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Abstract

In the recent years, the medical sector is getting increasingly interested in the wound dressing materials that contain medicinal herb instead of metal nanoparticles to impart antibacterial or other desirable properties. Herein, a novel multicomponent nanofibrous mat has been successfully prepared by electrospinning technique from a blended solution of polyvinyl alcohol, honey and Curcumin longa (turmeric) extract for potential application as the wound dressing material. Ethyl acetate extraction was followed to obtain the restorative components of turmeric. The fabricated nanofibrous materials were characterized by scanning electron microscope, moisture management tester, bacterial assay and Fourier-transform infrared spectroscopy (FTIR) to evaluate their morphological, moisture, antibacterial and chemical behavior, respectively. Nanofibers of fabricated mat show an average diameter of 340 nm with better moisture management properties compared to polyvinyl alcohol nanomat alone. The agar diffusion method has been used to evaluate the antibacterial activity against Staphylococcus aureus bacteria showing the formation of inhibition zone with a value of 29 mm and 38 mm. The presence of characteristic peaks in Fourier-transform infrared spectra reveals that all the desired components are present in the developed nanofibrous mats.

Keywords

Electrospinning turmeric honey antibacterial wound dressing

Introduction

The practice of using medicinal plants to treat human skin including healing of wounds and burn injuries, antifungal, antiviral, antibacterial applications against skin infections has long history and is going on for primary healthcare even in the modern age [1]. Usually, wound dressing materials are broadly used for the treatment of skin wounds which are basically prepared from the textile fabric incorporating metal nanoparticles to impart antibacterial or other desirable properties. The requirements including maintaining a local moist environment, protect the wound from side-infection, minimize the wound surface necrosis, prevent the wound dryness, biocompatibility and biodegradability are some notable criteria for a material to be used as wound dressing materials [2 –4]. Due to the usage of metal nanoparticles in the existing wound dressing materials, formation of similar materials using natural herbs like Curcuma turmeric, honey, nigella, and so on has great significance because of their inherent medicinal properties. As a result, the development of electrospun nanofibrous mats by electrospinning technique from natural polymers as novel materials in biomedical applications, such as wound dressing, tissue engineering and drug delivery, leads to contemporary research field both in academic and industrial sectors for their biodegradability, biocompatibility, low toxicity, high porosity, light weight, excellent pore interconnectivity and most importantly the presence of a large surface area [5 –10]. The emulsions made for electrospinning contain a water phase which may dissolve bioactive protein, hydrophilic drug and biomaterial, while the oily phase is a polymer that is biocompatible, biodegradable and soluble in organic solvent [11]. The large surface of nanofibrous mat as well as their porous structure favors a cell adhesion, proliferation and differentiation [6,12 –14]. That is why a wound dressing electrospun nanofibrous mat can meet the requirements such as higher gas permeation and protection of wound from infection and dehydration. There are many available metal nanoparticles that are used to deter bacterial affect, but the harmful effects of these nanoparticles and manmade antibiotics on the environment, human health, have triggered the use of natural antimicrobial compounds which are known to be nontoxic, environmentally sustainable and less likely to create resistant bacteria [15 –18].

The importance of biodegradable-biocompatible polymers such as polyvinyl alcohol (PVA), alginate, starch and chitosan or their derivatives has grown significantly over the past two decades due to their renewable and desirable biological properties [19]. These days PVA, being usually used as a carrier polymer during electrospinning, is one of the most frequent and synthetic polymer hydrogels because of its good biocompatibility finds applications in an advanced biomedical field including wound dressing and wound management [20], drug delivery system, artificial organs [21] and so on. Besides, because of its hydrogel-forming properties and ability to provide controlled release of antibiotics, PVA has turned out as a potential wound dressing material [22]. The permeability of PVA to small molecules, low interfacial tension, soft consistency and transparency also contribute toward its promising application for wound dressing purpose [23].

Turmeric (Curcuma longa) is a tropical herb of the Zingiberaceae family [24] and being used in essential medicine as a carminative, anthelmintic, laxative and as a cure for liver ailment [25]. Turmeric contains protein (6.3%), fat (5.1%), minerals (3.5%), carbohydrates (69.4%) and moisture (13.1%). The main bioactive component of turmeric is curcumin (diferuloylmethane) (3–4%) which is responsible for yellow color and comprises curcumin I (94%), curcumin II (6%) and curcumin III (0.3%) [26] leading to a wide to spectrum of biological actions. This bioactive component includes its various healing properties such as anti-inflammatory, antioxidant, anticarcinogenic, antimutagenic, anticoagulant, antifertility, antidiabetic, antibacterial, antifungal, antiprtozoal, antiviral, antifibrotic, antivenom, antiulcer, hypotensive and hypocholestermic activities [27]. The turmeric powder is also used in diabetic wounds, hepatic disorders [28] and has effects on both aseptic and septic wounds in rats and rabbits [29]. The ethanol extracts of curcumin have a highly anti-inflammatory activity against carrageenan-induced rat paw edema [30,31]. Curcumin has been shown as antibacterial antiamoebic [32,33] and also reported as anticarcinogenic [34,35]. Both turmeric and curcumin decrease blood sugar level in alloxan-induced diabetes in rat [36]. Moreover, the extract of turmeric holds antimicrobial activity [37 –39]. Turmeric has been described as a cancer remedy in Indian medical literature because it shows cytotoxic activity to mammalian cell that is effective in reducing animal tumors, indicating its potential for use to cancer treatment [40]. It has also been used in traditional medicine as a household remedy for various diseases, for instance, biliary disorders, anorexia, cough, diabetic wounds and hepatic disorders.

The prominent wound healing as well as antibacterial properties of honey has made it another potential candidate for wound treatment of skin [41]. Honey has long been used for injury dressing [42] and has significant therapeutic and dietary properties as it demonstrates antimicrobial action, deriding and freshening up activity against inflammation [43], cell reinforcement and wound recuperating activities [44]. The physical properties of honey impact the injury mending condition and the recuperating procedure, particularly on the grounds that honey is acidic and has pH of around 3.2–5.0 [45]. Honey being rich in sugar, however, its low water content and acidic nature disfavor microbial development. Honey produces hydrogen peroxide with the actuating of the catalyst glucose oxidase, which oxidizes glucose to gluconic acid and hydrogen peroxide [46]. Utilization of sugar to enhance wound mending has been accounted for a few hundred patients [47]. In addition, honey has been viewed as having explicit antibacterial properties against Staphylococcus aureus [48,49]. Honey’s antibacterial movement is halfway because of high osmolarity due to its sugar content. Honey makes a wet environment by attracting exudate to the injury surface, making a non-adherent interface between the dressing and wound bed [50].

The formation of nanofibrous mats from the individual solution system of turmeric extract and honey mixing with different synthetic and natural ingredients has been enriched by several studies [51 –53], but their synergistic effects on nanofibrous mat has not yet been studied thoroughly. Thus, the newness of the present study is to fabricate nanofibrous mat with honey and turmeric extract to explore their combined medicinal properties.

Experimental

Materials

First, turmeric was collected from local market in Gazipur city, Bangladesh. Locally produced pure honey was purchased from Sawpna super shop located in Gazipur, Bangladesh. PVA with molecular weight (MW) of 115,000, Degree of polymerization (DP) of 1700–1800, viscosity: 26–32 cps, 99% hydrolyzed granules were sourced from Loba Chemical Pvt. Ltd. (India). Reagent grade acetic acid (99.7%) and ethyl acetate with purity of 99% were purchased from Merck, Germany. All the chemicals and solvents were used without further purification.

Preparation of turmeric extract

Prior to extraction, turmeric was checked to ensure that materials are free from dirt and impurities. Then, a small piece of turmeric was oven-dried at 45°C and was ground using an electronic grinder at room temperature. The powder samples were soaked three times in ethyl acetate overnight at a ratio of 1:10 (w/v%) [54]. The crude extract of turmeric was then filtered through nylon mesh three times. Finally, the solvent was removed from the turmeric extracts at 40°C by magnetic stirrer and was kept it at 4°C for further use.

Preparation of PVA solution

PVA (10 g) was mixed with 90 mL deionized water to obtain 10 wt % (w/v) solution. The mixture was stirred and heated to 80°C to get a clear and transparent solution.

Preparation of electrospinning solution

Two electrospinning solutions were prepared to develop nanofibrous mat from the mixer of PVA-honey-turmeric at two mixing ratios. First solution being identified as CL-1 was prepared by adding 1 g of turmeric extract to 10 mL honey and 20 mL PVA solution and stirred at warming condition until a homogeneous solution is obtained. Another solution being identified as CL-2 was made by adding 2 g turmeric extract to 15 mL PVA and 15 mL honey and stirred as before to make it homogeneous. After preparation, both solutions were cooled to at room temperature. Although it is possible to produce nanofibrous mats using less than 1 g and more than 2 g turmeric extract, they are less potential and sticky which render their applications. Besides, the nanomat which contains more extract provides more antibacterial actions.

Electrospinning process

The prepared two solutions were loaded into a pump of electrospinning machine (model: TL-Pro-BM, company: Tong Li Tech, origin: China). The electrospining setup consists of a high voltage supply (–20 kV and +50 V), a syringe pump (TL-F6, Tong Li Tech, China), a rotary drum collector (diameter: 158 mm, length: 500 mm, 500 r/min), a syringe (30 mL), a heater (0.5 kW) and 5 needles (20 Gauge). The collector drum was wrapped with aluminium foil upon which nanomats was collected and connected with negative voltage. Optimum electrospinning condition was adjusted by trial and error method at ambient condition of 65% relative humidity and at 27°C.

Characterization

Morphological analysis

The orientation of nanofibers along with their diameter was measured by scanning electron microscope (SU 1510, Hitachi, Japan) at different magnifications. At least five different positions on the fibrous mats were captured to determine the diameter of the electrospun fibers. The fiber diameters were analyzed where 30 fibers were taken as a sample and frequency was measured with their particular diameter.

Measurement of moisture management properties

Moisture management properties of the developed mats were evaluated by a moisture management tester (MMT; M290, SDL Atlas, UK) according to the method of AATCC 195-2009. Wetting time, absorption rate, maximum wetted radius and spreading speed of inner and outer surface including accumulative one-way transport capacity (R) and overall moisture management capacity (OMMC) were evaluated following the above-mentioned standard to categorize the mat according to its interaction with liquid. The test was conducted using a saline solution which consists of 0.9% sodium chloride and 120-s measuring time to evaluate the moisture properties.

Antibacterial assay

Antibacterial activity of the electrospun nanofibrous mat was studied using disc diffusion method against S. aureus bacteria (as Gram-negative like Escherichia coli is less and Gram-positive like S. aureus is more responsible for wound infection) and the zone of inhibition was measured. A qualitative disk diffusion test using 1.5 × 10⁵ colony forming units (CFUs)/mL of the S. aureus was cultured on TSA plates while PVA nanomat was used as a control. The sample for the study was prepared by pelletizing the nanofibrous mat of 13 mm diameter disc on the agar plate and placed in the incubator overnight at 37°C. Next, the zone of inhibition formed by sample was measured.

Fourier-transform infrared spectroscopy analysis

The chemical structure of PVA nanofiber, CL-1 and CL-2 was characterized using Fourier-transform infrared spectroscopy (FTIR; IRPrestige21, Shimadzu Corporation, Japan). The spectra of the samples were recorded at 698–4000cm⁻¹ range with 4 cm⁻¹ resolution.

Result and discussion

Parameters optimization and morphological observation

The parameters of electrospinning were considered as voltage, pressure, ambient condition, heater power and collector distance. The optimum electrospinning conditions were achieved by trial and error method at –12.3 kV, +23 kV, 0.45 kW with a flow rate of 1.5 mL per hour under ambient condition of 65% relative humidity and at 27°C, respectively. No fiber is formed when the applied voltage is less than 12 kV because it cannot overcome the surface tension value of the solution which restricts the solution to flow towards the collector. Hence, a higher than 12 kV voltage difference was maintained for all samples. However, a constant collector distance was maintained (which is 15 cm for all samples) as collector distance affect the diameter of fibers. The scanning electron microscope (SEM) images of the developed samples are shown in Figure 1 which reflects the formation of smooth nanofiber for all samples having an average diameter of 340 nm and Figure 2 indicates for route of forming mats.

Figure 1.

SEM images at (a) 5 kV, ×5k; (b) 5 kV, ×10k; (c) 15 kV, ×5K and (d) fiber diameter [55] distributions with respective frequency.

Figure 2.

Sequence of developed nanofibrous mats. PVA: polyvinyl alcohol.

Analysis of moisture management properties

Along with other properties, the moisture behavior of a nanofibrous mat has great importance to be used as wound dressing materials since a moist condition is necessary for wound healing purpose. Hence, it is important to evaluate the capacity for transferring liquids from microclimate to external environment. The results of moisture management properties of PVA nanomat, CL-1 and CL-2 have been presented in Figure 3. The wetting time (s) of the PVA, CL-1 and CL-2 shows the same pattern in both surface of the mats which may be due to their favorable interaction with liquids. But in case of inner surface, i.e., the side of the mat which is in touch with skin, the absorption rate, wetted radius and spreading speed is higher for CL-1 and CL-2. This may be attributed to the reduction in adhesive property of the PVA polymer because of mixing honey and turmeric extract. The greater spreading speed indicates their fast absorbing nature which is very crucial for wound healing activity. This nature of the developed mat can prevent it from being wetted very quickly due to the absorption of liquids from wound area. On the other hand, in case of outer surface (which is exposed to external environment); the result of all above-mentioned parameters follow the same pattern excluding the wetted radius for CL-2. This may be happening due to poor one-way transport capacity of CL-2 which might arose due to the higher content of honey and turmeric extract. However, the value of one-way transport capacity and OMMC has been calculated using all parameters that indicate that the PVA mat is poor in moisture transport property and thus should be considered as water repellent whereas CL-1 and CL-2 have been considered as fast absorbing and slow drying as well as fast absorbing and quick drying fabric, respectively. This fast absorbing nature will allow them to absorb the wound liquid very quickly from the wound area and transfer it to outer surface leading to enhanced healing performances.

Figure 3.

Water location and time diagram (a to c) and quantitative grading of moisture behavior of PVA nanomat, CL-1 and CL-2 (a1 to c1). PVA: polyvinyl alcohol.

Antibacterial assay

The bacterial invasion into human tissue and cells is one of the most remarkable mechanisms in skin infection [56]. Among all, S. aureus is one of the Gram-positive pathogens cause various infections including infective endocarditis [19], bacteremia, skin and soft tissue, osteoarticular, pleuropulmonary infections and susceptible to curcumin mediated inhibition. The determination of inhibition zones by disc diffusion method of CL-1 and Cl-2 exhibits the outstanding antibacterial activities against S. aureus as shown in Figure 4 through the formation of inhibition zone of 29 mm and 38 mm, respectively, while no inhibition zone was found for PVA nanomat for same the strain (Table 1). This formation of inhibition zone indicates the vital role of honey and turmeric extract toward bacteria killing. The antibacterial property of honey is attributed to its high osmolarity, acidity (low pH) and the presence of hydrogen peroxide (H₂O₂) as well as non-peroxide components, i.e., the presence of phytochemical components like methylglyoxal where hydrogen peroxide is the predominant antimicrobial constituent [41]. Most types of honey generate H₂O₂ when diluted, because of the activation of the enzyme glucose oxidase that oxidizes glucose to gluconic acid and H₂O₂, which thus confers the antimicrobial activity. In addition to H₂O₂, several other non-peroxide factors are responsible for the unique antibacterial activity of honey [13] such as the presence of methyl syringate and methylglyoxal, which are largely present in manuka honey that is derived from the manuka tree (Leptospermum scoparium). Honey is characteristically acidic with pH between 3.2 and 4.5, which is low enough to be inhibitory to several bacterial pathogens. The antibacterial property of honey is also derived from the osmotic effect of its high sugar content and low moisture content, along with its acidic properties of gluconic acid and the antiseptic properties of its H₂O₂ [42,57,58]. On the other hand, Curcumin which is present in turmeric extract interacts with FtsZ (prokaryotic homologue of eukaryotic cytoskeletal protein tubulin) and inhibits bacterial cell proliferation. It binds to peptidoglycan (PGN), and an increasing concentration of PGN blocks the curcumin antibacterial activity (Figure 5). Curcumin also inhibits the growth of S. aureus by perturbing the bacterial membrane integrity [59]. The dimension of inhibition zone for CL-2 is higher than CL-1 which is undoubtedly due to the increased amount of honey and turmeric extract since both have significant antibacterial properties [27,60]. Along with bacterial resistance, the mesho structures of the developed nanomat can prevent the penetration of any bacteria because of the presence of tiny pores and thus can nullify the surrounding infections effectively. The obtained results are resembled with previous results and analysis [61,62].

Figure 4.

Formation of zone of inhibition of (a) PVA nanofiber and (b) CL-1 and CL-2.

Table 1.

Antibacterial activity (zone of inhibition) of the samples.

Samples code	ZOI	Bacteria name
PVA nanomat	No ZOI	Staphylococcus aureus
CL-1	29 mm
CL-2	38 mm

PVA: polyvinyl alcohol; ZOI: zone of inhibition.

Figure 5.

Mechanism of killing S. aureus by curcumin [59].

Fourier-transform infrared spectroscopy

Infrared spectroscopy confirmed the presence of functional group of PVA, honey and turmeric extract in the developed sample as shown in Figure 6. The functional group region is identified in the range of 4000–1450cm⁻¹ whereas the finger print region corresponds to the region of 1450–500 cm⁻¹.

Figure 6.

FTIR spectra of CL-1 and CL-2, respectively.

Besides the attributable peaks have been found at 3433 cm⁻¹ (O–H stretching), 2927 cm⁻¹ (C–H stretching) and 1095 cm⁻¹ (C–O–C stretching) in case of PVA nanofiber. This is in agreement with the signature peaks of PVA as reported in previous studies [63]. The band spectra at 1600–1650 cm⁻¹ and 1510 cm⁻¹ show the presence of carbonyl group and the ethylene group of turmeric extract. The peaks at 725 cm⁻¹, 817 cm⁻¹ and 967 cm⁻¹ indicated the bending vibrations of –CH bond of alkene group. Besides, the peak at 1250 cm⁻¹ corresponds to C–O stretching of ether group in curcumin. The peak around 1500–1400 cm⁻¹ indicated the –C–O elongation frequency of –OH groups in curcumin [64]. Without this, the band spectra at 1270 cm⁻¹ corresponds to C–O stretching of honey [65].

Conclusion

In this research, the optimum processing condition for the fabrication of PVA–honey nanofibrous mats laden with turmeric extract have been presented. Here, samples were developed through electrospinning method at two mixing ratios and then they were characterized. SEM, MMT, antibacterial assay and FTIR were used to characterize the prepared nanofibrous mats. The result of FTIR confirmed a presence of PVA, honey and turmeric extract compounds by showing their characteristic peaks. The PVA–honey nanofibrous mats loaded with turmeric extract exhibited enhanced moisture management properties. Due to the presence of antibacterial constituents of honey and turmeric extract, the developed samples showed the formation of inhibition of 29 mm and 38 mm, whereas no inhibition zone was formed for PVA nanofiber alone for the same bacteria. These properties indicate that the developed nanomats could be used as potential wound dressing materials and in tissue engineering. This multicomponent combination could lead to a wide range of applications in various engineering fields.

Footnotes

Acknowledgements

The authors are grateful to the Department of Textile Engineering, Dhaka University of Engineering and Technology, Gazipur, for permitting to use its lab facility. A special gratitude is also extended to the Department of Biochemistry, University of Dhaka for providing antibacterial test facilities for this research.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest regarding this research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Md. Abdus Shahid

Md. Nur Uddin

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Antibacterial wound dressing electrospun nanofibrous material from polyvinyl alcohol,honey and Curcumin longa extract

Abstract

Keywords

Introduction

Experimental

Materials

Preparation of turmeric extract

Preparation of PVA solution

Preparation of electrospinning solution

Electrospinning process

Characterization

Morphological analysis

Measurement of moisture management properties

Antibacterial assay

Fourier-transform infrared spectroscopy analysis

Result and discussion

Parameters optimization and morphological observation

Analysis of moisture management properties

Antibacterial assay

Fourier-transform infrared spectroscopy

Conclusion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

ORCID iDs

References