Development and characterization of Abutilon indicum (Indian mallow) leaf extract loaded electrospun nanofibres

Abstract

Herbal functionalized nanofibres are widely used in wound healing, tissue engineering, and air filters due to their efficient antimicrobial properties. Spinning nanofibres using herbs requires initial studies to check the compatibility of the herb, nanofibre formation, and spinnability. Efforts are being made to spin nanofibres using Abutilon indicum. Nanofibres produced using electrospinning techniques excel in unique structural and functional properties. Their structure aids in encapsulating active substances and controlled release of these substances. Abutilon indicum, also known as Indian mallow or Thuthi leaf, is a plant with various medicinal claims and antimicrobial properties. The current study focuses on developing nanofibres using the electrospinning technique with Polyvinyl Alcohol (PVA) polymer and Indian mallow extract (IME). Nanofibres were developed with PVA/IME in different proportions. The developed nanofibres were characterized for surface morphology, Fourier-transform infrared spectrum, material thickness, swelling ratio, tensile strength, and antimicrobial property. The addition of IME to PVA did not affect the spinnability of the nanofibres. Among the samples developed with different ratios, the 50:50 PVA/IME sample was observed to have enhanced antimicrobial activity due to the higher proportion of IME within the polymer.

Keywords

Nanospinning herbal antibacterial Indian mallow Thuthi leaf

Introduction

Nanofibres are widely researched due to their diameter being 1000 times smaller than human hair, offering better flexibility, porosity, stiffness, and tensile strength.¹ Natural polymers based on plant and animal sources, such as alginate, silk fibroin, and chitosan, have been used in the production of nanofibres.² Similarly, synthetic polymers like Polyvinyl Alcohol (PVA), polycaprolactone, and polyvinylpyrrolidone have also been utilized in nanofibre production. Nanofibres are produced using various techniques, including electrospinning, bicomponent spinning, melt-blowing, and flash spinning.³ Among these, electrospinning is the most versatile and cost-effective nanofibre production technique. Invented in the 1930s, electrospinning can produce fibers with diameters of less than 1 µm. With the growth of nanotechnology over the past four decades, nanofibres have gained significant potential.^4,5 In electrospinning, nanofibres are formed by the application of an electrostatic force.

The methods of electrospinning include blend electrospinning, side-by-side electrospinning, emulsion electrospinning, and co-axial electrospinning. Blend electrospinning is the simplest among these methods. In this method, drug encapsulation is achieved by dispersing or dissolving the drug in the polymeric solution. The encapsulation of the drug within the polymer is highly influenced by the interaction of the drug with the polymer, the solubility of the drug, and drug migration within the fiber.

Janus nanofibres are formed using a side-by-side nanospinning setup, producing nanofibres with two different sides. By altering the structure of the spinneret, the interface areas, width, etc., can be controlled in this technique; however, fluid compatibility is the most challenging aspect. Core-sheath nanostructures can be successfully produced by emulsion and co-axial electrospinning. These setups produce hybrid nanofibres with high drug loading efficiency. In emulsion spinning, immiscible bioactive compounds are emulsified into a solution using surfactants and later mixed with a polymer solution.⁶

PVA polymer is water-soluble and serves as an excellent driving polymer in the electrospinning process. It is a synthetic semi-crystalline polymer derived from the hydrolysis of polyvinyl acetate. PVA possesses remarkable properties, including good mechanical strength, ease of processing, biocompatibility, gas permeability, and favorable thermal characteristics. Its non-toxic nature makes it widely accepted for medical and filtration applications.⁷ In electrospinning, PVA produces high-quality nanofibres. Its biocompatibility, drug compatibility, good swelling properties, mechanical strength, and film-forming abilities make it an ideal polymer for nanofibre fabrication through electrospinning.^8,9

Herbal functionalized nanofibres are currently being researched for their enhanced antimicrobial properties, making them effective for medical and filtration applications.^10,11 Incorporating herbal extracts into PVA nanofibres improves their antimicrobial capabilities. One such herb, Abutilon indicum (commonly known as Indian mallow or Thuthi), belongs to the Malvaceae family.^12,13

Abutilon indicum is traditionally used to treat various ailments.¹³ Different parts of the plant, such as the root, seed, leaves, and bark, have medicinal applications. The edible leaves are applied to wounds and boils to promote rapid healing and their extract serves as a demulcent and diuretic. Crushed leaves mixed with water are used as a mouthwash to treat tooth and gum issues and are also beneficial for vaginal infections. The plant’s roots are known to clear chest infections, while the flowers are consumed directly to treat ulcers, colds, and coughs.¹⁴ The current study aims to develop PVA nanofibres functionalized with Indian mallow herbal extract in different proportions and to analyze their morphology, mechanical properties, and antimicrobial activity.

Materials and methods

Herbal and chemicals

The Indian Mallow leaf powder was sourced from the southern parts of Tamil Nadu. The Polyvinyl alcohol polymer (Degree of polymerization: 1700–1800; Degree of hydrolysis: 98–99 mol %) was obtained from Isochem Laboratories, India.

Preparation of IME

The stock solution of the IME was prepared by mixing 3 g of Indian mallow leaf powder with 10 ml of ethanol to achieve a 30% w/v concentration. The solution was stirred with a magnetic stirrer at 250 rpm at room temperature for 2 h, as shown in Figure 1. The leaf extract was filtered using filter paper and stored for further use without any modifications.

Preparation of PVA

Polyvinyl Alcohol (PVA) powder was mixed with distilled water and stirred using a magnetic stirrer at 250 rpm and 90°C for 2 h. The resulting mixture was used without any further modifications.

Preparation of nanofibre

Nanofibre mats were prepared using the electrospinning technique with a PSG Electrospinning Machine, manufactured by PSG Industrial Institute, Coimbatore, India, as shown in Figure 1. The polymer blend solution (PVA/IME) was mixed in volume ratios of 100/0, 90/10, 80/20, 70/30, and 50/50. A 10 ml solution was taken in a syringe. The distance between the needle tip and the collector was set to 18 cm to achieve a uniform nanosheet. A voltage of 21 kV was used for the current study, as no nanofibre formation occurred below this voltage. A feed rate of 0.1 ml/h was set to obtain the nanofibre sheet.

Characterization

Dynamic light scattering

The particle size of Indian mallow was measured using a Malvern Panalytical Zetasizer Nano ZS, United Kingdom. The particles were dispersed in methanol, and the movement of the dispersed particles in methanol was combined with light scattering to measure the particle size using the Malvern Panalytical Zetasizer Nano ZS.¹⁵

Surface morphology

Morphological studies of the prepared nanofibre sheets were conducted using a Field Emission Scanning Electron Microscope (FESEM) by coating the nanofibres with gold. The FESEM used for the analysis of the nanofibres was manufactured by CARL ZEISS (USA). The structure of the nanofibre sheets was analyzed.¹⁶

Fourier-transform infrared spectroscopy (FTIR)

FTIR analysis was conducted using a Horiba FluroMax 4L spectrofluorometer, which has a maximum resolution of 0.7 cm⁻¹ and a wavenumber range from 7800 to 350 cm⁻¹. The functional groups present in the PVA/IM nanofibres were identified using the ASTM E168 test method.

Thickness test

The thickness of the nanofibrous mat was measured using a digital thickness meter with a Workzone digital coating thickness gage from India. The probe of the thickness meter was placed around the material, and the thickness was read digitally.¹⁷

Swelling ratio

The swelling ratio of the nanofibres was measured using the JIS K8150 test method. The nanofibrous sheets were cut into squares with dimensions of 20 mm × 20 mm and weighed in their dry state. The weighed material was immersed in 30 ml of distilled water for 30 min at room temperature. After 30 min, the nanofibres were placed on a filter paper to remove the water adhered on the surface before weighing. The swelling ratio was calculated using equation (1).¹⁸

S w e l l i n g r a t i o = \frac{(W_{w e t} - W_{d r y})}{W_{d r y}}

(1)

Where. W_wet is wet weight of the nanofibre immersed in distilled water, W_dry is the dry weight of the nanofibre immersed in distilled water.

Tensile test

The tensile strength of the nanofibrous sheets was analyzed using an Instron Universal Testing Machine, India, which has a maximum load capacity of 100 N for testing the nanofibres. The nanofibrous samples were cut into rectangular shapes measuring 50 mm × 10 mm. The samples were clamped between the jaws of the Universal Testing Machine, and they were slowly extended until they fractured. The elongation was measured against the applied load.

Antimicrobial test

The antimicrobial property of the nanofibres was evaluated using the disk diffusion method (AATCC 147). The Gram-negative bacterium Escherichia coli (E. coli) was used to assess the antibacterial property of the nanofibre mats. The sample size of the nanofibre mats for the disk diffusion method was 8 mm × 8 mm. The developed PVA/IME nanofibre mats were placed on the surface of agar plates inoculated with E. coli and were incubated overnight at 37°C. The zone of inhibition was measured for all the incubated samples.

Result and discussion

Indian mallow particle size

Indian mallow leaf powder dispersed in methanol was analyzed using Dynamic Light Scattering (DLS) spectroscopy and was found to have an average particle size of 3618 nm. The DLS histogram of leaf powder is shown in Figure 2.

Surface morphology

The SEM images of the developed nanofibres are shown in Figure 3. The images indicate that 100% PVA, 90:10, and 80:20 proportions of PVA/IM nanofibres produced more uniform and bead-free nanofibres compared to the 70:30 and 50:50 PVA/IM nanofibres, which exhibited one or two beads in their structure. Therefore, the presence of Indian mallow leaf extract in the nanofibres does not affect the electrospinnability of the nanofibres; however, the morphology of the nanofibres is slightly affected by the presence of Indian mallow leaf extract. Fiber diameter were studied using Image software (Image J, National Institutes of Health, USA).⁹ Approximately 100 random measurements of the fiber diameter were taken from the SEM images. The average diameter of the developed nanofibres is presented in Table 1.

Table 1.

Average diameter of the developed nanofibers.

Sample	Average diameter (nm)
100% PVA	206 ± 23.52
90:10 PVA/IME	307 ± 25.73
80:20 PVA/IME	278 ± 31.71
70:30 PVA/IME	252 ± 45.61
50:50 PVA/IME	273 ± 24.22

PVA: polyvinyl alcohol; IME: Indian mallow extract; SD: standard deviation.

Data expressed as mean + SD (n = 100).

It can be inferred from Figure 3 and Table 1 that the 90:10 PVA/IM nanofibres exhibited a maximum average diameter of 307 nm. However, the diameter decreased with the increase in the amount of Indian mallow leaf extract, likely due to the higher conductivity of the polymer solution in the presence of the leaf extract. In the case of 50:50 PVA/IM nanofibres, the diameter slightly increased compared to the 90:10, 80:20, and 70:30 PVA/IM nanofibres, possibly due to the formation of beads in the nanofibrous structure. Among the five nanofibres analyzed, 100% PVA showed the smallest average diameter of 206 nm. Since the diameter of nanofibres affects their surface area, smaller diameters result in a larger surface area. The 70:30 and 50:50 PVA/IM nanofibres exhibited smaller diameters compared to the others.

Fourier-transform infrared spectrum

The FTIR spectrum for Indian Mallow, PVA and PVA/IM nanofibres is shown in Figure 4.

In PVA, a broad peak at around 3323.17 cm⁻¹ corresponds to -OH stretching vibration of hydroxyl group. A peak at 2916.40 cm⁻¹ is attributed to CH₂ asymmetric vibration. A weak peak at around 2851.72 cm⁻¹ indicates C-H stretching vibration. A medium peak at 1730.58 cm⁻¹ shows C = O carbonyl stretch. Two weak peaks at 1427.30 and 1374.11 cm⁻¹ corresponds to C-H bending vibration of CH₂ and C-H deformation vibration respectively. Peak value of 1246.19 cm⁻¹ shows C-O stretching vibration. Peaks at 1090.95 and 843.73 cm⁻¹ are attributed to C-O stretching vibration of acetyl group and C-C stretching vibrations respectively.^19,20

In Indian Mallow leaf powder, a weak peak at 2922.15 cm⁻¹ shows C-H stretching vibration in alkanes group. Peak value of 1732.02 cm⁻¹ corresponds to carbonyl groups which is mainly associated with amides and they are the attributes of proteins and enzymes. Peak at 1320.93, 1237.56, 1032.02 and 796.91 cm⁻¹ indicates C-H stretching of alkanes group, esters, sulfur compounds, mono substituted alkanes respectively.

In PVA / IM nanofibre, a broad peak at 3323.17 cm⁻¹ indicates –OH stretching vibration found in PVA. Peak value of 2922.15 cm⁻¹ corresponds to C-H stretching vibration of Indian Mallow. A peak at around 2851.72 cm⁻¹ indicates C-H stretching vibration in PVA. Carbonyl group is present at the peak value 1732.02 cm⁻¹ which conforms the presence of both PVA and Indian Mallow. Peak Values of 1320.93, 1237.56, 1032.02, 896.91 cm⁻¹ are attributes to C-H stretching vibration, esters, sulfur compounds, mono substituted alkanes respectively. The presence of these compounds is seen in Indian Mallow. Hence, the FTIR spectrum results conveys the presence of Indian Mallow extract in PVA/IM nanofibre.

Thickness

The thickness of the nanofibrous mats was measured at three different regions, and the average value is shown in Table 2. The thickness of the nanofibrous mat is determined by the electrospinning time and the type of collector used. Drum-type collectors provide more uniformly oriented fibers compared to plate-type collectors. In this research work, a plate-type collector was used. The nanofibres obtained were randomly oriented, leading to variations in thickness between the central and corner regions. According to the test results, the 80:20 PVA/IM nanofibre sample had the highest thickness value, while the 50:50 PVA/IM nanofibre sample had the lowest thickness value due to the presence of Indian mallow within the PVA polymer.

Table 2.

Characteristics of nanofibre mats.

Sample	Thickness (mm)	Swelling ratio (%)	Load at break (N)	Tensile strength (MPa)	Modulus (MPa)
100% PVA	0.156 ± 0.04	183 ± 3.61	0.640 ± 0.02	3.020 ± 0.10	17.491 ± 0.86
90:10 PVA/IME	0.123 ± 0.11	250 ± 3.06	0.019 ± 0.03	4.101 ± 0.11	7.760 ± 0.23
80:20 PVA/IME	0.093 ± 0.08	350 ± 2.65	7.055 ± 0.58	3.257 ± 0.14	39.563 ± 1.05
70:30 PVA/IME	0.076 ± 0.02	425 ± 3.21	3.964 ± 0.27	3.360 ± 0.37	5.484 ± 0.30
50:50 PVA/IME	0.073 ± 0.33	525 ± 4.58	5.343 ± 0.51	2.480 ± 0.09	11.204 ± 0.65

PVA: polyvinyl alcohol; IME: Indian mallow extract; SD: standard deviation.

Data expressed as mean + SD.

Swelling ratio

The average swelling ratio measured with three samples of the nanofibre samples developed is shown in Table 2. The swelling ratio increases with an increase in Indian mallow extract in proportion to PVA. The 90:10 PVA/IM nanofibres show a swelling ratio of 250% and gradually increase by 2.1 times for the 50:50 PVA/IM nanofibres. The -OH groups of PVA are hydrophilic and can absorb more water. The Indian mallow leaf powder has polar groups, like esters, which contribute to the hydrophilicity of the samples. The swelling ratio results indicate that the 50:50 PVA/IM nanofibre has high hydrophilic areas for water absorption.

Tensile behavior

The tensile behavior measured with three samples of the nanofibre samples developed is shown in Table 2. The mechanical behavior of nanofibrous mats is an important parameter that conveys the material’s response to the application of load or force. The load-extension behavior of the developed nanofibres is shown in Figure 5. The figure shows that the tensile property increases with the increase in Indian mallow extract (IME) up to 80:20 PVA/IM nanofibres, followed by a gradual decrease. This may be due to the presence of IM randomly distributed between the continuous polymeric chains. The tensile strength and modulus of 100% PVA nanofibre and PVA/IM nanofibre mats are key characteristics. Among them, the 80:20 PVA/IM nanofibres exhibited a high tensile strength of 7.055 N, while the 90:10 PVA/IM nanofibres showed the lowest tensile strength of 0.019 N.

Antimicrobial activity

An antimicrobial test was carried out for 100% PVA, 90:10, 80:20, 70:30, and 50:50 PVA/IME nanofibrous mats. The test was conducted with a gram-negative bacteria called Escherichia coli as thuthi does not have antimicrobial activity against gram-positive bacteria. The zone of inhibition was measured and recorded in millimeters.²¹ The zone of inhibition caused by Escherichia coli is shown in Table 3. Among the test results, 100% PVA had no zone of inhibition. The 50:50 PVA/IM nanofibres showed the highest zone of inhibition of about 23 mm, as shown in Figure 6. This may be due to the increase in the amount of Indian mallow leaf extract in the nanofibres, which contributes to the increase in antibacterial activity.

Table 3.

Antibacterial property of nanofibre mats.

Sample	Antimicrobial zone of inhibition (mm)
100% PVA	No zone
90:10 PVA/IME	10 ± 0.06
80:20 PVA/IME	20 ± 0.10
70:30 PVA/IME	22 ± 0.15
50:50 PVA/IME	23 ± 0.61

PVA: polyvinyl alcohol; IME: Indian mallow extract; SD: standard deviation.

Data expressed as mean + SD.

Conclusion

The PVA/IM nanofibre mats were successfully fabricated using the electrospinning technique. The addition of IM extract to PVA did not affect the spinnability of the nanofibres. The fabricated 80:20 PVA/IM nanofibre mat was superior in terms of mechanical and antimicrobial properties compared to other proportions. The 50:50 PVA/IM nanofibre mat exhibited the highest zone of inhibition for gram-negative E. coli bacteria. Due to their fiber surface morphology, strength, and microbial properties, the fabricated nanofibre mats can be used in medical and filtration applications.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the 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 iD

S. Tharakeswari

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