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Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect
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Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect Shahnaz Majeed a,*, Mohmmad Danish b, Aifa Husna Binti Zahrudin a, Gouri Kumar Dash a b
a Faculty of Pharmacy and Health Sciences, Universiti Kuala Lumpur, Royal College of Medicine Perak, 30450, Malaysia Universiti Kuala Lumpur Malaysian Institute of Chemical and Bioengineering Technology, Lot 1988, Kawasan Perindustrian Bandar Vendor, 78000, Alor Gajah, Melaka, Malaysia
Received 15 August 2017; revised 6 November 2017; accepted 7 November 2017
Abstract Silver nanoparticles have gained considerable importance in recent years due to their diverse medicinal activities. In the present study, we have explored filamentous fungi Penicillium italicum for the extracellular biosynthesis of silver nanoparticles (AgNPs) and evaluated its antibacterial and anticancer effects. The nanoparticles were characterized by using UVeVisible and Fourier transform infra-red (FTIR) spectroscopy and transmission electron microscopy (TEM) analysis. UVeVisible spectra showed specific absorption peak at 422.67 nm which confirmed the presence of nanoparticles. FTIR spectroscopy analysis revealed the presence of alcohols, phenols, alkenes, and amines that play major roles in stabilizing the synthesized AgNPs. Transmission electron microscopy (TEM) analysis showed spherical shape of AgNPs with size ranges from 14.5 nm to 23.3 nm. Antibacterial studies against Staphylococcus aureus, Salmonella enterica, Bacillus cereus, and Escherichia coli through disc diffusion method revealed 20 mm (for 40 mg/ml) of inhibition of zone especially for S. enterica and exhibited excellent synergistic effect when combined with moxifloxacin and streptomycin. Further, in anticancer studies, these nanoparticles demonstrated good anticancer effect against HEp-2 cancer cell line with IC50 value at 30 mg/ml through MTT assay. © 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Penicillium italicum; FTIR; TEM; Antibacterial activity; Anticancer activity
1. Introduction Nanotechnology is a branch of science that embodies biological, chemical, physical, and electrical and electronics engineering. The field offers a promising way to improve the properties of metals by * Corresponding author. E-mail address: [email protected] (S. Majeed). Peer review under responsibility of University of Kerbala.
transforming them into nanoparticles with in a size range of 1e100 nm [1,2]. Inorganic metal and metal oxide nanoparticles have paved a way to discover promising antimicrobial and anticancer candidates in recent years. Bacterial resistance towards antibiotics has emerged as a global issue nowadays due to excessive use of antibacterial agents. The bacterial resistance facilitates the re-emergence of diseases and is known as superbugs. Metallic nanoparticles have
https://doi.org/10.1016/j.kijoms.2017.11.002 2405-609X/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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S. Majeed et al. / Karbala International Journal of Modern Science xx (2017) 1e7
always acted as novel antimicrobial agents to resolve antibiotic resistance issues [3]. Various physical and chemical methods are used to prepare metallic nanoparticles [4]. However, these methods offer considerable toxic wastes and not environmental friendly. Further, the properties of nanoparticles can be enhanced when synthesized in a greener route since no toxic chemicals are used during the process. Green synthesis of nanoparticles is beneficial compared to chemical and physical approaches as the nanoparticles produced are nontoxic, economical and more stable [5,6]. In this study, the cell filtrate of Penicillium italicum was used for the production of silver nanoparticles (AgNPs). The nanoparticles were characterized by using a UVeVisible spectrophotometer, FTIR, and TEM analysis to confirm their particles size, shape, distribution and stability. The synthesized AgNPs were further evaluated for their antibacterial and anticancer activities. The antibacterial activity was performed using disc diffusion method against selected bacterial pathogens followed by studies on the synergistic effect with standard antibiotics such as moxifloxacin and streptomycin respectively. Evaluation of anticancer activity was carried out by HEp-2 cell line by MTT assay. 2. Material and methods 2.1. Soil sample collection
The soil sample was collected from Universiti Kuala Lumpur Royal College of Medicine Perak (UniKL RCMP), Tasek Premise. The soil sample was collected from 2 cm to 3 cm depth by using a sterile spatula and was transferred to the sterile plate with a cover. Then, the covered plate was placed at Research and Development Laboratory of UniKL RCMP, Tasek Premise and incubated at room temperature to dry the soil sample for 3e4 days for further process. 2.2. Isolation of fungal culture
Isolation of fungal culture was carried out by using serial dilution technique and spread plate method. About 1 g of dried soil sample was diluted in 10 mL of sterilized distilled water. Then, the solution was serially diluted to prepare a dilution with concentration range from 101 mL until 105 mL. 0.5 mL volume of solution from each concentration range of 103 mL until 105 mL was transferred aseptically onto PDA plates and was spread respectively to the concentration.
The plates were then placed in a dark environment and incubated at room temperature for 3 days. Then, for the preparation of a pure culture of Penicillium italicum the isolated fungal were sub-cultured on PDA plates. The purely isolated Penicillium italicum was maintained at 4 C for further studies [7]. 2.3. Microscopic and colony characterization
The author observed Penicillium italicum in mycology, and the colony morphology was recorded concerning colour, size, shape, and nature of colony. 2.4. Biosynthesis of AgNPs
The purely isolated Penicillium italicum was used to synthesis the AgNPs by extracellular biosynthesis. Fungal biomass was grown aerobically by adding the fungal spore in a liquid nutrient media and incubated at 30 C on an orbital shaker at 160 rpm for 72 h. After 72 h, the produced fungal biomass was filtered by using Whatman filter paper No. 1 and thoroughly washed 2e3 times by using distilled water to remove the residual media part and other debris. The fungal biomass was put into a conical flask which contains 100 mL of double distilled water and incubated at 25 C on rotary shaker at 160 rpm for another 72 h. After 72 h of incubation on the rotary shaker, the biomass was filtered by using Whatman filter paper No. 1 and washed thoroughly with distilled water 2e3 times to remove a residual part until a clean biomass was produced. The fresh, clean, clear and cell-free extract was taken into the clean conical flask for further study purpose. 1 mM of AgNO3 was added into the conical flask containing the clear cell-free extract and kept at 25 C on a rotary shaker at 160 rpm for 72 h in a dark condition. 2.5. Characterization of AgNPs
The colour of the solution becomes more darken and cloudy after 72 h of incubation on the rotary shaker 160 rpm at 25 C indicated the formation of silver nanoparticles. These AgNPs were further investigated by using UVeVisible Spectrophotometer, and analysis was carried out from the wave length 300e600 nm to check the maximum absorbance (lmax). FTIR analysis was used to reveal the proteins, and functional group contained in the AgNPs which responsible for the stability of the nanoparticles and FTIR powder form of the sample was mixed with potassium bromide and observed the spectra by FTIR spectroscopy. The
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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particle size, shape and electrostatic charge of the AgNPs are characterized by using TEM analysis. For TEM the nanoparticles solution was diluted, and one drop of diluted nanoparticles was put on the carboncoated grid and subjected to TEM analysis. 2.6. Antibacterial activity of AgNPs
The eco-friendly synthesized silver nanoparticles were evaluated for its antibacterial properties against Staphylococcus aureus, Escherichia coli, Salmonella enterica and Bacillus cereus by disc diffusion method. For the antibacterial test, each antibiotic disc was treated with 20 mg/disc AgNPs and for blanks which had been impregnated with 40 mg/disc to check the synergism. The synergistic effect of silver nanoparticles with antibiotic moxifloxacin and streptomycin were investigated against above bacterial pathogen. The zone of inhibition was measured after 24 h incubation at 37 C. 2.7. Cytotoxic effect of AgNPs
Human laryngeal carcinoma (HEp-2) was brought from national center of cell science (NCCS) Pune. The biologically synthesized AgNPs were used to check the cytotoxic effect on HEp-2 cell line at different concentrations by using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium Bromide (MTT) assay. HEp-2 cells were properly grown in minimal essential medium (MEM) on 96 well plate and incubated at 37 C for 24 h with 5% carbon dioxide. The plate was incubated with different concentration of silver nanoparticles from 5 mg/ml to 50 mg/ml to check the cytotoxic effect on the cells. After 24 h of incubation all wells of plate were washed with phosphate buffer saline (PBS) thoroughly, then each well of the plate was treated with MTT solution and incubated at 37 C for 2e3 h. After the incubation, the wells were treated with 100 ml of DMSO to solubilize the crystals formed by MTT solution, and the whole plate was put on the shaker to solubilize the crystals so that colour will develop. The intensity of colour was measured by using spectrophotometer at 570 nm to check the viability of cells by using following formula. Cell Viability ð%Þ ¼
Absorbance of AgNPs treated cells Absorbance of Untreated cells 100
The above formula was used to determine the IC50 value of cells that killed 50% cells upon using a different concentration of AgNPs and compared with untreated cells, where DMSO was used as negative control.
3
3. Result and discussion In the present study, we biosynthesized the AgNPs using the aqueous extract of Penicillium italicum. The change of colour in the cell filtrate from light yellow to dark brown after addition of silver nitrate indicated the formation of AgNPs which was due to the reduction of silver ions and effect of surface plasmon resonance [8]. The colour of the fungal extract remained unchanged even after the being incubated for 48 h indicating that the synthesized AgNPs were well dispersed and the nanoparticles did not aggregate with each other (Fig. 1) [9,10]. Characterization of the AgNPs from UVeVisible Spectrophotometric analysis revealed strong absorption peak at 422 nm indicating the formation of AgNPs due to inter band transition and plasmon resonance of nanoparticles (Fig. 2) [11]. FTIR analysis of AgNPs shown in Fig. 3 revealed nine bands at 3401.18, 1653.91, 1638.48, 1627.89, 1509.70, 1384.71, 1076.09, 825.42, and 672.38 cm1. The band at 3401.18 cm1 showed the presence of alcohol and phenol functional groups with OeH stretching and H-bonded, while the 1653.91 cm1 represented eC]Ce stretching vibration of alkenes functional group. The bands observed at 1638.48 and 1627.89 cm1 matches to the bending vibrations of 1 amines with NeH bend. The functional group of nitro compounds appeared at 1509.70 cm1. The bands observed at 1384.71 and 672.38 cm1 represented CeH bend of alkanes and alkynes functional groups respectively. Meanwhile, the functional group of aliphatic amines also involved in the reduction of Agþ into Ag0 when the band was observed at 1076.09 cm1 with CeN stretching vibrations. The band at 825.42 cm1 could be assigned to CeCl stretch of alkyl halides. The results from FTIR graph revealed the presence of different biomolecules that acted as reducing and capping agents that were involved in the stabilization of the AgNPs (Fig. 3). TEM analysis was carried out to determine the morphology and size of the synthesized AgNPs which showed that the silver nanoparticles produced were dispersed well with no agglomeration. Besides, the AgNPs were spherical in shape and size ranged from 14.5 nm to 23.3 nm (Fig. 4). Screening for antibacterial activity was carried out by using disc diffusion method against S. aureus, S. enterica, B. cereus and E. coli. The synthesized nanoparticles showed good activity alone and also when combined with antibiotics. Highest zone of inhibition was observed by S. enterica (38 mm) and E. coli (38 mm),
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Fig. 1. Colour change from light yellow to dark brown indicates formation of AgNPs (A) Before addition of AgNO3 (B) After addition of AgNO3.
Fig. 2. UVeVis spectra analysis of silver nanoparticles synthesized from Penicillium italicum.
followed by S. aureus (29 mm) and B. cereus (28 mm) respectively when the AgNPs were used in combination with moxifloxacin to study the synergistic effect. Similarly, with streptomycin, the AgNPs demonstrated the highest zone of inhibition by E. coli (30 mm) followed by S. aureus (28 mm), B. cereus (26 mm) and S. enterica (25 mm) respectively, as presented in Table 1. The results of the study conclude that the synthesized silver nanoparticles from Penicillium italicum both alone or when in combination with moxifloxacin and streptomycin have good antibacterial activity against human pathogens (Fig. 5) [12].
The antibacterial activity may be attributed from the fact that the synthesized nanoparticles may bind to the bacterial surface and alter the cell membrane permeability thereby interfering with bacterial respiration due to the production reactive oxygen species (ROS) [13]. However, the attraction of these nanoparticles to the surface of cell membrane depends on the particle surface area. The small size of silver nanoparticles will offer a greater surface area that can interact with cell membrane surface; this will give a significant bactericidal effect and cause the bacterial cell death [14].
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Fig. 3. FTIR graph of AgNPs synthesized from Penicillium italicum.
Fig. 4. TEM analysis of AgNPs synthesized from Penicillium sp. showed size ranging from 14.5 to 23.3 nm with spherical in shape. TEM images at different magnification.
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Table 1 Zone of inhibition (mm) of Moxifloxacin (Mox) and Streptomycin (Strep) against four different bacterial pathogens in the absence and presence of silver nanoparticles (AgNPs). No
Pathogens
AgNPs (40 ml/disc)
Mox (5 mcg/disc)
Moxi þ AgNPs (20 mg)
Strept. (25 mcg/disc)
Strept þ AgNPs (25 mcg/disc)
1 2 3 4
Staphylococcus aureus Salmonella enterica Bacillus cereus Escherichia coli
16 13 15 17
27 34 26 34
29 38 28 38
25 23 24 28
28 25 26 30
Fig. 5. Synergistic effect in combination with Moxifloxacin and Streptomycin against bacterial pathogens, (A) S. aureus (B) S. enterica (C) B. cereus (D) E. coli.
The cytotoxic effect of the AgNPs on HEp-2 cells can be visualized from the morphological changes observed in the cells induced by nanoparticles. The HEp-2 cells, when treated with different concentrations of nanoparticles, demonstrated the activity in a concentration-dependent manner with IC50 value at 30 mg/ml (Fig. 6). The altercation within the cell morphology can be explained by shrinkage of the cell membrane and cell blebbing due to generation ROS by the nanoparticles. The generated ROS destroys the essential enzymes of cells especially reductase hence,
in turn, damages the base pair of DNA and causes the cell death [15]. 4. Conclusion Silver nanoparticles synthesized from Penicillium italicum showed good antibacterial activity and anticancer activity against the HEp-2 cancer cells. Hence these silver nanoparticles could become good anticancer and antibacterial agent but needs further cytotoxic and animal model before brought into the market.
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Fig. 6. Cytotoxic effect of AgNPs synthesized Penicillium italicum on HEp-2 cell line.
Conflict of interest There is no conflict of interest among authors. Acknowledgement The author would thank Centre of Research and innovative Universiti Kuala Lumpur for providing financial assistance to carry out this study. References [1] M. Sriramulu, S. Sumathi, Fungal based synthesis of silver nanoparticles and their antimicrobial activity, Int. J. Chem Tech Res. 10 (1) (2017) 367e377. [2] M.N. Moore, Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 32 (8) (2006) 967e976. [3] J. Mohammad Haipour, M. Katharina Fromm, Ali Akbar Askarran, Dorleta Jimenez de Aberasturi, Idoia Ruiz de Larramendi, Teofilo Rojo, Vahid Serpooshan, Wolfgang J. Parak, Morteza Mahmoudi, Antibacterial properties of nanoparticles, Trends Biotechnol. 10 (2012) 1016. [4] M.S. Mastuli, R. Rusdi, A.M. Mahat, N. Saat, N. Kamarulzaman, Sol-gel synthesis of highly stable nano sized MgO from magnesium oxalate dihydrate, Adv. Mater. Res. 545 (2012) 137e142. [5] P. Azmath, S. Baker, D. Rakshith, S. Satish, Mycosynthesis of silver nanoparticles bearing antibacterial activity, Saudi Pharm. J. 24 (2015) 140e146. [6] R. Bharathidasan, A. Panneerselvam, Biosynthesis and characterization of silver nanoparticles using endophytic fungi Aspergillus concius, Penicillium janthinellum and Phomosis sp. Int. J. Pharm. Health Res. 3 (9) (2012) 3163e3169.
[7] K.S. Hemath Naveen, Gaurav Kumar, L. Karthik, K.V. Bhaskara Rao, Extracellular biosynthesis of silver nanoparticles using the filamentous fungus Penicillium sp. Arch. Appl. Sci. Res. 2 (6) (2010) 161e167. [8] D.K. Rahi, A.S. Parmar, Mycosynthesis of silver nanoparticles by an endophytic Penicillium species of Aloe vera root, evaluation of their antibacterial and antibiotic enhancing activity, Int. J. Nanomater. Biostructures 4 (3) (2014) 46e51. [9] S. Majeed, A. Nanda, Biosynthesis and assessment of silver nanoparticles with sparfloxacin and ofloxacin synthesized from Penicillium sp. on some bacterial pathogens, Int. J. Appl. Biol. Pharm. Technol. 5 (2) (2014) 108e114. [10] M. Sastry, K.S. Mayya, K. Bandyopadhyay, pH dependent changes in the optical properties of carboxylic acid derivatized silver colloidal particles, Colloids Surfaces A Physicochem. Eng. Aspects 127 (1997) 221e228. [11] K. Kathiresan, S. Manivannan, M.A. Nabeel, B. Dhivya, Studies on silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sediment, Colloids Surfaces B Biointerfaces 71 (1) (2009) 133e137. [12] S.S. Birla, V.V. Tiwari, A.K. Gade, A.P. Ingle, A.P. Yadav, M.K. Rai, Fabrication by Silver nanoparticles by Phoma glomerate and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, Lett. Appl. Microbiol. 48 (2009) 173e179. [13] M. Danilczuk, A. Lund, J. Saldo, H. Yamada, J. Micha-lik, Conduction electron spin resonance of small silverparticles, Spectrochim. Acta. Part A Mol. Biomol. Spectrosc 63 (2006) 189e191. [14] A.D. Mudasir, A. Ingle, M. Rai, Enhanced antimicrobial activity of silver nanoparticles synthesized by Cryphonectria sp. evaluated singly and in combination with antibiotics, J. Nanomed. 9 (2013) 105e110. [15] M.I. Sriram, S.M. Kanth, K. Kalishwaralal, S. Gurunathan, Antitumor activity of silver nanoparticles in Dalton's lymphoma ascites tumour model, Int. J. Nanomed. 5 (2010) 753e762.
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ScienceDirect Karbala International Journal of Modern Science xx (2017) 1e7 http://www.journals.elsevier.com/karbala-international-journal-of-modern-science/
Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect Shahnaz Majeed a,*, Mohmmad Danish b, Aifa Husna Binti Zahrudin a, Gouri Kumar Dash a b
a Faculty of Pharmacy and Health Sciences, Universiti Kuala Lumpur, Royal College of Medicine Perak, 30450, Malaysia Universiti Kuala Lumpur Malaysian Institute of Chemical and Bioengineering Technology, Lot 1988, Kawasan Perindustrian Bandar Vendor, 78000, Alor Gajah, Melaka, Malaysia
Received 15 August 2017; revised 6 November 2017; accepted 7 November 2017
Abstract Silver nanoparticles have gained considerable importance in recent years due to their diverse medicinal activities. In the present study, we have explored filamentous fungi Penicillium italicum for the extracellular biosynthesis of silver nanoparticles (AgNPs) and evaluated its antibacterial and anticancer effects. The nanoparticles were characterized by using UVeVisible and Fourier transform infra-red (FTIR) spectroscopy and transmission electron microscopy (TEM) analysis. UVeVisible spectra showed specific absorption peak at 422.67 nm which confirmed the presence of nanoparticles. FTIR spectroscopy analysis revealed the presence of alcohols, phenols, alkenes, and amines that play major roles in stabilizing the synthesized AgNPs. Transmission electron microscopy (TEM) analysis showed spherical shape of AgNPs with size ranges from 14.5 nm to 23.3 nm. Antibacterial studies against Staphylococcus aureus, Salmonella enterica, Bacillus cereus, and Escherichia coli through disc diffusion method revealed 20 mm (for 40 mg/ml) of inhibition of zone especially for S. enterica and exhibited excellent synergistic effect when combined with moxifloxacin and streptomycin. Further, in anticancer studies, these nanoparticles demonstrated good anticancer effect against HEp-2 cancer cell line with IC50 value at 30 mg/ml through MTT assay. © 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Penicillium italicum; FTIR; TEM; Antibacterial activity; Anticancer activity
1. Introduction Nanotechnology is a branch of science that embodies biological, chemical, physical, and electrical and electronics engineering. The field offers a promising way to improve the properties of metals by * Corresponding author. E-mail address: [email protected] (S. Majeed). Peer review under responsibility of University of Kerbala.
transforming them into nanoparticles with in a size range of 1e100 nm [1,2]. Inorganic metal and metal oxide nanoparticles have paved a way to discover promising antimicrobial and anticancer candidates in recent years. Bacterial resistance towards antibiotics has emerged as a global issue nowadays due to excessive use of antibacterial agents. The bacterial resistance facilitates the re-emergence of diseases and is known as superbugs. Metallic nanoparticles have
https://doi.org/10.1016/j.kijoms.2017.11.002 2405-609X/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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S. Majeed et al. / Karbala International Journal of Modern Science xx (2017) 1e7
always acted as novel antimicrobial agents to resolve antibiotic resistance issues [3]. Various physical and chemical methods are used to prepare metallic nanoparticles [4]. However, these methods offer considerable toxic wastes and not environmental friendly. Further, the properties of nanoparticles can be enhanced when synthesized in a greener route since no toxic chemicals are used during the process. Green synthesis of nanoparticles is beneficial compared to chemical and physical approaches as the nanoparticles produced are nontoxic, economical and more stable [5,6]. In this study, the cell filtrate of Penicillium italicum was used for the production of silver nanoparticles (AgNPs). The nanoparticles were characterized by using a UVeVisible spectrophotometer, FTIR, and TEM analysis to confirm their particles size, shape, distribution and stability. The synthesized AgNPs were further evaluated for their antibacterial and anticancer activities. The antibacterial activity was performed using disc diffusion method against selected bacterial pathogens followed by studies on the synergistic effect with standard antibiotics such as moxifloxacin and streptomycin respectively. Evaluation of anticancer activity was carried out by HEp-2 cell line by MTT assay. 2. Material and methods 2.1. Soil sample collection
The soil sample was collected from Universiti Kuala Lumpur Royal College of Medicine Perak (UniKL RCMP), Tasek Premise. The soil sample was collected from 2 cm to 3 cm depth by using a sterile spatula and was transferred to the sterile plate with a cover. Then, the covered plate was placed at Research and Development Laboratory of UniKL RCMP, Tasek Premise and incubated at room temperature to dry the soil sample for 3e4 days for further process. 2.2. Isolation of fungal culture
Isolation of fungal culture was carried out by using serial dilution technique and spread plate method. About 1 g of dried soil sample was diluted in 10 mL of sterilized distilled water. Then, the solution was serially diluted to prepare a dilution with concentration range from 101 mL until 105 mL. 0.5 mL volume of solution from each concentration range of 103 mL until 105 mL was transferred aseptically onto PDA plates and was spread respectively to the concentration.
The plates were then placed in a dark environment and incubated at room temperature for 3 days. Then, for the preparation of a pure culture of Penicillium italicum the isolated fungal were sub-cultured on PDA plates. The purely isolated Penicillium italicum was maintained at 4 C for further studies [7]. 2.3. Microscopic and colony characterization
The author observed Penicillium italicum in mycology, and the colony morphology was recorded concerning colour, size, shape, and nature of colony. 2.4. Biosynthesis of AgNPs
The purely isolated Penicillium italicum was used to synthesis the AgNPs by extracellular biosynthesis. Fungal biomass was grown aerobically by adding the fungal spore in a liquid nutrient media and incubated at 30 C on an orbital shaker at 160 rpm for 72 h. After 72 h, the produced fungal biomass was filtered by using Whatman filter paper No. 1 and thoroughly washed 2e3 times by using distilled water to remove the residual media part and other debris. The fungal biomass was put into a conical flask which contains 100 mL of double distilled water and incubated at 25 C on rotary shaker at 160 rpm for another 72 h. After 72 h of incubation on the rotary shaker, the biomass was filtered by using Whatman filter paper No. 1 and washed thoroughly with distilled water 2e3 times to remove a residual part until a clean biomass was produced. The fresh, clean, clear and cell-free extract was taken into the clean conical flask for further study purpose. 1 mM of AgNO3 was added into the conical flask containing the clear cell-free extract and kept at 25 C on a rotary shaker at 160 rpm for 72 h in a dark condition. 2.5. Characterization of AgNPs
The colour of the solution becomes more darken and cloudy after 72 h of incubation on the rotary shaker 160 rpm at 25 C indicated the formation of silver nanoparticles. These AgNPs were further investigated by using UVeVisible Spectrophotometer, and analysis was carried out from the wave length 300e600 nm to check the maximum absorbance (lmax). FTIR analysis was used to reveal the proteins, and functional group contained in the AgNPs which responsible for the stability of the nanoparticles and FTIR powder form of the sample was mixed with potassium bromide and observed the spectra by FTIR spectroscopy. The
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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S. Majeed et al. / Karbala International Journal of Modern Science xx (2017) 1e7
particle size, shape and electrostatic charge of the AgNPs are characterized by using TEM analysis. For TEM the nanoparticles solution was diluted, and one drop of diluted nanoparticles was put on the carboncoated grid and subjected to TEM analysis. 2.6. Antibacterial activity of AgNPs
The eco-friendly synthesized silver nanoparticles were evaluated for its antibacterial properties against Staphylococcus aureus, Escherichia coli, Salmonella enterica and Bacillus cereus by disc diffusion method. For the antibacterial test, each antibiotic disc was treated with 20 mg/disc AgNPs and for blanks which had been impregnated with 40 mg/disc to check the synergism. The synergistic effect of silver nanoparticles with antibiotic moxifloxacin and streptomycin were investigated against above bacterial pathogen. The zone of inhibition was measured after 24 h incubation at 37 C. 2.7. Cytotoxic effect of AgNPs
Human laryngeal carcinoma (HEp-2) was brought from national center of cell science (NCCS) Pune. The biologically synthesized AgNPs were used to check the cytotoxic effect on HEp-2 cell line at different concentrations by using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium Bromide (MTT) assay. HEp-2 cells were properly grown in minimal essential medium (MEM) on 96 well plate and incubated at 37 C for 24 h with 5% carbon dioxide. The plate was incubated with different concentration of silver nanoparticles from 5 mg/ml to 50 mg/ml to check the cytotoxic effect on the cells. After 24 h of incubation all wells of plate were washed with phosphate buffer saline (PBS) thoroughly, then each well of the plate was treated with MTT solution and incubated at 37 C for 2e3 h. After the incubation, the wells were treated with 100 ml of DMSO to solubilize the crystals formed by MTT solution, and the whole plate was put on the shaker to solubilize the crystals so that colour will develop. The intensity of colour was measured by using spectrophotometer at 570 nm to check the viability of cells by using following formula. Cell Viability ð%Þ ¼
Absorbance of AgNPs treated cells Absorbance of Untreated cells 100
The above formula was used to determine the IC50 value of cells that killed 50% cells upon using a different concentration of AgNPs and compared with untreated cells, where DMSO was used as negative control.
3
3. Result and discussion In the present study, we biosynthesized the AgNPs using the aqueous extract of Penicillium italicum. The change of colour in the cell filtrate from light yellow to dark brown after addition of silver nitrate indicated the formation of AgNPs which was due to the reduction of silver ions and effect of surface plasmon resonance [8]. The colour of the fungal extract remained unchanged even after the being incubated for 48 h indicating that the synthesized AgNPs were well dispersed and the nanoparticles did not aggregate with each other (Fig. 1) [9,10]. Characterization of the AgNPs from UVeVisible Spectrophotometric analysis revealed strong absorption peak at 422 nm indicating the formation of AgNPs due to inter band transition and plasmon resonance of nanoparticles (Fig. 2) [11]. FTIR analysis of AgNPs shown in Fig. 3 revealed nine bands at 3401.18, 1653.91, 1638.48, 1627.89, 1509.70, 1384.71, 1076.09, 825.42, and 672.38 cm1. The band at 3401.18 cm1 showed the presence of alcohol and phenol functional groups with OeH stretching and H-bonded, while the 1653.91 cm1 represented eC]Ce stretching vibration of alkenes functional group. The bands observed at 1638.48 and 1627.89 cm1 matches to the bending vibrations of 1 amines with NeH bend. The functional group of nitro compounds appeared at 1509.70 cm1. The bands observed at 1384.71 and 672.38 cm1 represented CeH bend of alkanes and alkynes functional groups respectively. Meanwhile, the functional group of aliphatic amines also involved in the reduction of Agþ into Ag0 when the band was observed at 1076.09 cm1 with CeN stretching vibrations. The band at 825.42 cm1 could be assigned to CeCl stretch of alkyl halides. The results from FTIR graph revealed the presence of different biomolecules that acted as reducing and capping agents that were involved in the stabilization of the AgNPs (Fig. 3). TEM analysis was carried out to determine the morphology and size of the synthesized AgNPs which showed that the silver nanoparticles produced were dispersed well with no agglomeration. Besides, the AgNPs were spherical in shape and size ranged from 14.5 nm to 23.3 nm (Fig. 4). Screening for antibacterial activity was carried out by using disc diffusion method against S. aureus, S. enterica, B. cereus and E. coli. The synthesized nanoparticles showed good activity alone and also when combined with antibiotics. Highest zone of inhibition was observed by S. enterica (38 mm) and E. coli (38 mm),
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Fig. 1. Colour change from light yellow to dark brown indicates formation of AgNPs (A) Before addition of AgNO3 (B) After addition of AgNO3.
Fig. 2. UVeVis spectra analysis of silver nanoparticles synthesized from Penicillium italicum.
followed by S. aureus (29 mm) and B. cereus (28 mm) respectively when the AgNPs were used in combination with moxifloxacin to study the synergistic effect. Similarly, with streptomycin, the AgNPs demonstrated the highest zone of inhibition by E. coli (30 mm) followed by S. aureus (28 mm), B. cereus (26 mm) and S. enterica (25 mm) respectively, as presented in Table 1. The results of the study conclude that the synthesized silver nanoparticles from Penicillium italicum both alone or when in combination with moxifloxacin and streptomycin have good antibacterial activity against human pathogens (Fig. 5) [12].
The antibacterial activity may be attributed from the fact that the synthesized nanoparticles may bind to the bacterial surface and alter the cell membrane permeability thereby interfering with bacterial respiration due to the production reactive oxygen species (ROS) [13]. However, the attraction of these nanoparticles to the surface of cell membrane depends on the particle surface area. The small size of silver nanoparticles will offer a greater surface area that can interact with cell membrane surface; this will give a significant bactericidal effect and cause the bacterial cell death [14].
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Fig. 3. FTIR graph of AgNPs synthesized from Penicillium italicum.
Fig. 4. TEM analysis of AgNPs synthesized from Penicillium sp. showed size ranging from 14.5 to 23.3 nm with spherical in shape. TEM images at different magnification.
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002
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Table 1 Zone of inhibition (mm) of Moxifloxacin (Mox) and Streptomycin (Strep) against four different bacterial pathogens in the absence and presence of silver nanoparticles (AgNPs). No
Pathogens
AgNPs (40 ml/disc)
Mox (5 mcg/disc)
Moxi þ AgNPs (20 mg)
Strept. (25 mcg/disc)
Strept þ AgNPs (25 mcg/disc)
1 2 3 4
Staphylococcus aureus Salmonella enterica Bacillus cereus Escherichia coli
16 13 15 17
27 34 26 34
29 38 28 38
25 23 24 28
28 25 26 30
Fig. 5. Synergistic effect in combination with Moxifloxacin and Streptomycin against bacterial pathogens, (A) S. aureus (B) S. enterica (C) B. cereus (D) E. coli.
The cytotoxic effect of the AgNPs on HEp-2 cells can be visualized from the morphological changes observed in the cells induced by nanoparticles. The HEp-2 cells, when treated with different concentrations of nanoparticles, demonstrated the activity in a concentration-dependent manner with IC50 value at 30 mg/ml (Fig. 6). The altercation within the cell morphology can be explained by shrinkage of the cell membrane and cell blebbing due to generation ROS by the nanoparticles. The generated ROS destroys the essential enzymes of cells especially reductase hence,
in turn, damages the base pair of DNA and causes the cell death [15]. 4. Conclusion Silver nanoparticles synthesized from Penicillium italicum showed good antibacterial activity and anticancer activity against the HEp-2 cancer cells. Hence these silver nanoparticles could become good anticancer and antibacterial agent but needs further cytotoxic and animal model before brought into the market.
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Fig. 6. Cytotoxic effect of AgNPs synthesized Penicillium italicum on HEp-2 cell line.
Conflict of interest There is no conflict of interest among authors. Acknowledgement The author would thank Centre of Research and innovative Universiti Kuala Lumpur for providing financial assistance to carry out this study. References [1] M. Sriramulu, S. Sumathi, Fungal based synthesis of silver nanoparticles and their antimicrobial activity, Int. J. Chem Tech Res. 10 (1) (2017) 367e377. [2] M.N. Moore, Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 32 (8) (2006) 967e976. [3] J. Mohammad Haipour, M. Katharina Fromm, Ali Akbar Askarran, Dorleta Jimenez de Aberasturi, Idoia Ruiz de Larramendi, Teofilo Rojo, Vahid Serpooshan, Wolfgang J. Parak, Morteza Mahmoudi, Antibacterial properties of nanoparticles, Trends Biotechnol. 10 (2012) 1016. [4] M.S. Mastuli, R. Rusdi, A.M. Mahat, N. Saat, N. Kamarulzaman, Sol-gel synthesis of highly stable nano sized MgO from magnesium oxalate dihydrate, Adv. Mater. Res. 545 (2012) 137e142. [5] P. Azmath, S. Baker, D. Rakshith, S. Satish, Mycosynthesis of silver nanoparticles bearing antibacterial activity, Saudi Pharm. J. 24 (2015) 140e146. [6] R. Bharathidasan, A. Panneerselvam, Biosynthesis and characterization of silver nanoparticles using endophytic fungi Aspergillus concius, Penicillium janthinellum and Phomosis sp. Int. J. Pharm. Health Res. 3 (9) (2012) 3163e3169.
[7] K.S. Hemath Naveen, Gaurav Kumar, L. Karthik, K.V. Bhaskara Rao, Extracellular biosynthesis of silver nanoparticles using the filamentous fungus Penicillium sp. Arch. Appl. Sci. Res. 2 (6) (2010) 161e167. [8] D.K. Rahi, A.S. Parmar, Mycosynthesis of silver nanoparticles by an endophytic Penicillium species of Aloe vera root, evaluation of their antibacterial and antibiotic enhancing activity, Int. J. Nanomater. Biostructures 4 (3) (2014) 46e51. [9] S. Majeed, A. Nanda, Biosynthesis and assessment of silver nanoparticles with sparfloxacin and ofloxacin synthesized from Penicillium sp. on some bacterial pathogens, Int. J. Appl. Biol. Pharm. Technol. 5 (2) (2014) 108e114. [10] M. Sastry, K.S. Mayya, K. Bandyopadhyay, pH dependent changes in the optical properties of carboxylic acid derivatized silver colloidal particles, Colloids Surfaces A Physicochem. Eng. Aspects 127 (1997) 221e228. [11] K. Kathiresan, S. Manivannan, M.A. Nabeel, B. Dhivya, Studies on silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sediment, Colloids Surfaces B Biointerfaces 71 (1) (2009) 133e137. [12] S.S. Birla, V.V. Tiwari, A.K. Gade, A.P. Ingle, A.P. Yadav, M.K. Rai, Fabrication by Silver nanoparticles by Phoma glomerate and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, Lett. Appl. Microbiol. 48 (2009) 173e179. [13] M. Danilczuk, A. Lund, J. Saldo, H. Yamada, J. Micha-lik, Conduction electron spin resonance of small silverparticles, Spectrochim. Acta. Part A Mol. Biomol. Spectrosc 63 (2006) 189e191. [14] A.D. Mudasir, A. Ingle, M. Rai, Enhanced antimicrobial activity of silver nanoparticles synthesized by Cryphonectria sp. evaluated singly and in combination with antibiotics, J. Nanomed. 9 (2013) 105e110. [15] M.I. Sriram, S.M. Kanth, K. Kalishwaralal, S. Gurunathan, Antitumor activity of silver nanoparticles in Dalton's lymphoma ascites tumour model, Int. J. Nanomed. 5 (2010) 753e762.
Please cite this article in press as: S. Majeed et al., Biosynthesis and characterization of silver nanoparticles from fungal species and its antibacterial and anticancer effect, Karbala International Journal of Modern Science (2017), https://doi.org/10.1016/j.kijoms.2017.11.002