Accepted Manuscript Mycosynthesis: Antibacterial, Antioxidant and antiproliferative activities of silver nanoparticles synthesized from Inonotus obliquus (Chaga mushroom) extract P.C. Nagajyothi, T.V.M. Sreekanth, Jae-il Lee, Kap Duk Lee PII: DOI: Reference:
S1011-1344(13)00274-1 http://dx.doi.org/10.1016/j.jphotobiol.2013.11.022 JPB 9622
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
7 August 2013 4 November 2013 21 November 2013
Please cite this article as: P.C. Nagajyothi, T.V.M. Sreekanth, J-i. Lee, K.D. Lee, Mycosynthesis: Antibacterial, Antioxidant and antiproliferative activities of silver nanoparticles synthesized from Inonotus obliquus (Chaga mushroom) extract, Journal of Photochemistry and Photobiology B: Biology (2013), doi: http://dx.doi.org/10.1016/ j.jphotobiol.2013.11.022
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Mycosynthesis: Antibacterial, Antioxidant and antiproliferative activities of silver nanoparticles synthesized from Inonotus obliquus (Chaga mushroom) extract P. C. Nagajyothi1, T.V.M. Sreekanth2, Jae-il Lee3, Kap Duk Lee3* 1
Department of Physiology, College of Oriental Medicine, Dongguk University, Gyeongju,
South Korea 2
Department of Life Chemistry, Catholic University of Daegu, Gyeongsan 712-702, South Korea.
Department of Nanomaterial Chemistry, Dongguk University, Gyeongju, South Korea
Corresponding author: Kap Duk Lee, Tel.: +82 54 770 2217; Fax: +82 054 770 2386; E-mail: [email protected]
______________________________________________________________________________ ABSTRACT In the present study, silver nanoparticles (AgNPs) were rapidly synthesized from silver nitrate solution at room temperature using Inonotus obliquus extract. The mycogenic synthesized AgNPs were characterized by UV-Visible absorption spectroscopy, Fourier transform infrared (FTIR), Scanning electron microscopy (SEM) with Energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM) and Atomic force microscopy (AFM). SEM revealed mostly spherical nanoparticles ranging from 14.2 to 35.2 nm in size. All AgNPs concentrations showed good ABT radical scavenging activity. Further, AgNPs showed effective antibacterial activity against both gram negative and gram positive bacteria and antiproliferative activity toward A549 human lung cancer (CCL-185) and MCF-7 human breast cancer (HTB-22) cell lines. The samples demonstrated considerably high antibacterial, and antiproliferative activities against bacterial strains and cell lines.
1. Introduction The “green synthesis” of metal nanoparticles has received great attention due to their unusual optical, chemical, photochemical, and electronic properties . Metal nanoparticles can be synthesized via chemical [2,3] and biological methods. Chemical methods for metal nanoparticle fabrication usually involve toxic materials that are expensive and potentially harmful to the environment . However, green synthesis of nanoparticles is an easy, inexpensive, efficient and eco-friendly biological method of biosynthesis of AgNPs. Such methods use plants [5-7], bacteria [8,9], fungi [10,11] or yeast [12,13], which are known to reduce silver ions into silver nanoparticles both extra and intracellularly [14-16], as well as mushrooms such as Volvariella volvacea , Pleurotus sajor , Pleurotus florida , Ganoderma lucidum , Agaricus bisporus . In this study, we used extract of the Inonotus obliquus to synthesis of AgNPs. Mushrooms are known to have anti-inflammatory, antitumor, antiviral, antivaterial, hepatoprotective and hypotensive activities in biological systems [22-24]. Many varieties of naturally occurring mushrooms have long been known to have promising antioxidant and anticancer properties and prolong longevity , as well as to contain antitumor compounds [26, 27]. Accordingly, a variety of edible mushrooms have been taken as vitamin and mineral supplements. Inonotus obliquus (family: Hymenochaetaceae) is a black mushroom that grows on birch trees in northern climates such as Russia . Inonotus obliquus mushrooms have been used as a folk remedy in Russia and Eastern Europe since the 16th century for a wide variety of human diseases without any intolerable toxic side effects [29, 30]. These mushrooms are also used as traditional medicine for the treatment of diabetes [31, 32]. Additionally recent studies, have reported that the polyphenolic compounds produced by Inonotus obliquus can protect cells against oxidative stress . These mushrooms have been reported to show therapeutic benefits including anti-inflammatory, immune-modulatory and hepatoprotective effects . Despite its popular use, the mechanism underlying the pharmacological activity of Inonotus obliquus is yet to be elucidated.
2. Material and methods Inonotus obliquus mushrooms were obtained from a forest area of Siberia, Russia. Silver nitrate (AgNO3) was procured from Sigma-Sldrich Chemical Pvt. Ltd., Seoul, South Korea.
Preparation of mushroom extract A total of 10g mushrooms were washed repeatedly with distilled water to remove any
organic impurities. The cleaned mushrooms were then crushed into small pieces with a sterilized knife and placed into a 500 mL beaker containing 200 mL double distilled water and thoroughly stirred for about half an hour. This solution was then filtered through whatman filter paper no.41 and stored 4°C. 2.2.
Mycosynthesis of AgNPs An aqueous solution of silver nitrate (1mM) was prepared and used for the synthesis of
AgNPs. Specifically, 5 mL of mushroom extract was added into 95 mL of 1mM silver nitrate to reduce Ag+ to Ag0. This solution exposed to room temperature, which resulted in a change in color from orange color to dark orange within 80 min. 2.3.
Characterization UV-Vis spectral analysis was performed using a Cary- 4000 spectrophotometer. The
morphology of the prepared AgNPs was observed by SEM (Hitachi, S-4800), EDX (Horiba, 6853-H), TEM (Hitachi, H-7100) and AFM (Veeco dimension 3100 SPM). The structure and composition of AgNPs were analyzed by XRD (AXS D8 Advance). Further characterization was accomplished using FTIR (Bruker Germany). 2.4.
Disc diffusion method Synthesized AgNPs were tested for antibacterial activity against Gram-positive (S.
epidermis), and Gram-negative (E. coli and P. mirabilis) by the disc-diffusion method. To accomplish this, the bacterial test organisms were grown in LB broth for 24 hrs, after which 200 µ l of the LB broth culture of each bacterial organism were spread on Muller-Hinton agar using cotton swab and allowed to dry for 10 minutes. Paper discs containing the AgNPs at 30 µL volume were then placed on agar plates along with discs containing penicillin and tetracycline. The samples were incubated for 24 hrs at 37º C. The inhibition zones were measured at the end of the incubation period. 3
Assessment of increase in fold area The increase in fold area was assessed by calculating the mean surface area of the
inhibition zone of each tested antibiotic using the following equation: (b-a) ⁄a×100, where a and b are the inhibition zones for A (reference drug) and B (reference drug+ AgNPs) respectively. 2.6.
ABTS radical scavenging Assay ABTS radical cations were produced by reacting ABTS (7mM) and potassium persulfate
(2.45mM) on incubating the mixture at room temperature in dark place for 16 hours. The solution thus obtained was further diluted with PBS to give an absorbance of 1.000. Different concentrations of the test sample in 10µl were added to 50µl water and 140 µl of ABTS working solution to give a final volume of 200µl. The absorbance was recorded immediately at 734 nm. BHT was used as reference standard. The percent inhibition was calculated from the following equation: Absorbance of control – Absorbance of test sample % inhibition = ---------------------------------------------------------------- × 100 Absorbance control 2.7.
Cell culture The A549 human lung cancer cell line (CCL-185) and MCF-7 human breast cancer cell
line (HTB-22 cell line (CRL-5822) were purchased from ATCC (American Type Culture Collection, VA, USA), and cultured in RPMI-1640 (Gibco BRL, Grand Island, NY, USA) or Eagle’s minimal essential medium (EMEM, Gobco BRL) supplemented with 10-20% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37°C in a humidified incubator under 5% CO2. 2.8.
Cell proliferation assay The antiproliferative activity of all compounds was measured using a WST-1 Cell
Proliferation Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, cells were seeded at 1×105 cells/well in 24-well flat-bottom culture plates, after which they were incubated with AgNPs at 100 µL volume for 24 h. After the initial incubation period, reconstituted WST1 mixture (10 µL) was added to each well and samples were further incubated for 2 hrs at 37°C in a CO2 incubator. The plates were then gently mixed on an orbital shaker for 1 min to ensure
the homogenous distribution of color. Finally, the absorbance was read at 450 nm using an automated microplate reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA, USA). 2.9.
Statistical analysis All statistical analyses were performed using GraphPad Prism 5.0 for the Macintosh
(GraphPad Software, San Diego, CA, USA). Data signify the means plus or minus the standard error of mean (means ± S.E.M.) of three samples and are representative of three independent experiments. 3.
Results and discussion Inonotus obliquus mushroom extract was added to an aqueous silver nitrate solution,
resulting in a rapid change in color from orange to dark orange within 80 min (Fig.1) due to excitation of surface plasmon vibrations in metal nanoparticles . One of the most widely used techniques for characterization of AgNPs is UV–Vis spectrophotometry . The UV-Vis spectra of the mushroom extract with silver nitrate showed a strong broad peak at 427 nm (Fig. 2), which indicated the presence of AgNPs . According to the Mie’s theory , only a single SPR band is expected in the absorption spectra of spherical metal nanoparticles, whereas anisotropic particles could give rise to two or more SPR bands depending on the shape of the particles. The nanoparticle solution of silver was found to be very stable for more than eight months with no signs of aggregation even at the end of this period. FTIR measurements were carried out to identify possible biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced AgNPs synthesized from mushroom extract. The FTIR spectrum of the mushroom extract showed peaks at 3435, 1635, 2100 and 666 cm-1, while biosynthesized AgNPs showed peaks at 3435, 1635, 2100 and 685 cm-1 (Fig.3), and no significant difference was observed between the spectral positions of absorption bands in mushroom extract and biosynthesized AgNPs. A broad band was also observed at 3396 cm-1 corresponding to the N-H stretching of secondary amines. The stronger band at 1635cm−1 was attributed to amide I vibrations corresponding to stretching of carbonyl groups in amide linkages . The weaker band at 2100 cm-1 was assigned to –C=C- stretching of alkynes, while the medium band at 666 cm-1 corresponded to alkynes (-C=C-H; C-H bend).
The XRD technique was used to determine and confirm the crystal structure of AgNPs (Fig.4). There were three well-defined characteristic diffraction peaks at 38.3°, 44.1° and 64.8 corresponding to the (111), (200) and (220) crystallographic planes of the cubic face-centered silver, respectively. Elemental analysis of the AgNPs was performed using EDX of the SEM. The SEM images revealed shapes that were either spherical or near spherical and ranging in the size from 14.2 to 35.2 nm (Fig 5), confirming the development of silver nanostructures. Strong signals from the silver atoms were observed, as well as signals from C, O,and Si. From the EDX spectrum, it is clear that AgNPs reduced by mushroom have the weight percentage of silver as 6.66% (Fig. 6). Transmission electron microscopy (TEM) has been employed to characterize the size, shape and morphology of synthesized AgNPs. The TEM image of AgNPs is shown in Fig. 7. The TEM images have shown that the formed AgNPs were predominately spherical in nature, but that triangle, hexagonal, and uneven shaped nanoparticles were also formed. The morphology of AgNPs was also determined using atomic force microscopy (AFM), which clearly showed the formation of spherical shaped nanoparticles (Fig 8). The effects of AgNPs , penicillin and tetracycline against gram negative (E.coli and P.mirabilis,) and gram positive bacteria (S.epidermidis) were investigated using the disk diffusion method. The diameter of the inhibition zones (in mm) are shown in table 1. The antibacterial activity of penicillin and tetracycline increased in the presence of AgNPs against both gram negative and gram positive bacteria (Fig.9). The antibacterial activity of penicillin against gram negative and gram positive bacteria was greater in the presence of AgNPs than tetracycline. The largest increase in the fold area was observed against S.epidermidis (75%) followed by E. coli (46.66%) and P. mirabilis (13.63%). Tetracycline in combination with AgNPs produced the increase in fold area against S.epidermidis (42.85%) followed by E.coli (31.57%) and P.mirabilis (9.09%). No enhancement of antibacterial activities of AgNPs was observed against P.mirabilis and S. epidermidis. The antibacterial activities of penicillin and tetracycline increased in the presence of AgNPs against both gram positive and gram negative bacteria. AgNPs are decrease in size and large surface area than silver ions, which allows them to closely interact with antibiotics. The antibiotic molecules contain active groups such as hydroxyl and amido groups, which can easily react with AgNPs via chelation . Wang et al.  6
reported, AgNPs reacting with sulfur containing proteins in the interior of the cell as well as phosphorous containing compounds such as DNA will affect the respiratory chain and cell division in bacteria, ultimately causing the death of the cell. ABTS•+ generated from the oxidation of ABTS•+ by potassium persulphate, is a excellent tool for determining the antioxidant activity of hydrogen-donating and chain breaking antioxidants . Free radical scavenging activity of the AgNPs on ABTS radicals was found to increase with increase in the concentration, showing maximum inhibition (76.57%) at 1 mM and minimum inhibition (60.98%) at 0.125 mM solution (Fig.10). The antiproliferative activity of AgNPs (1mM) against A549 human lung cancer (CCL-185) and MCF-7 human breast cancer (HTB-22) cell lines was investigated. AgNPs significantly inhibited the cell proliferation in A549 (51.61%) and MCF-7 (59.11) cells, when compared with standard (Fig.11). The cytotoxic effects of silver are the result of active physicochemical interaction of silver atoms with the functional groups of intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA .
Conclusion A simple, stable and eco-friendly method of biosynthesizing AgNPs was successfully
developed using Inonotus obliquus (Chaga) mushroom extract. Mushrooms containing proteins played major roles as reducing as well as capping agents for use in synthesis of novel AgNPs, in acting as a reducing as well as a capping agent in order to synthesize a novel AgNPs. Furthermore, these functionalized AgNPs were more effective as antibacterial agents both against gram positive and gram negative bacteria. All AgNPs samples showed, 1Mm (76.57%), 0.5 mM (72.5%, 0.25 mM (64.09%) and 0.125 mM (60.98%) good ABTS free radical scavenging activity. Finally, the AgNPs showed significant cytotoxicity against A549 human lung cancer (CCL-185) and MCF-7 human breast cancer (HTB-22) cell lines.
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Figure 1. Color change of Inonotus obliquus extract containing silver before and after synthesis of AgNPs
Figure 2. UV-visible spectrum of AgNPs synthesized by treating 1mM aqueous AgNO3 solution with Inonotus obliquus extract after 80 min.
Figure 3. FTIR spectra of Inonotus obliquus extract and AgNPs from Inonotus obliquus extract.
Figure 4. XRD pattern of green synthesized AgNPs from Inonotus obliquus extract.
Figure 5. SEM images of spherical AgNPs synthesized by Inonotus obliquus extract.
Figure 6. EDX spectra of AgNPs synthesized from Inonotus obliquus extract.
Figure 7. TEM image showing spherical, triangle, hexagonal and uneven shaped of green synthesized AgNPs from Inonotus obliquus extract.
Figure 8. AFM image of green synthesized AgNPs from Inonotus obliquus extract.
Figure 9. Antibacterial activity assay against gram negative and gram positive bacteria.
Figure 10. ABTS activity of green synthesized AgNPs from Inonotus obliquus extract.
Figure 11. Dose-dependent antiproliferative effect of AgNPS on cancer cells. Two cancer cells were treated with standard and AgNPS at dose of 100 µL for 24 hrs, and measured the cell proliferation by WST-1 assay. Results are represented as percentage of cell growth with the mean ± S.E.M. of three independent experiments
Table 1. Inhibition zones (mm) of antibiotics alone and in combination with AgNPs against gram negative and gram positive bacteria.
Zone of inhibition (mm) Bacteria
+ AgNPs (%)
(KCTC 2441) P.mirabilis (KCTC 2556) S.epidermi dis (KCTC 1917)
A simple, stable and eco-friendly method of biosynthesizing AgNPs was successfully
developed using Inonotus obliquus extract. The TEM images have shown that the formed AgNPs were predominately spherical in
nature, but that triangle, hexagonal, and uneven shaped nanoparticles were also formed. AgNPs showed significant cytotoxicity against A549 human lung cancer (CCL-185) and
MCF-7 human breast cancer (HTB-22) cell lines.