Non-wettable antibacterial thin film: PS/ Aloe vera and PS/ Acalypha indica

Abstract

The water and liquid repelling materials due to their low surface energy and specific nanometer and micrometer scale roughness have particular interest due to the great variety of potential applications ranging from self-cleaning surfaces to microfluidic devices. Natural plant-based polymer hydrophobic coatings have several engineering and biomedical applications. In this study, natural plants such as Aloe vera and Acalypha indica were used for preparation of polystyrene hydrophobic film by the dip-coating method. The effects of Aloe vera and Acalypha indica extract on the morphological, structural, optical and antibacterial properties of as-prepared polystyrene thin films were studied. FTIR spectra were carried out to ascertain the presence of functional groups in the prepared polymeric film. Contact angle measurement showed that the PS/Aloe vera has higher anti-wetting property (CA = 112°) compared to PS/Acalypha indica (CA = 104°), which could be confirmed by the surface morphology analysis. Furthermore, the effective antibacterial activity against Klebsiella pneumoniae and Staphylococcus aureus was compared by agar-well diffusion method. The observed result reports, PS with Aloe vera film exhibit higher hydrophobic with antibacterial activity compared to PS with Acalypha indica film. In addition, the PS/Aloe vera coated cotton maintained their repellent properties against various liquids for 10 h, while PS/Acalypha indica coated cotton exhibit anti-wetting property for 4 h. These findings open up new avenues for the preparation of hydrophobic film for various biomedical applications.

Introduction

Over the course of time, with the development of civilization and the evolution among new technologies humans began modernizing their lifestyles, which lead to the loss of information about numerous natural products. However, still some plants which are currently used for the purposes as they were in ancient days. Green synthesis of polymer films can overcome the problems related to physical and chemical methods. The preparation of polymer film based on plant extract is one of the cleanest, biocompatible, nontoxic and eco-friendly methods. ¹ Aloe vera and Acalypha indica are such plants, which have many applications in this modern world. ² Aloe vera is the oldest and most used medicinal plant worldwide. It has a short stem with thick green leaves like structure that grows from central point. ³ A cut leaf of Aloe vera reveals an orange-yellow sap from the opening known as the juice. ⁴ The Aloe vera leaf contains clear, gluey mucilaginous substance known as gel. The gel consists of 99.3% water and the remaining 0.7% containing a range of active compounds including polysaccharides, vitamins, amino acids, phenolic compounds and organic acids. ⁵

Acalypha indica is an annual erect herb commonly called “Kuppaimeni.” It belongs to the family Euphorbiaceae. It is a common shrub in Indian gardens, backyards of houses and waste places through the plains of India. The root, stem and leaf of Acalypha indica possess herbal activity. ⁶ Acalypha indica can be used against many diseases such as diarrhea, ⁷ asthma, ⁸ gum-teeth disease, ⁹ mouth ulcer, ¹⁰ and pimples. ¹¹ The fresh Acalypha indica plant has a wide variety of nutrients such as carbohydrates, proteins, vitamins and fat. This plant has high iron content, followed by zinc, copper, nickel and chromium, which are useful for patients with mineral deficiency problems. ⁶ A substantial number of research papers have been published about the use of Aloe vera and Acalypha indica, along with their applications in both synthetic and natural materials for wound healing, drug delivery systems, super-oleophobic and hydrophobic thin films and tissue engineering.^12-18 Researchers have observed that the addition of natural plant has extensively improved the overall efficacy of the resultant product.

The hydrophobicity of surface can be determined from the water contact angle (WCA), which is the angle between the water droplets and the surface of an object on a contact line. If the water contact angle on the surface of the material is more than 90°, the surface of the material is called the hydrophobic surface. The static water contact angle more than 130° and sliding angle less than 10° are known as superhydrophobic surface. ¹⁹ The superhydrophobic behavior of the surface is mainly influenced by two factors such as roughness in micro-nano order and chemical composition.^20-22 Controlled wettability modification of polymer is exceedingly alluring for different applications such as self-cleaning, antireflection, antifogging and other surface coating applications. Recently, surface modification of polymers has been widely investigated. ²³ Polystyrene is one of the most widely used polymers in microfluids and biotechnology applications. The objective of the present work is to prepare a novel polystyrene-based thin film coatings employing a green approach from the leaf extracts of Aloe vera and Acalypha indica precursors. The prepared film effectively coated on cotton pad and then, its functional properties such as hydrophobicity, surface roughness, optical behavior and antibacterial activity have been explored. ¹

Methodology

Materials

Polystyrene with molecular weight of 500,000 purchased from Alfa-Aesar and toluene procured from Sigma-Aldrich was used without further purification. Aloe vera (L. Burm. F. (=Aloe barbadensis Mill.)—LILIACEAE, and Acalypha indica L- EUPPHORBIACEAE were taken from the local area of Coimbatorecity and identified by Botanical Survey of India, Southern regional center, Coimbatore.

Cleaning and drying process of Aloe vera and Acalypha indica

The collected fresh leaves of plants were thoroughly washed with sterile distilled water to remove dirt and their thick epidermis of Aloe vera was then dissected longitudinally into pieces. The colorless parenchymatous tissue (aloe gel) collected in a container and dried in a hot air oven for 7–8 h at 40°C, where the leaves of Acalypha indica are shade dried and powdered.

PS/Aloe vera and PS/Acalypha indica solution preparation

The sol-gel method was used to prepare the PS/Aloe vera and PS/Acalypha indica solutions. To obtain a solution of PS, 1.2 g polystyrene was mixed with 30 ml toluene and stirred for an hour at 60°C.

The Aloe vera and Acalypha indica solutions were obtained by vigorous stirring of 5 g of both plant powders separately in 20 ml toluene for 4 h. The obtained solutions were filtered using Whatman filter paper no.1. To prepare PS/Aloe vera solution, Aloe vera solution was drop-wisely added into the prepared PS solution and kept in ultrasonicator. The solution was stirred for an hour followed by aging process of 1 day at room temperature to obtain homogeneous solution. On the other hand, PS/Acalypha indica solution was prepared by blending of Acalypha indica and PS solution with continuous stirring for an hour followed by 1 day aging. The resulting homogeneous gels were dip-coated on microscopic glass slides and cotton pads. The schematic representation of the experimental procedure adopted to obtain the blend solution is shown in Figure 1.

Figure 1.

Schematic of experimental procedure.

Agar-well diffusion method

The prepared solutions were experimented with two bacterial strains of gram-negative Klebsiella pneumoniae and gram-positive Staphylococcus aureus for antibacterial test by agar-well diffusion method. The bacterial culture was seeded on nutrient agar plate. The wells were bored with 8 mm borer in seeded agar and then 50 µl of all prepared solutions were added in each well. Soon after, the plates were kept at 10°C for 30 min. After the plates normalized to room temperature it was incubated at 37°C for 24 h. Then, the zone of inhibition was measured and recorded. Standard antibiotic disc of ciprofloxacin was used as the reference drug for antibacterial assay and all the data are expressed as mean ± SD (standard deviation).

Results and discussion

Fourier transform infrared spectroscopy

FTIR spectroscopy was carried out to identify the predominant functional groups present in the polymeric film by SHIMADZU JASCO (4600) instrument with wave number range between 400 cm⁻¹ and 4500 cm⁻¹, as shown in Figure 2. The peaks at 3026 cm⁻¹ and 752 cm⁻¹ correspond to C–H stretching. The symmetric and asymmetric stretching vibrations of –CH₂ attributed to the vibration band at 2850 cm⁻¹ and 2926 cm⁻¹. The vibration band at 1805 cm⁻¹ was due to the characteristic of C=O stretching, indicating the carbonyl groups in the Aloe vera sample. ²⁴ The aromatic C=C stretching vibration in the benzene ring of PS appears at 1598 cm⁻¹. ²⁵ The peaks at 1492 cm⁻¹ and 1448 cm⁻¹ correspond to the C–H bending vibration. ²⁶ The absorption peak at 1066 cm⁻¹ represents the C–O stretching associated with rhamnogalacturonam, a side-chain constituent of pectins. ²⁴ The band at 1384 cm⁻¹ was assigned to the primary amine (N–H) bending mode, which is the appropriate characteristic peak of flavonoids and terpenoids present in Acalypha indica plant leaves. The vibration peaks at 1317 cm⁻¹ and 1243 cm⁻¹ signify the carboxylic acid (C–O) group. ²⁷ The major plant and polymer compounds present in the prepared films were confirmed by FTIR study.

Figure 2.

FTIR spectra of (a) Aloe vera blended polystyrene and (b) Acalypha indica blended polystyrene film.

UV-visible analysis

To analyze the absorbance range of the prepared thin films, the UV-Vis-NIR spectra were conducted using JASCO (V670) spectrometer with a wavelength range between 250 nm and 700 nm. Figure 3 shows the UV-visible spectra of the PS/Aloe vera and PS/Acalypha indica film. The optical absorption spectra of PS/Aloe vera exhibited strong absorption below 300 nm. ²⁸ In this study, PS/Acalypha indica film in the UV region at around 418 nm favors the antireflective and UV-adsorption properties of textile applications. The above result revealed that the PS with Aloe vera and Acalypha indica were predominant and exhibit good absorbance, and thus, good optical properties. ²⁷

Figure 3.

UV-visible absorbance spectra of Aloe vera blended polystyrene and Acalypha indica blended polystyrene film.

Antibacterial performance

The qualitative antibacterial activity of the prepared PS/Aloe vera and PS/Acalypha indica solutions were tested by a agar-well diffusion method. The prepared culture plates were inoculated with urinary tract infection pathogen of S. aureus and nosocomial infection pathogen of K. pneumoniae using the streak-plate method. ²⁹ Fresh bacterial inocula was prepared by inoculating loopful of test organisms into nutrient broth, followed by incubation at 37°C for 7–8 h until moderate turbidity was developed. A loop of culture was swabbed onto the Mueller-Hinton agar for testing the antibacterial activity.^1,30,31 The zone of inhibition was seen after 24 h of incubation in the dark at 37°C. Ciprofloxin was used as standard antibiotic for antibacterial activity. Pristine polystyrene thin film does not have any antibacterial effect.^32,33 The water-repellent PS/Aloe vera and PS/Acalypha indica samples demonstrated ability to inhibit the growth of bacteria. However, clear zone of inhibition can be seen around PS treated with Aloe vera and Acalypha indica Figure 4 and the standard deviation of inhibition values are tabulated in Table 1. The well loaded with PS/Aloe vera and PS/Acalypha indica showed the maximum zone of inhibition against K. pneumoniae (13.33 ± 0.13, 11.16 ± 0.20). In contrast, respectively PS/Aloe vera and PS/Acalypha indica exhibited lesser inhibition zone against S. aureus (08.2 ± 0.26, 09 ± 0.21). It showed that the plant extract treated samples have significant zone of inhibition against S. aureus than K. pneumoniae. This result is in accordance with Mahendran et al. have reported fabrication of antibacterial effects of polycarbonate/leaf extract-based thin film. Azadarichta indica, Psidium guajava, Acalypha indica, Andrographis paniculata and Ocimum sanctum leaf extracts were used for this study. In addition, the as-prepared Psidium guajava film exhibited significant antibacterial activity against Escherichia coli and S. aureus compared to Azadarichta indica, Acalypha indica, Andrographis paniculata and Ocimum sanctum-based polycarbonate films.¹⁷ The above assessment showed the natural plant-based polymer film has significant antibacterial activity with lower toxicity.

Figure 4.

Antibacterial activity of Aloe vera blended polystyrene and Acalypha indica blended polystyrene.

Table 1.

Antibacterial activity of PS/Aloe vera and PS/Acalypha indica.

S. no	Micro organisms	Zone of inhibition (mm)
		PS/Aloe vera	PS/Acalypha indica	Control
1.	Klebsiella pneumoniae	13.33 ± 0.13	11.16 ± 0.20	15.10 ± 0.17
2.	Staphylococcus aureus	08.20 ± 0.26	09 ± 0.21	12.13 ± 0.15

Surface anti-wetting properties

Wettability is one of the significant properties of a solid surface, and the contact angle of water droplets on the surface is a direct description of the wettability of a solid.^34,35 Chemically, wetting can occur because the molecules making up the surface tends to be polar. So that there will be a tug-of-war between the surface and H₂Omolecules that are also polar. Hydrophobic material coating can able to modify the surface, making the surface tends to be non-polar. ³⁶ The static contact angle of the surface was measured by Kruss instrument. The contact angle of PS/Aloe vera and PS/Acalypha indica was found to be 112° and 104° and hence the hydrophobic nature of the prepared film is shown in Table 2. The hydrophobicity of a polymer surface is an important property that underlies many applications such as environmentally resistant coatings, waterproof fabrics, low friction devices and inert biomaterials. The effect of surface roughness on hydrophobicity can be explained by two distinct wetting behaviors depending upon the geometrical surface and the extent of roughness.

Table 2.

Roughness, thickness and contact angle of PS/Aloe vera and PS/Acalypha indica film.

Sample name	Roughness (µm)	Thickness (µm)	Contact angle (°)	Drop figure
PS/Aloe vera	3.82	12.92	112
PS/Acalypha indica	0.61	4.25	104

To confirm the hydrophobicity of the prepared film, the roughness and thickness was measured by 3D laser profilometry with 0.2 µm step size and profile of surface roughness is shown in Figure 5(a) and (b). In general, low roughness surface have wettable property while high roughness surface have non-wettable property in micro or nanometer range. Of all two films, PS/Aloe exhibits high surface roughness 3.83 µm, which enhances the non-wettable behavior of the surface. The results reported that the contact angle is influenced by roughness of the surface and also properties of the liquids. In this manner, higher rougher surface of PS/Aloe exhibits more hydrophobic nature than PS/Acalypha.

Figure 5.

Surface roughness profile of Aloe vera blended polystyrene and Acalypha indica blended polystyrene film.

Anti-wetting test on cotton pad

It is well known that increase in roughness of hydrophobic surface will enhance non-wettability. In our work, natural plant-based polymeric coating was initiated on the cotton surface by simple dip-coating method according to the literature, ³⁷ the outer surface of the cotton was changed by polystyrene may improve surface non-wettability. The wettability of uncoated cotton pad, PS/Aloe vera and PS/Acalypha indica coated cotton pads were determined using various liquids such as water, coke, milk and coffee droplets over the samples using standard single use syringe. The non-wettability test on cotton pads ensures hydrophobic nature of PS/Aloe vera and PS/Acalypha indica. So, the hydrophobization of cotton is necessary for the medical field. Notably, the PS/Aloe vera modified cotton exhibited higher repellent property till 10 h compared to the 4 h repellent property of PS/Acalypha indica modified cotton, which can be judged from the surface roughness and static contact angle analysis, as shown in Figure 6. Because of the lower surface roughness, the PS/Acalypha indica coated cotton pad showed lesser repellent effect compared to the PS/Aloe vera coated cotton pad.

Figure 6.

Uncoated, Aloe vera/Acalypha blended polystyrene coated cotton pad.

Conclusion

The non-wettable and antibacterial coating was prepared using polystyrene with Aloe vera and Acalypha indica by a simple dip-coating method. The structure, morphology, optical, non-wettability and antibacterial activity of the prepared films were examined by FTIR, UV, laser profilometry, contact angle and agar-well diffusion analysis. FTIR spectra confirmed that the presence of phytochemicals in the plant-based film. The contact angle measurement proved that the prepared films were exhibited non-wettable property, which is also confirmed by the surface roughness analysis. Furthermore, the plant-based polystyrene film showed a potential antibacterial activity against the tested pathogens, in which the PS/Aloe vera exhibited more efficient bactericidal activity than PS/Acalypha indica film because of the presence of a large number of phytochemicals such as alkaloids, flavonoids, phenolic compounds and terpenoids. Additionally, PS/Aloe vera coated cotton substrate exhibited excellent non- wettable behavior against various liquids (water milk, coke and coffee) upto 10 h. The above results showed the prepared natural plant-based thin film property validates thin film coating for medical applications on cotton gauze and surgical fabrics.