Experimental Study on Antimicrobial Activity of Silk

Fibers and Polymers 2018, Vol.19, No.9, 1880-1886 DOI 10.1007/s12221-018-8239-3

ISSN 1229-9197 (print version) ISSN 1875-0052 (electronic version)

Experimental Study on Antimicrobial Activity of Silk Fabric Treated with Natural Dye Extract from Neem (Azadirachta indica) Leaves Abeer A. Abd El Aty1#, Gehan T. El-Bassyouni2, Nabawia A. Abdel-Zaher3, and Osiris W. Guirguis4* 1

Chemistry of Natural and Microbial Product Department, National Research Centre (NRC), Giza 12622, Egypt Refractories, Ceramics and Building Materials Department, National Research Centre (NRC), Giza 12622, Egypt 3 Textile Metrology Lab, National Institute for Standards, Giza 12211, Egypt 4 Biophysics Department, Faculty of Science, Cairo University, Giza 12613, Egypt (Received March 28, 2018; Revised June 29, 2018; Accepted July 4, 2018)

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Abstract: In the present study, a novel eco-friendly production of silk fabrics dyed with different natural dye bath concentrations (40, 80, 120, 160, 200 and 240 g/l) extracted from neem (Azadirachta indica) leaves was developed. The surface morphology of the fabrics was examined by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) spectroscopy to characterize the chemical structure of the fabrics. The SEM images of the undyed fabric show that the fabric was tightly woven with little porosity between the fibres with dozens of silk threads in orthogonal directions. By increasing the neem concentration, a scale of fine particles grew on the surface of the silk fabrics with little macroscopical defects was demonstrated. The fiber diameters and tightness between filaments were significantly increased. The FTIR displayed that, neem dye does not change the characteristic peaks of the silk fabrics. Also, the evaluation of the antimicrobial activity of the undyed and neem dyed silk fabrics was monitored for Gram positive and Gram negative bacteria in addition to yeasts and fungi by using the agar diffusion method. The comparison between the different dye bath concentrations was based on the inhibition zones obtained after incubation. The antimicrobial activity in leaf extract of neem was estimated in Staphylococcus aureus, Bacillus subtilis and Lactobacillus cereus (Gram positive bacteria); Escherichia coli (Gram negative bacteria); Candida albicans and Candida tropicalis (yeasts); and Aspergillus niger and Fusarium solani (fungi). The results emphasized that, the highest neem dye bath concentration (240 g/l) was found to display good inhibitory effect against the Gram positive and reasonable activity against the Gram negative bacteria. Furthermore, the different dye bath concentrations possess no activities against yeast and fungi. In conclusion, the data reveal that the increase of neem dye concentration does not damage the silk fabric; however, it improves its antimicrobial activity by incorporating with antimicrobial agent. The current study highlighted that using neem leaves had beneficial effect in controlling the pathogenic microbial organisms. Keywords: Natural dye, Azadirachta indica, Silk fabrics, Surface morphology, Structure configuration, Antimicrobial activity

textile materials was mentioned previously [4]. The biocidal effect of plant extracts was triggered by its constituent types, such as alcohols, ethers, phenols, aldehydes, ketones, which renders them highly efficient against a wide range of microbial strains microbial strains [5]. Recently, there has been an interest in the use of natural dye in textile coloration. In textile industry, natural dyes with antimicrobial properties were extracted from several plants [6]. Extraction of color component from natural sources is important for dyeing any textile substrate in order to evaluate their dyeing characteristics and to maximize the color yield [7]. Neem plant (Azadirachta indica) belongs to the family of Meliaceae, also named as Holy Tree, bead tree and Indian Lilac. This tree was found in tropical and semi-tropical countries like Burma and India [8]. The main constituents of neem leave include: protein (7.1 %), carbohydrates (22.9 %), minerals, calcium, phosphorus, vitamin C, carotene, etc. Also, The chemical composition of the extract contains: glutamic acid, tyrosine, aspartic acid, alanine, praline, glutamine, cystine like amino acids, and fatty acids such as dodecanoic, tetradecanoic, elcosanic, etc. Neem is a nontoxic, biocompatible to humans and having a diversity of medicinal and germicidal properties. As a traditional medicine against various human ailments, neem was used

Introduction Internationally, growing realization about organic value of eco-friendly products has created new interest of users towards consumption of natural fiber, dyed with natural dyes. Natural dyes are identified for their use in coloring of natural fibers like silk, wool and cotton as foremost areas of application since prehistoric times [1]. A fabric with antimicrobial properties containing biocides is an important challenge for the protection against pathogenic microorganisms. Most modern antimicrobial finished textiles were established using synthetic products. Environmental friendly products are recently recommended [2]. Fabrics are regularly exposed to microorganisms, thus functionalization of these materials can verify an efficient method to achieve the antimicrobial active barriers. Fungi are microorganisms of highly pathogenic potential to human hosts, as some of them are significant infectious agents to immune-compromised individuals [3]. The use of natural dyes for antimicrobial finishing of *Corresponding author: [email protected] # Present address: Biology Department, Faculty of Education, Hafr Al Batin University, Saudi Arabia 1880

Antimicrobial Activity of Silk Treated with Natural Dye

since ancient times in Egypt. All parts of the tree were used as traditional medicine for domestic medication against different human diseases [9]. Most of the parts of the plant such as fruits, seeds, leaves, bark and roots enclose compounds with recognized number of human ailments such as; anti-inflammatory, anxiolytic, anti-androgenic, antistress, humoral and cell-mediated immune stimulant, antihyperglycemic, liver-stimulant, anti-viral, antiulcer, antifungal and anti-malarial activities [10]. In addition, they are used as a promoting adsorbent for dyes in aqueous solutions and as a household pesticide [11]. Furthermore, one of the most widely recognized benefits of neem oil, leaves, tea, and every other derivative is its strong antibacterial and antimicrobial effects [12]. Also, neem is used as an active factor in different industries ranging from cosmetics to agriculture [13]. Neem leaves comprises relatively important minerals required by the biochemical system. Al-Hashemi and Hossain revealed that the biochemical screening of the crude extracts of neem leaves exposed positive results of flavonoids, saponins, steroids, alkaloids, amino acid and tannins [14,15]. Neem has been found to be highly effective in the treatment of periodontal diseases, thus demonstrating its biocompatibility with human PDL fibroblasts. In the present work, the surface morphology of the fabrics was examined by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) spectroscopy to identify the chemical structure of the fabrics. Likewise, the present study aimed to compare the antimicrobial efficacy of silk fabrics dyed with different neem dye bath concentrations (40, 80, 120, 160, 200 and 240 g/l) of natural aqueous dyeing solution attained by extraction from neem leaves. The agar diffusion method was followed to evaluate the antimicrobial activities against different pathogenic strains of bacteria such as: Gram positive bacteria (S. aureus ATCC29213, B. subtilis ATCC6633, L. cereus ATCC14579); Gram negative bacteria (E. coli ATCC25922); yeasts (C. albicans ATCC10321, C. tropicalis ATCC750); and fungi (A. niger NRC53, F. solani NRC15). Growing studies on the extraction processes of natural dyes and their subsequent antimicrobial application on textiles validate their restoration. Therefore the neem plant can be considered as a good source of natural dye with antimicrobial properties and can be used in commercial dyeing and protective finishing of textile fabrics.

Experimental A 100 % white silk fabric of weight 55 g/m2 and thickness 0.06 cm was supplied from Akhmeem city, Sohag Governorate of Upper Egypt and used without any further purification. Dried green leaves of neem plant were used to obtain the neem dye without using any mordant during the dyeing process. 50 g of neem leaves were crushed and immersed in

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Figure 1. The absorption spectrum of the aqueous extract of the neem leaves.

500 ml of distilled water and allowed to boil for one hour. The solution was filtered and its optical density was investigated in the range from 200 to 700 nm by using Shimadzu Double Beam Spectrophotometer model V-530 (with band width 2.0 nm and accuracy ±0.05 %) and was taken as a measure of concentration. From the absorption spectrum of the aqueous extract neem leaves (Figure 1) three peaks were detected: the first peak at λmax=320 nm of optical density 3.765; the second peak at λmax=370 nm of optical density 3.402; and the third one at λmax=400 nm of optical density 3.569. In addition, the dried green leaves of neem plant were found to easily discharge color into hot water. Color strength and depth in color of the aqueous extract neem leaves was increased by increasing the quantity of leaves to change the concentration of dyeing. Silk fabrics were dyed using different dye bath concentrations (40, 80, 120, 160, 200 and 240 g/l) ranging from hell to dull yellow color using a liquor ratio of 1:50 at pH=5 for 45 minutes and at temperature 60 ºC for each concentration; noting that the pH of the liquor extracted from the dye was pH=5, i.e., acidic in nature [16]. The samples were thoroughly washed with cold water and then dried at ambient temperature. Scanning electron microscopy (SEM) was performed using a Quanta FEG 250 Electron Scanning Microscope (Holland). The observation of samples was conducted at an accelerating voltage of 20 kV under 100× (undyed sample only), 3000× and 6000× magnifications. Fourier-transform infrared (FTIR) was used to identify the available functional groups. The FTIR spectra of undyed and neem dyed silk fabrics with different dye bath concentrations were recorded on a Vertex FTIR Spectrometer (Bruker Daltonics Inc., Germany) in the attenuated total reflection (ATR) mode covering the wavenumber range 4000-400 cm-1. The resolution and the scan number were 2 cm-1 and 32, respectively. The agar diffusion method was used to assess the antimicrobial activity of the silk fabrics impregnated with the different neem dye bath concentrations in comparison with the undyed one. Antimicrobial activity was screened

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via in-vitro testing against different pathogenic strains of bacteria, yeasts and fungi [17-19]. All silk fabrics were tested against Gram positive bacteria (Staphylococcus aureus ATCC29213, Bacillus subtilis ATCC6633 and Lactobacillus cereus ATCC14579), Gram negative bacteria (Escherichia coli ATCC25922), yeasts (Candida albicans ATCC10321 and Candida tropicalis ATCC750), Fungi (Aspergillus niger NRC53 and Fusarium solani NRC15). Bacteria and yeast strains are American type culture collection and fungal

Figure 2. SEM image of the microstructure of the undyed silk fibres (100×).

Abeer A. Abd El Aty et al.

isolates were obtained from the culture collection of the Department of Chemistry of Natural and Microbial Products, National Research Center (NRC), Cairo, Egypt. Spores suspension of pathogenic strains were prepared and adjusted to be approximately (1×108 of bacteria and 1×106 of fungi). Approximately 1 ml of fungal and bacterial spore suspensions was inoculated into each plate containing 50 ml of sterile PDA and nutrient agar medium, respectively. Discs of equalsized round pieces (15 mm diameter) of silk fabrics were applied on the surface of the wet inoculated agar plates and left over night at 4 ºC to allow the release of the active substances from the surface of silk fabric. Plates were incubated for 24 h at 30 ºC for bacteria and 72 h at 28 ºC for fungi. An inhibition zone becomes noticeable when the antimicrobial agents diffuse into the agar. Efficiency of the antimicrobial activity or the discharge rate of the active agent was patterned by the size of the inhibition zone. The inhibition zone diameter of the clear zone behind the real control disc was measured with a digital caliper in mm. All the antibacterial tests were conducted under sterile conditions in duplicates and each sample was tested three times to get more accurate results. The average diameter zone of inhibition was calculated.

Figure 3. Scanning electron microscope images of: (a, b) undyed silk fabrics (3000× and 6000×); (c, d) neem dyed bath silk fabrics with concentration 120 g/l (3000× and 6000×); and (e, f) neem dyed bath silk fabrics with concentration 240 g/l (3000× and 6000×).

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Results and Discussion Scanning electron microscopy image of the structure of the undyed silk fabric is presented in Figure 2. It demonstrates that the fabric is tightly woven with little porosity between the fibres through dozens of silk threads in orthogonal directions [20]. The SEM images of the neem dyed silk fabrics together with the undyed one are shown in Figure 3. Images (a) and (b) exhibit that, the morphology of the undyed silk is smooth and nothing appear besides the smooth surface [21-23]. Images (c) and (d) demonstrate variations in the surface morphology due to the neem dyeing effect, a scale of fine particles grow on the surface of silk fabrics with little macroscopically defects. It can be detected that at higher neem concentration of 240 g/l, the neem sheet act as a thin film at the edge of the silk fabrics and crumpled onto the surface with a large scale (Images e and f). Therefore, it could be decided that the fine particles and sheets of neem are entrapped in between the yard structures in a strong condition [24]. Likewise, after dyeing both the fiber diameters and tightness between filaments significantly increased [22]. FTIR is used to identify the structure characterization of silk fabrics. Figure 4 shows FTIR-ATR spectra of undyed and neem dyed silk fabrics. It is clear that, undyed silk fabric is identified by three characteristic peaks at 3276 cm-1 (N-H and O-H stretching), 1619 cm-1 (amide I) and 1512 cm-1 (amide II), as well as a weak shoulder at 1265 cm-1 which attributed to the β-sheet structure of silk [22,25,26]. The peak at 1227 cm-1 assigned to amide III. The peak at around 3072 cm-1 assigned to =CH, aromatic as well as the peak at 2924 cm-1 due to the C-H stretching vibration. The peaks at 1442 cm-1 corresponded to CH2 shear vibration; 1163 cm-1 corresponded to C-O-C stretching; and 1065 cm-1 attributed to C-O stretching. The peak at the wavenumber range from 700 to 500 cm-1 assigned to C-C stretching vibration. It was also noted that the major absorption in the amide I and amide II band of dyed silk fabrics with neem is the same as that of the undyed silk fabric. As the neem concentration was increased up to 240 g/l, the bands at 1619 and 1512 cm-1 shifted and covered the ranges of wavenumbers 1621-1617 cm-1 and 1514-1511 cm-1, respectively, which approve that the β-sheet crystallite was deformed by the neem dye [27]. In addition, neem dye does not change the characteristic peaks of silk fabrics, i.e., neem dyeing has no effect on the chemical structure of the silk fabric. The obtained results are in agreement with that previously reported in the literature by many researchers who studied the modifications of the chemical structure of silk fabrics upon treatment using different herbal extracts [22,25-30]. The antibacterial activities of the undyed and dyed silk fabrics were tested against the four common bacterial strains. Figure 5 shows the inhibition zones of neem dyed silk fabrics using different dye bath concentrations against

Figure 4. Variations in FTIR-ATR spectra of undyed and neem dyed bath silk fabrics.

the Gram positive bacteria (S. aureus, B. subtilis, and L. cereus) and Gram negative bacteria (E. coli). Figure 6 designates that all dyed fabrics validated significantly different antimicrobial effects (p