacterial properties and was reported as a suitable scaffold for cell adhesion, growth, and proliferation of fibroblast populations (Hussein et al. 2021). Furthermore, when high concentrations of AuNPs are induced in the human body, body weight, red blood, and hematocrit are reduced (Zhang et al. 2010). These nanoparticles' negative effects are limiting their utility in biomedical applications.

Avoiding the harmful effects of metallic nanoparticles could be as simple as using plant extract-based medicinal medicines. Because of their strong antimicrobial and antibacterial capabilities, bio-based components are frequently used in biomedical applications. The use of bio-based medicinal herbs to treat human skin illnesses, such as wound healing, burn injuries, antifungal, antiviral, and antibacterial applications, has a long history of use in primary health care (Bihani and Mhaske, 2020; Freitas et al. 2020; Shahabuddin et al. 2022). Because of their biodegradability, biocompatibility, and low toxicity, these natural components are commonly employed in electrospun nanofibers (Charernsriwilaiwat et al. 2010 and Elsabee et al. 2012). The following biocomponents were used in this study: nigella sativa, garlic, olive oil, and honey. When examining the therapeutic characteristics of nigella, the antibacterial and antimicrobial activity should be examined, as it has been used for a long time for health illnesses, as nigella oil can be used as an antiseptic and anesthetic. The nigella extract was used to generate antibacterial nanocomposite materials for wound-dressing materials blending with PVA, as reported by Ali and colleagues (Ali et al. 2020a, b). They obtained beadless fine fiber manufacturing using acetic acid as a solvent. They also discovered that PVA blended nanomat has excellent antibacterial, moisture, and thermal properties, suggesting that it might be employed in wound-dressing materials. This is due to the presence of functional components in nigella, such as thymoquinone (TQ) and thymohydroquinone (THQ). These groups are susceptible to the gram-positive bacteria.

Honey is also a common bio-based component that is commonly employed in electrospinning biodegradable antimicrobial compounds (Shahid et al. 2020). Honey contains a wide range of medicinal and nutritional properties, and it has long been used for wound healing (Khan et al. 2007), antibacterial, and anti-inflammation activities (Vandamme et al. 2013). It has low water content and an acidic pH (3.2–5.0). It also contains plenty of sugar and H₂O₂, which have antibacterial properties against S. aureus bacteria (Cooper et al. 1999). Honey also contains a number of non-peroxides, such as methyl syringate and methylglyoxal, which have antibacterial properties (Mandal and Mandal, 2011). All this exposure to antibacterial agents leads it to implement in nanocomposite material to develop wound-healing nanomats. Shahid et al. developed a nanomat using honey with curcumin longa having tremendous antibacterial properties suggested for wound-healing materials (Shahid et al. 2020).

The essential bio-based medicinal substances used in this investigation were garlic and olive oil, and both elements displayed exceptional antibacterial activity in the nanocomposite created. Olive oil contains phenolic and polyphenolic functional groups such as oleuropein and hydroxytyrosol (Heidari-Soureshjani et al. 2016), resulting in antioxidant, anti-inflammatory (Heidari-Soureshjani et al. 2016), and antibacterial activity against S. aureus and S.typhimurium bacteria (Guo et al. 2020). Similarly, garlic has been reported as an excellent antibacterial agent against human pathogenic bacteria. It can resist both gram-positive and gram-negative bacteria since it contains phytochemical constituents, allicin derived from the alliin–alliinase system (Uchida et al. 1975). This substance has a high susceptibility to gram-positive bacteria. Furthermore, the presence of flavonoid components and essential oil demonstrated antimicrobial properties (Ali et al. 2020b; Salima et al. 2015). However, the ingredients are not electrospun alone, therefore several biopolymers are being used.

The experiment was carried out through PVA; a highly biodegradable, biocompatible, and non-toxic polymer (Kamoun et al. 2015) as a carrier of medicinal constituents for ease of consistent electrospinning (Raghunathan & Marx, 2019). This PVA is extensively used in various fields like cosmetics, food, pharmaceutical, and medical industries (Kamoun et al. 2021 and Raghunathan and Marx 2019). Moreover, PVA can be used as a wound-dressing material due to its hydrogel-forming properties and provide a controlled release of antibiotics. Apart from this, many properties like small molecules, low interfacial tension, soft consistency, and transparency, making it prominent to be used as a wound-dressing material (Abou-Okeil et al. 2012 and Park et al. 2015).

A great deal of previous research was carried out using PVA, chitosan, and other biopolymers dissolving in organic solvents. These solvents include acetic acid, hexafluoroisopropanol (HFIP), and trifluoroacetic acid (TFA) (Hadipour-Goudarzi et al. 2014; Liu et al. 2014). Several organic solvents are used in electrospinning toxic causes of severe environmental and health crises during evaporation. Moreover, the remaining solvents in the fibers are restricted from being used in biomedical applications (Amalraj et al. 2018; Ghosal et al. 2018; Lv et al. 2018; Zhang et al. 2021). However, green solvents such as ionic liquid (Angaiah et al. 2018) and deep eutectic solvents are also being utilized, especially for biopolymer dissolution.

The electrospun nanocomposite may generate a platform for protecting wounds, and these polymeric materials may have the potential to heal infectious bacteria due to their medicinal properties. Kecong et al. developed a polymer-drug core-sheath nanohybrid material to compare the drug-polymer nanocomposites, showed improved antibacterial properties, and suggested to be used in drug delivery (Ghosal et al. 2021; Joseph et al. 2017; Zhou et al. 2022). According to the author search, even though the PNH and PGHO nanocomposites are made from novel multi-components such as nigella, honey, garlic, and olive oil, which have tremendous magical properties; antibacterial, wound-healing potentiality, and enhanced absorbance behaviors properties have not yet been thoroughly studied using a green approach. However, in terms of PNH, it was difficult to form nanofibers due to the extremely viscous nature of honey and the oily nature of nigella polymers, although PVA carrier polymers made it easier. Similarly, the PGHO nanocomposite demonstrated an oily surface since it was made from garlic and olive oil.

The purpose of this research is to develop hybrid biodegradable nanofibrous materials from bio-based components for biomedical applications, and a water-soluble PVA solution was used to enable electrospinning with diverse plant-based medicinal components easier. Because hazardous organic solvents and metallic nanoparticles are a concern for human wellness, we investigated the experiments utilizing green electrospinning (Sen et al. 2022). This fabrication approach does not involve any toxic chemical process. From all the above, based on green technology, antibacterial properties, cytotoxicity, thermal, and morphological characteristics, the developed PNH and PGHO can be suggested to be used in potential biomedical applications.

Materials and methods

Materials

Nigella sativa, Sundarbans raw honey, garlic, and extra virgin olive oil were purchased from the local shopping center (Swapna Super Shop, Gazipur, Bangladesh). Polyvinyl alcohol (PVA) (MW 115000, Viscosity 26–32, DP 1700–1800) was purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Absolute methanol (99%) was sourced from Merck (Darmstadt, Germany). All the chemicals and reagents were utilized as received.

Preparation of nigella extract

The extraction process was carried out following our previous research (Ali et al. 2020a, b). The nigella sativa (black seed) was properly cleaned, washed, and dried. A grinder was used to grind the dried clean black seeds into powder. Ground nigella was steeped in methanol overnight to extract the therapeutic component (nigella: methanol = 1: 2) (w/w). The soaked extract was filtered (3X) using a quadrupled nylon mesh fabric. The filtered solution was evaporated at 60 °C in a hot plate equipped with a magnetic stir bar (Isotemp, Fisher Scientific, Waltham, MA, US) until it transformed into an oily-gel form, then it was kept in a refrigerator at 4 °C.

Preparation of garlic extract

The clean garlic granules were crushed into paste form and mixed with olive oil (garlic paste: olive = 1:2) (w/w). The mixture was heated at 60° C in a hot plate equipped with a magnetic stir bar (Isotemp, Fisher Scientific, Waltham, MA, US) until it became a homogeneous solution. The homogeneous mixture was stored at room temperature.

Preparation of PVA solution

PVA solution of 10% (w/v) was prepared using DI water. The mixture of PVA solution was heated in a hot plate equipped with a magnetic stir bar (Isotemp, Fisher Scientific, Waltham, MA, US) at 70–80 °C until it became a transparent homogeneous solution.

Preparation of PNH and PGHO electrospinning solution

Honey was mixed with PNH and PGHO to develop hybrid nanocomposites. Nigella extract of 2.8% (by wt) was mixed with 6 mL honey and 20 mL PVA solution and stirred for around 2 h to obtain a homogeneous solution of PNH. Similarly, 25 mL PVA solution was blended into 5 mL honey and 5 mL garlic/olive oil mixture to develop PGHO samples using the same protocol as PNH.

PVA, PNH, and PGHO electrospinning process

An electrospinning machine (TL–Pro–BM, Tong LI Tech, China) with parameters of (applied voltage 20–24 kV), a heater (0.45–0.5 kW), a syringe pump (TL–F6, Tong Li Tech, China), a rotary drum collector (diameter:158 mm, 500 mm and 500 rpm), and 5 needles of 20 gauge were used in the experiment. The feed rate was maintained between 1.50 mL/h and 2.50 mL/h. An aluminum foil was wrapped around a rotating drum with a needle tip to a collector distance of 15 cm to obtain the deposited nanofiber. The experiment was carried out in a room with a relative humidity of 65% and a temperature of 27 °C.

Characterizations

Morphology

Scanning electron microscopy (SEM) (SU 1510, Hitachi, Japan) was used to examine the morphological structures at a magnification of × 5 k and × 10 k and a voltage of 5 kV and 10 kV, respectively. ImageJ software was used to determine and analyze the fiber diameter (n∼ 30) at the nanoscale.

Antibacterial assay

The antibacterial activity of the developed samples against gram-positive S. aureus bacteria was determined using the disc diffusion method, and the zone of inhibitions was analyzed. TSA plates were used with 0.5 × 10⁶ colony forming units (CFUs) Per mL of S. aureus using the standard disc diffusion method. Pelletizing with a disc diameter of 13 mm on an agar plate, which was placed in the incubator overnight at 37 °C, was used to prepare the samples. The inhibitory zones that formed were measured in millimeters.

Cytotoxicity

To analyze the cytotoxicity of the developed samples, a biological safety cabinet and CO₂ incubator, Nuaire, USA, a trinocular microscope with a camera, and a hemocytometer were required. On the Vero cell line, cytotoxic effects were detected. An African green monkey's kidney epithelial cells were cultured in Dulbecco's Modified Eagles' medium containing 10% fetal bovine serum, 0.2% gentamycin, and 1% penicillin–streptomycin (1:1). Following a 24 h incubation period at 37 °C and 5% CO₂, 1.5104/100 µL of cells were sown onto 48-well plates. The following day, an autoclaving procedure was performed on each well. After 48 h of incubation, the cytotoxicity of duplicate wells was evaluated using an inverted microscope.

Thermal analysis

The thermogravimetric analysis (TGA) to evaluate the thermal behavior of the PNH and PGHO nanocomposites was conducted by a thermal analyzer (SDT 650, Discovery, DE, USA) within the temperature range from 50 to 300 °C at a constant heating rate of 5 °C/min. The PNH and PGHO solutions were taken in the amount of 6.5 mg, while a PVA nanomat was used as a control sample.

Absorbance behavior analysis

The developed samples were tested using a moisture management tester (MMT) (M290, SDL Atlas, UK) following AATCC 195-2009 method to evaluate their absorbance properties. Several parameters were evaluated like wetting time, absorption rate, maximum wetted radius, outer and inner surface spreading speed, accumulative one–way transport capacity (R), and overall absorbance behaviors capacity (OMMC). A saline liquid containing 0.9% sodium chloride was utilized in the test, and the duration taken was 120 s.

Fourier transform infrared spectroscopy (FTIR)

The PNH and PGHO samples were analyzed by FTIR (IR Prestige21, Shimadzu Corporation, Japan) to evaluate their chemical nature; sample spectra were recorded at 650–4000 cm^–1 range with 4 cm^–1 resolution. The findings were plotted using Sigma plot software (Systat software Inc., IL, US).

Results and discussion

Morphological analysis

Table 1 lists the electrospinning parameters used in the experiment, including applied voltage, pressure/feed rate, ambient temperature, heater, and needle tip to collector distance. The heater evaporates the solvents into the extruded fibers (Ali et al. 2021a, b). Under a magnification of × 5 k and × 10 k, the PVA, PNH, and PGHO samples were assessed using an SEM, and it was shown that PVA had an average diameter of 230 ± 20 nm, PNH had an average diameter of 150 ± 10 nm, and PGHO had an average diameter of 170 ± 10 nm, with mesh and tiny porous structures, as shown in Fig. 1. However, the developed fibers demonstrated slight stickiness due to the addition of honey. Very similar work was conducted by Ali et al. using acetic acid as a solvent obtained fibers diameter was around 245 nm (Ali et al. 2021a, b). A series of plant-extract-based nanofibrous production has been carried out using the same parameters incorporating aloe vera (Garcia-Orue et al. 2017 and Ghorbani et al. 2020), neem (Ali and Shahid 2019), and curcumin (Sun et al. 2013) having fiber diameters between 112 and 420 nm. The optimum fiber formation was carried out for PNH at an applied voltage of 23 kV, maintaining a feed rate 1.50–2.00 mL/h, while the value was 24 kV for PGHO and the feed rate was 1.50–1.80.mL/h. This could be related to the fact that the viscosity of PGHO solution was higher than the viscosity of PNH solution due to the addition of garlic extraction. Several PVA-loaded nanofibrous materials were developed using plant-based extract where flow rates were between 0.20 and 3.0 mL/h (Charernsriwilaiwat et al. 2013; Mouro et al. 2019; Shokrollahi et al. 2020).

Table 1.

Parameter optimizations and morphological observations

Sample name	Feed rate (mL/h)	Applied voltage (kV)	Needle tip to collector distance (cm)	Heater (kV)	Fiber diameter (nm)
PVA	2.50	20	15	0.45	230 ± 20
PNH	1.50–2.00	23	15	0.50	150 ± 10
PGHO	1.50–1.80	24	15	0.50	170 ± 10

Fig. 1.

Morphological observation using SEM and fiber diameter distribution with respective frequency (Mahendran et al. 2016) (a1) visualization of PVA nanofibers and fiber distribution at 5 kV, ×5 k; (b1) visualization PNH nanofibers and fiber distribution at 5 kV, ×10 k; (c1) visualization of PGHO nanofibers and fiber distribution at 5 kV, ×5k

Antibacterial assay and cytotoxicity

The PNH and PGHO both have demonstrated outstanding antibacterial properties against the gram-positive Staphylococcus aureus (S. aureus) bacteria, but at the same time, there was no activity of only PVA nanomat illustrated in Fig. 2. PNH showed a 36 mm inhibition zone, while PGHO exhibited a 35 mm inhibition zone (Table 2), indicating that PNH is slightly more antibacterial than PGHO and resists bacterial growth.

Fig. 2.

Antibacterial activity of nanofibrous mats against S. aureus bacteria at 37 °C for overnight (a) the zone of inhibition of PNH showed 36 mm and no inhibition for PVA; (b) the zone of inhibition for PGHO showed 35 mm while PVA demonstrated no inhibition

Table 2.

Antibacterial activity against Staphylococcus aureus (S. aureus)

Name	Bacterial strain	Zone of inhibition
PNH	Staphylococcus aureus (S. aureus)	36 mm
PGHO	Staphylococcus aureus (S. aureus)	35 mm
PVA	Staphylococcus aureus (S. aureus)	X

Forouzanfar et al. investigated that nigella is highly antibacterial resistant since it contains thymoquinone (TQ) and thymohydroquinone (THQ) components (Fig. 3), and S. aureus; a gram-positive bacteria, is susceptible to these two constituents (Ali et al. 2020a, b and Forouzanfar et al. 2014). Furthermore, nigella consists of many other functional constituents like carvacrol, thymol, and terpenoids which are reported as potential antimicrobial agents reported by Ali and colleagues (Ali et al. 2020a, b). When a bacterial cell is broken down, then the cell constituents are released, resulting in bacterial cell death. (Ali et al. 2020a, b; Mohammed et al. 2019). Apart from this, biofilm formation is one of the considerable crucial aspects of several pathogens, including S.aureus.

Fig. 3.

Medicinal constituents of Nigella (a) thymoquinone (TQ), (b) thymohydroquinone (THQ) (Gilani et al. 2004), Olive (c) hydroxytyrosol, (d) oleuropein (Rahmani et al. 2014), Honey (e) methyl syringate (f) methylglyoxal (Jeffrey and Echazarreta 1996), and Garlic (g) Allicin (Gebreyohannes and Gebreyohannes 2013)

TQ may resist biofilm formation in several bacterial strains, including S.aureus, S.epidermidis, and E. faecalis. Even more, this TQ not only inhibits oxidative activities of biofilms microbial cells but also mitigates the number of living cells. Moreover, the mesostructured developed hybrid nanocomposites may resist the penetration of any bacterial growth due to having tiny pores which can set aside the infectious area (Ali et al. 2020a, b).

In the PGHO sample, garlic and olive oil were employed, both of which are bacteria-killing natural agents. The major phenolic compounds with antibacterial and antioxidant characteristics examined by Bubonja-Sonje et al. (2011) are hydroxytyrosol (Fig. 3). Furthermore, secoiridoides (oleuropein and derivatives) (Fig. 3) have been shown to inhibit or delay harmful bacterial growth (Bisignano et al. 1999). Additionally, olive oil is a source of a high proportion of single fatty acid and fat-soluble A, D, E, and K vitamins. As a result, this oil contains crucial natural components, and it's possible that this ointment could be utilized to treat wounds (Ünsal et al. 2001). Garlic, on the other hand, contains allicin (Fig. 3), the most abundant organosulfur, and its decomposition ajoene. It has a variety of biological actions, including antibacterial, antioxidant, antibiotic, anticancer, and antiviral properties (Edikresnha et al. 2019; Focke et al. 1990; Prasad et al. 1995). As a result, garlic extract is highly susceptible to gram-positive bacteria. Furthermore, flavonoid and essential oil-containing materials have been shown to have potent antibacterial properties (Salima 2015). Polyurethane-loaded garlic has been electrospun using the toxic solvent dimethylformamide for wound-healing applications (Mani & Jaganathan 2019), while no toxic solvent was incorporated into the PGHO nanocomposite. The inclusion of honey increases the wettability of the olive and PU, which are naturally hydrophobic. The developed scaffold had a lower hemolytic index percentage (PU/olive oil: 1.41% and PU/olive oil/honey/propolis: 0.95%) than the control (2.48%), indicating that it was safe to use with RBC (Jaganathan et al. 2019).

Honey, which has been employed in both PNH and PGHO, is another component; the most readily available medicinal materials in the environment have several features, including high osmolarity, acidity, and, more importantly, the presence of H₂O₂. Some prior research has identified H₂O₂ as the primary antibacterial ingredient in phytochemical elements such as methylglyoxal (Molan 1992 and Shahid et al. 2020). When honey is diluted, H₂O₂ is produced. Due to the activation of the enzyme glucose oxidase, it oxidizes the glucose into H₂O₂ and gluconic acid. This H₂O₂ and gluconic acid demonstrate antibacterial activities. Besides the H₂O_2, honey consists of many non-peroxides like methyl syringate and methylglyoxal (Fig. 3), which have antimicrobial activities. PVA-loaded honey has been electrospun using several biocomponents like Carica papaya (Balaji et al. 2016), curcumin (Shahid et al. 2020), and nepeta dschuparensis (Naeimi et al. 2020). These all developed fibrous exhibited outstanding antibacterial properties and were suggested to be used in wound healing and burn injury.

Additionally, having a low pH (3.2 and 4.5) which is sufficient to resist certain pathogens. Antibacterial activity is attributed to the osmotic action, high sugar content, and moisture properties, in addition to the acidic characteristics (Mandal and Mandal, 2011; Molan, 1992; Molan & Rhodes, 2015). The synergistic effect of these natural resource-based restorative elements was employed to create PNH and PGHO nanocomposites that can be used in biomedical applications, particularly as a wound-dressing materials.

Figure 4 depicts the deactivation mechanism of S. aureus bacteria. The oxidative stress produced by the nigella, garlic, and olive oil–loaded samples can destroy the cell wall, protein, lipids, amino acids, carbohydrates, and DNA (Forouzanfar et al. 2014). The therapeutic components released on the surface and inside the bacteria cause cell death by damaging the cell wall and organelles. However, antibacterial activity is triggered by a series of events, such as peptidoglycan and cytoplasmic membrane depletion, protein motive force loss, ATP generation depletion with DNA replication breakdown, intracellular outflow, and so on (Mamonova et al. 2015). Although these bioactive components are sound against antibacterial growth, the mechanism of bacterial killing could have been a further study.

Fig. 4.

Inactivation of S. aureus bacteria by disrupting membrane proteins due to the antibacterial compounds of PNH and PGHO

Utilizing in-vitro cytotoxicity testing, potential drug candidates or biomaterials can be evaluated in cell culture. Using these assay methods, researchers may rapidly test a large number of nanofiber membranes, providing crucial information for future animal investigations. Biocompatibility of nanofibrous mats was determined by determining how well they maintained cell survival (Saleemi et al. 2020; Shahid et al. 2022). To determine the cytotoxicity of the nanofiber membrane, kidney epithelial cells were cultured in culture for 48 h using African green monkeys.

 Figure 5 illustrates the cell viability of PNH, PGHO, and PVA, where PVA was considered as a control. When seen at 10 × magnification, three samples were shown in Fig. 5 to compare favorably to the control membranes. When the number of viable cells dropped below 5%, the PNH and PGHO became cytotoxic. Even if the control sample showed around 100% cell viability, the methanol extract might have caused for PNH and PGHO nanofibers.

Fig. 5.

Cell viability in microscopic analysis (a) PNH (b) PGHO (c) PVA (d) comparison chart

Thermal analysis

Thermogravimetric analysis was utilized to determine the thermal properties of PNH and PGHO, with PVA nanomat used as a control, as shown in Fig. 6. The small initial peak at 50 °C may be due to moisture evaporation in the materials (Abidi et al. 2008). While the TG curve of both PNH and PGHO shows good thermal stability, PGHO showed the maximum decomposition at 245 °C, and for PNH, this temperature was at 225 °C, while neem-loaded PVA/chitosan nanomat showed degradation temperature above 250 °C (Ali et al. 2019). The PVA nanomat, on the other hand, begins to degrade at 210 °C (Lee et al. 2009). Another previous research work published by Naeimi et. al. reported that the first and second degradation of PVA/chitosan/nepeta dschuparensis nanomat occurred at 250 °C and 350 °C, respectively (Naeimi et al. 2020). Generally, the PVA nanofibers melt at 190 °C or 227 °C due to their less crystalline region, however, the inclusion of multiple substances such as honey, nigella, garlic, and olive oil has improved the stability of PGHO and PNH nanofibrous because of an increased number of H-bonding. Beyond 275 °C, three materials were decomposed rapidly, and 100% weight loss was noticed at 300 °C. An increased concentration of several biocomponents not only helped in forming more nanostructures and helped in binding the strong bond with PVA. Because of the reduction in segmental motion, the addition of diverse components may resist polymer chain movements (Naeimi et al. 2020).

Fig. 6.

Thermogravimetric analysis of (a) pure PVA, (b) PNH, and (c) PGHO

Absorbance behaviors analysis

The developed samples have undergone an absorbance behavior test to study the moisture properties wherein top and bottom (outer and inner) surfaces were tested, as shown in (Fig. 7). While the wetting time of both PNH and PGHO on the outer surface was found to be somewhat similar, on the inner surface, PGHO takes less than one second, which is five times quicker than PNH. This means that PGHO is preferable to use as a wound-healing material since its inner side may interact with human skin that can absorb the wound liquid quickly, while the time is taken by only PVA nanomat likewise PNH. In terms of absorption rate, the inner surface of PNH is nine times higher than the PGHO, while the outer surfaces are nearly similar. The very similar wound-healing potential results obtained in PVA/curcumin/honey nanocomposites were reported by Shahid et al. (2021).

Fig. 7.

(a, a1) Water location and time diagram of PNH; (b, b1) water location and time diagram of PGHO

Furthermore, while PNH has the same maximum wetted radius on both sides, PGHO's outer surface is significantly larger than the inner surface. PGHO is faster than PNH in terms of spreading speed on both sides, which is a more important factor in wound-dressing activities (Ali et al. 2021a, b). However, in terms of one-way transport capability, PNH and PGHO showed opposite sceneries, while PNH is more transferable than PGHO. This may happen due to the oilier behavior nature of garlic and olive oil. When it comes to discussing the OMMC, PNH accounted for more than doubled than PGHO. This meant that the overall moisture performance of PNH is preferable as a wound dressing. PVA-nigella nanomat also revealed outstanding healing properties, and the researcher applied this nanomat in vivo analyses to reveal fast healing effects (Ali et al. 2021a, b). Therefore, PNH, in comparison with PGHO, shows higher water absorbency. These fast-absorbing natures of developed PNH and PGHO nanocomposites allow them to be used in wound-dressing area since it transfers the liquid to the outer surface, leading to enhanced healing performance.

Fourier transform infrared spectroscopy (FTIR) analysis

The presence of functional groups of PVA, nigella, garlic, honey, and olive oil in the developed samples is demonstrated by infrared spectroscopy (Fig. 8).While the identification region covers a range of 4000–650 cm^–1, the fingerprint region in respect to the region of 1000–650 cm^–1 (Islam et al. 2021). The broadband results from hydrogen bonding in PVA, with the main peako bserved in FTIR spectra at wavenumbers 3297 cm^–1 (O–H stretching) and 2922 cm^–1 (C–H₂ asymmetric stretching) (Mansur et al. 2008). Moreover, the wavenumber at 1664 cm^–1 is due to the C=O carbonyl stretch. Therefore, the presence of PVA in both PNH and PGHO nanocomposites has been confirmed.

Fig. 8.

FTIR spectra of PNH and PGHO show individual peaks

In terms of PNH, the nigella extract consists of various functional groups, including thymoquinone, dithymoquinone, thymohydroquinone, and thymol. The FTIR spectra of this PNH sample revealed multiple weak peaks and functional groups that correspond to C–H, –CH₂, CH₃, C=O, C–O, and C=C (Doolaanea et al. 2014). The presence of these functional groups can be seen in the main phenolic chemicals, thymoquinone, dithymoquinone, thymohydroquinone, and thymol. A weak peak indicates the presence of methyl and isopropyl substituents at 2860 cm^–1 assigned to the C–H stretching of the aliphatic group (alkenes), which implies the existence of methyl and isopropyl substituents.The weak peak represents the aromatic compounds at 1402 cm^–1. The peaks at 1242 cm^–1 and 1026 cm^–1 belong to aliphatic amines in nigella (Doolaanea et al. 2014). Moreover, the peak at 916 cm^–1corresponds to carboxylic acid in nigella sativa. There is also a weak peak at wavenumber 1700 cm⁻¹ that can be attributed to the C=O stretching of the forester and ketone groups (Doolaanea et al. 2014).

Honey is also present in these two samples, despite the presence of nigella. The presence of hydroxyl groups in water and carbohydrates (glucose, fructose, and trehalose) results in a strong band with transmission at 3300 cm^–1 occurs due to having hydroxyl groups of water and carbohydrates (glucose, fructose, and trehalose) (Horvatinec and Svečnjak 2020). The vibration at 2922 cm^–1 corresponds to the C–H stretching of methyl and methylene groups of protein and lipids. The N–H deformation and C–N stretching vibrations (amide II) of peptides, enzymes, and other hemolymph protein-based constituents occur in the spectral range between 1510 cm^–1 and 1544 cm^–1 (Parin et al. 2021). The spectral regions between 1470 and 1280 cm^–1 are characterized by a series of weak broad signals at 1459 cm^–1 and 1407 cm^–1 due to CH₂ bending and C=O asymmetric stretching (COO–) at 1337 cm^–1 due to C–H deformation vibration, which overlaps with C–N stretching (amide III) (Parin et al. 2021).

On the other hand, in the case of PGHO, garlic and olive oil were used. These two materials consist of several functional groups. While olive oil consists of oleuropein and hydroxythyrosol groups, garlic contains an allicin group. These two groups are also present in the developed nanocomposites. The allicin contains a diallyl disulfide group where S–S linkage is present, while the oleuropein and hydroxythyrosol contain polyphenolic compounds; phenolic hydroxyl OH, carboxylic COOH, carbonyl C=O, and methoxyl groups –CH₃ with an aromatic ring (Yeganeh et al. 2022). The wide bade confirmed the O–H stretch of COOH at 3397 cm^–1.

Furthermore, the peak at 1715 cm^–1 is associated with C=O stretching, as are the peaks at 1240 cm^–1 and 1636 cm^–1 with organic molecules containing nitrogen and sulfur. Sulfur-containing compounds were found at a wavelength of 1242 cm^–1, which corresponds to S=O stretching (Barreto et al. 2017). The strong peak at 1026 cm^–1 corresponds to the C–O stretching of olive oil. The proposed hydrogen bonding has been illustrated in Fig. 9. Thefunctional groups of components containing PNH and PGHO samples might react through hydrogen bonding with PVA and make the electrospun nanocomposites.

Fig. 9.

(a) Proposed Hydrogen bonding between PVA and thymoquinone (TQ) (b) Hydrogen bonding between PVA and thymohydroquinone (THQ) (Ali et al. 2021a, b) (c) Hydrogen bonding between PVA and hydroxytyrosol (d) Hydrogen bonding between PVA and oleuropein (Nediani et al. 2019) (e) Hydrogen bonding between PVA and methyl syringate (f) Hydrogen bonding between PVA and methylglyoxal (g) allicin (Liu et al. 2020a, b)

Conclusion

The synergistic effects of multi-component nanofibers followed by the green approach result in outstanding antibacterial, moisture, thermal, and cell viability. Furthermore, because FTIR confirmed the presence of all constituents contained in the nanocomposites and these constituents were shown to have antibacterial activity against S. aureus bacteria, it can be applied to various biomedical applications.

Importantly, our results demonstrate that green electrospun can be produced using water-soluble synthetic biopolymers without the use of metallic compounds in addition to the synergistic effect of biocomponents, even though hazardous solvents and metallic compounds are frequently used in biomedical electrospun. Here we demonstrated that using PVA with bioactive agents could be a promising nanofibers development for health care applications, there is no need for hazardous solvents and metallic compounds.

However, more investigation, such as assessing these nanocomposites on animal or human, would have been essential for further study. To the best of our knowledge, the mechanical properties of these materials may be the biggest obstacle to utilize in biomedical applications. However, all these opportunities to work with environmentally friendly procedures that toxic, harmful solvents and metallic compounds, moisture, cell viability, and antibacterial properties suggested that these hybrid nanocomposites could be used in biomedical application, such as materials for dressing wounds. Briefly, bioactive compounds embedded in nanofibers have great potential as biomaterials for treating injuries. This environmentally friendly strategy might help to stay away from harmful substances.

Author contributions

MNU: concept, experimental, and writing the draft; MM and SMI: experimental; MAU, MJ: proofread and approved.

Funding

The research team did not receive grants or funds from any public, private, or commercial organizations.

Declarations

Conflict of interest

The authors declare no potential conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any authors.