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Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications
Journal Pre-proofs Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications N. Valarmathi, Fuad Ameen, A. Almansob, P. Kumar, S. Arunprakash, M. Govarthanan PII: DOI: Reference:
S0167-577X(19)31876-2 https://doi.org/10.1016/j.matlet.2019.127244 MLBLUE 127244
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
22 October 2019 10 December 2019 23 December 2019
Please cite this article as: N. Valarmathi, F. Ameen, A. Almansob, P. Kumar, S. Arunprakash, M. Govarthanan, Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications, Materials Letters (2019), doi: https://doi.org/10.1016/j.matlet.2019.127244
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Elsevier B.V. All rights reserved.
Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications N. Valarmathi1, Fuad Ameen2, A. Almansob2, P. Kumar3, S. Arunprakash1*, M. Govarthanan4* 1Department
of Botany, Arignar Anna Government Arts College, Namakkal-637 002, Tamil Nadu, India.
2Department
of Botany & Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
3Department
of Animal Health and Management, Alagappa University, Karaikudi – 630003, Tamil Nadu, India
4Department
of Environmental Engineering, Kyungpook National University, Daegu – 41566, South Korea.
*Corresponding authors: S. Arunprakash, [email protected]; M. Govarthanan, [email protected]; https://orcid.org/0000-0001-8725-3059
Abstract The present study describes an environment-friendly biosynthesis of silver nanoparticles from marine red algae Spyridia filamentosa through the bio-reduction process. S. filamentosa have significantly high amounts of phytochemicals and secondary metabolites responsible for the formation of AgNPs. UV-Vis spectra results showed corresponding AgNPs peak at 420 nm. Transmission electron micrographs showed well dispersed spherical particles with the 20-30 nm in size. FT-IR and XRD results revealed the characteristics of AgNPs. Further, the AgNPs displayed potential antibacterial activity against Staphylococcus sp. (KC688883) and Klebsiella sp. (KC899845), and anticancer activity against MCF-7 cells.
Keywords: Antibacterial; apoptopic; MCF-7 cells: seaweed; Spyridia filamentosa
1. Introduction Green nano-biotechnology deals for the formation of silver nanoparticles (AgNPs) from various biological sources [1-4] with different morphology and sizes with potential industrial applications [5]. Presently, there is an increasing attention to the synthesis of AgNPs from marine seaweeds. Among the marine seaweeds, red algae have received considerable attention due to their abundance, biologically active substances and ease of use. Recent researches have displayed the production of AgNPs from seaweeds and their potential biological applications [68]. However, this is the first report on synthesis of AgNPs using red algae Spyridia filamentosa. S. filamentosa is one of the most common marine red algae present along tropical and warm temperature coasts and contain a broad variety of therapeutically important phytochemicals such as, alkaloids, phenols, flavonoids and terpenoids. It has been reported that the marine seaweeds possess potential antibacterial and antiviral activities against clinical pathogens [9-10]. Hence, the current research explores the biosynthesis of AgNPs from S. filamentosa and evaluates their potential antibacterial and cytotoxic properties 2. Materials and methods 2.1. S. filamentosa collection Red seaweed S. filamentosa was collected from coastal area of Kanyakumari District, Tamil Nadu, India. Freshly collected S. filamentosa was thoroughly washed with running tap water to remove all the impurities attached on the surface of the S. filamentosa. Later the seaweed was rinsed using distilled water and air dehydrated for 5-7 days under shade. The air dried seaweed was ground into fine powder and used for further study. All other chemicals used in this study were of analytical grade and purchased from Sigma –Aldrich (USA). 2.2. S. filamentosa extract preparation and AgNPs synthesis
Two gram of powdered S. filamentosar was mixed with 100 mL of distilled water, boil for 10-20 min, and filtered through filter paper (Whatman No.1). Filtered supernatant was used as an extract for the AgNPs synthesis. Briefly, 20 mL of S. filamentosa extract was dissolved in 80 mL of AgNO3 (1 MM) and incubated at 37 ˚C under static condition for 2 h. Formation of AgNPs was noticed from colorless to brown color. The resulting AgNPs were centrifuged at 8,000g for 10 min. The deposited AgNPs were washed several times (~3-4 times) with double distilled water, lyophilized and stored 4 °C. 2.3. Characterization of AgNPs The optical absorption of the AgNPs was measured by UV-Vis spectroscopy (Elico-SL 164, Hyderabad, India). The size and shape of the AgNPs was measured by transmission electron microscopy (TEM, FEI Tecnai TF 20 high resolution). Presence of silver compound in the AgNPs was identified using scanning electron micrograph-energy dispersive spectroscopy (SEM–EDS; Jeol JSM 6390). The bio-molecules of the S. filamentosa extract and AgNPs were identified by Fourier transform infrared spectroscopy (FTIR, Perkin-Elmer (IRAffinity-1S)). To determine crystalline nature of AgNPs, X-ray powder diffraction was performed (XPERT-Pro diffractometer using Cu-Ka radiation). 2.4. Antibacterial activity The antibacterial potential of AgNPs was tested against the clinical pathogens according to Govarthanan et al. [1]. Gram-negative Klebsiella sp. (KC899845) and Grampositive Staphylococcus sp. (KC688883) were used for the determination of antibacterial activity. Four different concentrations of AgNPs (5, 10, 15 and 20 mM) were tested with different time intervals (12, 24, and 48 h). The growth performances under AgNPs stress of the pathogens were
observed at 600 nm. Control experiments were performed without addition of AgNPs. Growth inhibition (%) was calculated by using absorption units according to Elegbede et al. [11] 2.5. In-vitro cytotoxicity and apoptotic staining Cytotoxic of potentials of AgNPs (0, 25, 50, 75 and 100 µg mL-1) were analyzed by MTT assay according to Mosmann et al. [12] using MCF-7 breast cancer cells. To understand the morphological features of the cell lines treated with AgNPs were measured by using Acridine orange/Ethidium bromide (AO/EB) and Hoechst 33342 staining and visualized under fluorescence microscope (Accuscope, EXI-310) at a magnification of 20X [13]. 3. Results and discussion Biosynthesis of AgNPs was initially observed by color change and UV-Vis spectroscopic analysis. The AgNPs formation was clearly observed within 20-30 min after S. filamentosa extract was added to the AgNO3 solution. The reaction mixture has been turned into brown color from colorless solution because of bio-reduction of silver ions to AgNPs [14]. The UV-Vis spectrum showed an absorbance band around 420 nm, which is assigned to the AgNPs. It has been well documented that the phytochemicals and/or secondary metabolites of S. filamentosa extracts are largely responsible for the bio-reduction of silver into AgNPs [15]. Fig.1 (a) represents the shape and dimension of the AgNPs. The TEM micrograph revealed the spherical nature of AgNPs 20-30 nm in size. SEM-EDS confirmed the presence of a characteristic silver peak along with signal of Cl. Fig. 1 (b) showed the EDS graph of elemental silver peak at 3 KeV [14]. FTIR spectrum of S. filamentosa extract (supplementary fig.1) and AgNPs are represented in Fig.2. The IR spectrum of AgNPs showed multiple absorbance bands at 3402,
2922, 2852, 1652, 1456, 1099 and 617 cm-1 (Fig.2). The broad band at 3402 cm-1 reveals the possibly presence of hydroxyl and carbonyl groups in the S. filamentosa. Small and sharp peaks at 2922, and 2852 cm-1 were represents the methyl groups present in the in the S. filamentosa. The peaks at 1652 and 1456 cm-1 were attributed stretching vibrations of carboxyl groups. The bands at 1099 and 617 cm-1 were assigned to the carboxylic acids, ether and alcoholic groups. Similar characteristic FTIR peaks of the AgNPs were obtained from AgNPs synthesized from Caulerpa scalpelliformis [15]. The results are consistent with other marine seaweeds such as, Sargassum wightii, Padina tetrastromatica, and Ulva lactuca [16, 17]. XRD results showed the face-centered cubic nature of Ag (Fig.3). The strong intensities of the ((111), (200), (311), (142), and (220)) diffraction peaks established the cubic structure of AgNPs synthesized from S. filamentosa. The antibacterial potential of S. filamentosa AgNPs was evaluated against Grampositive Staphylococcus sp. (KC688883) and Gram-negative Klebsiella sp. (KC899845) bacteria. The dose dependant AgNPs concentration showed significant growth inhibition of the pathogens. Briefly, 63.4% of Klebsiella sp., growth was inhibited by 20 mM concentration of AgNPs at 48 h. However, only 44.6% of Staphylococcus sp., growth was inhibited at the same concentration of AgNPs at 48 h (Fig.4 a and b). The results revealed that, the AgNPs posses strong antibacterial inhibition rate against Gram-negative Klebsiella sp. It has been also reported that the AgNPs have strong lethal effect to Gram-negative bacteria than the Gram-positive bacteria [18]. Song et al. [18] reported that the graphene oxide silver (Go-Ag) shown strong growth inhibition activity against gram negative Escherichia coli than the Gram-positive Staphylococcus aureus.
The cytotoxic potential of AgNPs was tested against human breast cancer cells (MCF-7). Viability of MCF-7 cells with the increasing concentrations of AgNPs (25, 50, 75 and 100 µg mL-1) and the results revealed that the viability of the cells was related to the quantity of AgNPs (data not shown). Vivek et al. [19] studied the cytotoxicity of AgNPs was considerably improved according to the quantity of AgNPs. Fig.5 (a) represents viable cancer cells and fig.5 (b) represents the death of cancer cells induced by AgNPs with orange to red by fluorescence inferring fragmented chromatin of cells. Fig. 5 (c) and (d) represents cancerous cell and nuclear fragmented apoptotic cells. The results are consistent with previous study reported impact of nanopartices on induced apoptosis [13]. Overall, the results demonstrated that the AgNPs have a better ability to pronounce apoptosis of cancer cells. Conclusion A marine source of S. filamentosa was successfully used to synthesize AgNPs for the first time. S. filamentosa mediated synthesis of AgNPs are sphere-shaped with the range between 20-30 nm in size. The S. filamentosa extract capped AgNPs exhibited a strong lethal effect to the both Gram-positive Staphylococcus sp. and Gram-negative Klebsiella sp. In addition, the AgNPs showed strong toxic activity to MCF-7 cell lines, which suggests that the AgNPs could be very useful in cancer treatments. Overall, AgNPs synthesized from the marine S. filamentosa could be used as an efficient antibacterial agent in food, textile industries as well as medical applications. Acknowledgement The authors Fuad Ameen and A. Almansob extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group NO (RGP-1438-029).
References [1] M. Govarthanan, T. Selvankumar, K. Manoharan, R. Rathika, K. Shanthi, K. J. Lee, M. Cho, S. Kamala-Kannan, B.T. Oh, Int. J. Nanomed., 9 (2014), 1593-1599 [2] M. Govarthanan, Y.S. Seo, K.J. Lee, I.B. Jung, H.J. Ju, J.S. Kim, M. Cho, S. Kamala-Kannan, B.T. Oh, Artif. Cells Nanomed. Biotechnol., 44 (2016), 1878-1882 [3] M. Govarthanan, T. Selvankumar, R. Mythili, C. Sudhakar, K. Selvam, Environ. Sci. Pollut. Res., 24 (2017), 19459-19464 [4] R. Mythili, T. Selvankumar, S. Kamala-Kannan, C. Sudhakar, F. Ameen, A.A. Sabri, K. Selvam, M. Govarthanan, H. Kim, Mater. Lett., 225 (2018), 101-104 [5] R.R.R. Kannan, W.A. Stirk, J. Van Staden, South A. J. Bot., 86 (2013) 1-4 [6] M. Vivek, P.S. Kumar, S. Steffi, S. Sudha, Avicenna J. Med. Biotechnol., 3 (2011), 143-148 [7]
K. Vijayaraghavan, A. Mahadevan, M. Sathishkumar, S. Pavagadhi, R. Balasubramanian
Chem. Eng. J., 167 (2011), 223-227 [8] S. Rajeshkumar, C. Kannan, G. Annadurai, Drug. Invent. Today, 4 (10) (2012), 511-513 [9] R. Cyril, R. Lakshmanan, A. Thiyagarajan, J. Coast. Life Med., 5 (10) (2017), 427-432 [10] G. Adaikalaraj, R.D. Patric, M. Johnson, N. Janakiraman, A. Babu, Asian Pac. J. Trop. Biomed., 2 (2) (2012), 1077-1080
[11] J.A. Elegbede, A. Lateef, M.A. Azeer, T.B. Asafa, T.A. Yekeen, I.C. Oladipo, D.A. Aina, L.S. Beukes, E. B. Gueguim-Kana, Waste Biomass Valorizat. (2018), 10.1007/s12649-0180540-2 [12] T. Mosmann, J. Immunol. Methods. 65 (1983) 55–63 [13] S. Jeyarani, N.M. Vinitha, P. Puja, S. Senthamilselvi, U. Devan, A.J. Velangani, M. biruntha, A. Pugazhendhi, P. Kumar, J. Photoch. Photobio B. 202 (2020) 111715. [14] R. Manikandan, B. Manikandan, T. Raman, K. Arunagirinathan, N.M. Prabhu, M. Jothi Basu, M. Perumal, S. Palanisamy, A. Munusamy, Spectrochim Acta A., 138 (2015), 120-129 [15] R. Manikandan, R. Anjali, M. Beulaja, N.M. Prabhu, A. Koodalingam, G. Saiprasad, P. Chitra, M. Arumugam, Process Biochem., 79 (2019) 135-141. [16] S. Rajeshkumar, C. Kannan, G. Annadurai, Drug. Invent. Today, 4 (10) (2012), 511-513 [17] J. Devi, B. Saraniya Bhimba, D.M. Valentin Peter, Indian J. Geo-Mar. Sci., 42 (2012), 125130 [18] B. Song, C. Zhang, G. Zeng, J. Gong, Y. Chang, Y. Jiang, Arch. Biochem. Biophys. 604 (2016) 167-176 [19] Vivek R, Thangam R, Muthuchelian K, Gunasekaran P, Kaveri K, Kannan S, Process Biochem. (2012), 47, 2405–2410 Figure legends Fig.1 (a) TEM imgae of AgNPs synthesized from S. filamentosa, (b) SEM-EDS of AgNPs. Fig.2. FT-IR Spectrum of AgNPs Fig.3. XRD pattern of AgNPs Fig.4. Antibacterial activity of AgNPs (a) Staphylococcus sp., (b) Klebsiella sp. Fig.5. Fluorescence based staining (a & b) and dual staining (c & d)
Highlights Silver nanoparticles (AgNPs) were successfully synthesized from the marine seaweed Spyridia filamentosa The adopted synthesis method was eco-friendly, and rapid. The AgNPs are well dispersed and spherical in shape 20-30 nm in size. AgNPs showed extensive antibacterial activity against both Gram-positive and Gramnegative bacteria AgNPs exhibited potential cytotoxic activity against MCF-7 cells.
Fig.1
Fig.2
Fig.3
Fig.4 (a)
Fig. 4 (b)
Fig.5
Author contributions All authors conceived the Study, N. Valarmathi and S. Arunprakash designed and performed the experiment, collected data, analysed data and wrote the draft manuscript. Fuad Ameen and Almansob contributed data analysis and revisions. P. Kumar contributed FT-IR analysis cytotoxicity experiments and revision. M. Goarthanan antibacterial activity experiments, data analysis, and revision. All authors commented on the manuscript.
Conflict of interest
The authors declare that they don’t have any conflict of interest in this manuscript
Declaration Statement The authors declared that they dont have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. any conflict of interest in this manuscript.
Supplementary Figures Fig.1. FT-IR spectrum of S. filamentosa extract
S0167-577X(19)31876-2 https://doi.org/10.1016/j.matlet.2019.127244 MLBLUE 127244
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
22 October 2019 10 December 2019 23 December 2019
Please cite this article as: N. Valarmathi, F. Ameen, A. Almansob, P. Kumar, S. Arunprakash, M. Govarthanan, Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications, Materials Letters (2019), doi: https://doi.org/10.1016/j.matlet.2019.127244
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Elsevier B.V. All rights reserved.
Utilization of marine seaweed Spyridia filamentosa for silver nanoparticles synthesis and its clinical applications N. Valarmathi1, Fuad Ameen2, A. Almansob2, P. Kumar3, S. Arunprakash1*, M. Govarthanan4* 1Department
of Botany, Arignar Anna Government Arts College, Namakkal-637 002, Tamil Nadu, India.
2Department
of Botany & Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
3Department
of Animal Health and Management, Alagappa University, Karaikudi – 630003, Tamil Nadu, India
4Department
of Environmental Engineering, Kyungpook National University, Daegu – 41566, South Korea.
*Corresponding authors: S. Arunprakash, [email protected]; M. Govarthanan, [email protected]; https://orcid.org/0000-0001-8725-3059
Abstract The present study describes an environment-friendly biosynthesis of silver nanoparticles from marine red algae Spyridia filamentosa through the bio-reduction process. S. filamentosa have significantly high amounts of phytochemicals and secondary metabolites responsible for the formation of AgNPs. UV-Vis spectra results showed corresponding AgNPs peak at 420 nm. Transmission electron micrographs showed well dispersed spherical particles with the 20-30 nm in size. FT-IR and XRD results revealed the characteristics of AgNPs. Further, the AgNPs displayed potential antibacterial activity against Staphylococcus sp. (KC688883) and Klebsiella sp. (KC899845), and anticancer activity against MCF-7 cells.
Keywords: Antibacterial; apoptopic; MCF-7 cells: seaweed; Spyridia filamentosa
1. Introduction Green nano-biotechnology deals for the formation of silver nanoparticles (AgNPs) from various biological sources [1-4] with different morphology and sizes with potential industrial applications [5]. Presently, there is an increasing attention to the synthesis of AgNPs from marine seaweeds. Among the marine seaweeds, red algae have received considerable attention due to their abundance, biologically active substances and ease of use. Recent researches have displayed the production of AgNPs from seaweeds and their potential biological applications [68]. However, this is the first report on synthesis of AgNPs using red algae Spyridia filamentosa. S. filamentosa is one of the most common marine red algae present along tropical and warm temperature coasts and contain a broad variety of therapeutically important phytochemicals such as, alkaloids, phenols, flavonoids and terpenoids. It has been reported that the marine seaweeds possess potential antibacterial and antiviral activities against clinical pathogens [9-10]. Hence, the current research explores the biosynthesis of AgNPs from S. filamentosa and evaluates their potential antibacterial and cytotoxic properties 2. Materials and methods 2.1. S. filamentosa collection Red seaweed S. filamentosa was collected from coastal area of Kanyakumari District, Tamil Nadu, India. Freshly collected S. filamentosa was thoroughly washed with running tap water to remove all the impurities attached on the surface of the S. filamentosa. Later the seaweed was rinsed using distilled water and air dehydrated for 5-7 days under shade. The air dried seaweed was ground into fine powder and used for further study. All other chemicals used in this study were of analytical grade and purchased from Sigma –Aldrich (USA). 2.2. S. filamentosa extract preparation and AgNPs synthesis
Two gram of powdered S. filamentosar was mixed with 100 mL of distilled water, boil for 10-20 min, and filtered through filter paper (Whatman No.1). Filtered supernatant was used as an extract for the AgNPs synthesis. Briefly, 20 mL of S. filamentosa extract was dissolved in 80 mL of AgNO3 (1 MM) and incubated at 37 ˚C under static condition for 2 h. Formation of AgNPs was noticed from colorless to brown color. The resulting AgNPs were centrifuged at 8,000g for 10 min. The deposited AgNPs were washed several times (~3-4 times) with double distilled water, lyophilized and stored 4 °C. 2.3. Characterization of AgNPs The optical absorption of the AgNPs was measured by UV-Vis spectroscopy (Elico-SL 164, Hyderabad, India). The size and shape of the AgNPs was measured by transmission electron microscopy (TEM, FEI Tecnai TF 20 high resolution). Presence of silver compound in the AgNPs was identified using scanning electron micrograph-energy dispersive spectroscopy (SEM–EDS; Jeol JSM 6390). The bio-molecules of the S. filamentosa extract and AgNPs were identified by Fourier transform infrared spectroscopy (FTIR, Perkin-Elmer (IRAffinity-1S)). To determine crystalline nature of AgNPs, X-ray powder diffraction was performed (XPERT-Pro diffractometer using Cu-Ka radiation). 2.4. Antibacterial activity The antibacterial potential of AgNPs was tested against the clinical pathogens according to Govarthanan et al. [1]. Gram-negative Klebsiella sp. (KC899845) and Grampositive Staphylococcus sp. (KC688883) were used for the determination of antibacterial activity. Four different concentrations of AgNPs (5, 10, 15 and 20 mM) were tested with different time intervals (12, 24, and 48 h). The growth performances under AgNPs stress of the pathogens were
observed at 600 nm. Control experiments were performed without addition of AgNPs. Growth inhibition (%) was calculated by using absorption units according to Elegbede et al. [11] 2.5. In-vitro cytotoxicity and apoptotic staining Cytotoxic of potentials of AgNPs (0, 25, 50, 75 and 100 µg mL-1) were analyzed by MTT assay according to Mosmann et al. [12] using MCF-7 breast cancer cells. To understand the morphological features of the cell lines treated with AgNPs were measured by using Acridine orange/Ethidium bromide (AO/EB) and Hoechst 33342 staining and visualized under fluorescence microscope (Accuscope, EXI-310) at a magnification of 20X [13]. 3. Results and discussion Biosynthesis of AgNPs was initially observed by color change and UV-Vis spectroscopic analysis. The AgNPs formation was clearly observed within 20-30 min after S. filamentosa extract was added to the AgNO3 solution. The reaction mixture has been turned into brown color from colorless solution because of bio-reduction of silver ions to AgNPs [14]. The UV-Vis spectrum showed an absorbance band around 420 nm, which is assigned to the AgNPs. It has been well documented that the phytochemicals and/or secondary metabolites of S. filamentosa extracts are largely responsible for the bio-reduction of silver into AgNPs [15]. Fig.1 (a) represents the shape and dimension of the AgNPs. The TEM micrograph revealed the spherical nature of AgNPs 20-30 nm in size. SEM-EDS confirmed the presence of a characteristic silver peak along with signal of Cl. Fig. 1 (b) showed the EDS graph of elemental silver peak at 3 KeV [14]. FTIR spectrum of S. filamentosa extract (supplementary fig.1) and AgNPs are represented in Fig.2. The IR spectrum of AgNPs showed multiple absorbance bands at 3402,
2922, 2852, 1652, 1456, 1099 and 617 cm-1 (Fig.2). The broad band at 3402 cm-1 reveals the possibly presence of hydroxyl and carbonyl groups in the S. filamentosa. Small and sharp peaks at 2922, and 2852 cm-1 were represents the methyl groups present in the in the S. filamentosa. The peaks at 1652 and 1456 cm-1 were attributed stretching vibrations of carboxyl groups. The bands at 1099 and 617 cm-1 were assigned to the carboxylic acids, ether and alcoholic groups. Similar characteristic FTIR peaks of the AgNPs were obtained from AgNPs synthesized from Caulerpa scalpelliformis [15]. The results are consistent with other marine seaweeds such as, Sargassum wightii, Padina tetrastromatica, and Ulva lactuca [16, 17]. XRD results showed the face-centered cubic nature of Ag (Fig.3). The strong intensities of the ((111), (200), (311), (142), and (220)) diffraction peaks established the cubic structure of AgNPs synthesized from S. filamentosa. The antibacterial potential of S. filamentosa AgNPs was evaluated against Grampositive Staphylococcus sp. (KC688883) and Gram-negative Klebsiella sp. (KC899845) bacteria. The dose dependant AgNPs concentration showed significant growth inhibition of the pathogens. Briefly, 63.4% of Klebsiella sp., growth was inhibited by 20 mM concentration of AgNPs at 48 h. However, only 44.6% of Staphylococcus sp., growth was inhibited at the same concentration of AgNPs at 48 h (Fig.4 a and b). The results revealed that, the AgNPs posses strong antibacterial inhibition rate against Gram-negative Klebsiella sp. It has been also reported that the AgNPs have strong lethal effect to Gram-negative bacteria than the Gram-positive bacteria [18]. Song et al. [18] reported that the graphene oxide silver (Go-Ag) shown strong growth inhibition activity against gram negative Escherichia coli than the Gram-positive Staphylococcus aureus.
The cytotoxic potential of AgNPs was tested against human breast cancer cells (MCF-7). Viability of MCF-7 cells with the increasing concentrations of AgNPs (25, 50, 75 and 100 µg mL-1) and the results revealed that the viability of the cells was related to the quantity of AgNPs (data not shown). Vivek et al. [19] studied the cytotoxicity of AgNPs was considerably improved according to the quantity of AgNPs. Fig.5 (a) represents viable cancer cells and fig.5 (b) represents the death of cancer cells induced by AgNPs with orange to red by fluorescence inferring fragmented chromatin of cells. Fig. 5 (c) and (d) represents cancerous cell and nuclear fragmented apoptotic cells. The results are consistent with previous study reported impact of nanopartices on induced apoptosis [13]. Overall, the results demonstrated that the AgNPs have a better ability to pronounce apoptosis of cancer cells. Conclusion A marine source of S. filamentosa was successfully used to synthesize AgNPs for the first time. S. filamentosa mediated synthesis of AgNPs are sphere-shaped with the range between 20-30 nm in size. The S. filamentosa extract capped AgNPs exhibited a strong lethal effect to the both Gram-positive Staphylococcus sp. and Gram-negative Klebsiella sp. In addition, the AgNPs showed strong toxic activity to MCF-7 cell lines, which suggests that the AgNPs could be very useful in cancer treatments. Overall, AgNPs synthesized from the marine S. filamentosa could be used as an efficient antibacterial agent in food, textile industries as well as medical applications. Acknowledgement The authors Fuad Ameen and A. Almansob extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group NO (RGP-1438-029).
References [1] M. Govarthanan, T. Selvankumar, K. Manoharan, R. Rathika, K. Shanthi, K. J. Lee, M. Cho, S. Kamala-Kannan, B.T. Oh, Int. J. Nanomed., 9 (2014), 1593-1599 [2] M. Govarthanan, Y.S. Seo, K.J. Lee, I.B. Jung, H.J. Ju, J.S. Kim, M. Cho, S. Kamala-Kannan, B.T. Oh, Artif. Cells Nanomed. Biotechnol., 44 (2016), 1878-1882 [3] M. Govarthanan, T. Selvankumar, R. Mythili, C. Sudhakar, K. Selvam, Environ. Sci. Pollut. Res., 24 (2017), 19459-19464 [4] R. Mythili, T. Selvankumar, S. Kamala-Kannan, C. Sudhakar, F. Ameen, A.A. Sabri, K. Selvam, M. Govarthanan, H. Kim, Mater. Lett., 225 (2018), 101-104 [5] R.R.R. Kannan, W.A. Stirk, J. Van Staden, South A. J. Bot., 86 (2013) 1-4 [6] M. Vivek, P.S. Kumar, S. Steffi, S. Sudha, Avicenna J. Med. Biotechnol., 3 (2011), 143-148 [7]
K. Vijayaraghavan, A. Mahadevan, M. Sathishkumar, S. Pavagadhi, R. Balasubramanian
Chem. Eng. J., 167 (2011), 223-227 [8] S. Rajeshkumar, C. Kannan, G. Annadurai, Drug. Invent. Today, 4 (10) (2012), 511-513 [9] R. Cyril, R. Lakshmanan, A. Thiyagarajan, J. Coast. Life Med., 5 (10) (2017), 427-432 [10] G. Adaikalaraj, R.D. Patric, M. Johnson, N. Janakiraman, A. Babu, Asian Pac. J. Trop. Biomed., 2 (2) (2012), 1077-1080
[11] J.A. Elegbede, A. Lateef, M.A. Azeer, T.B. Asafa, T.A. Yekeen, I.C. Oladipo, D.A. Aina, L.S. Beukes, E. B. Gueguim-Kana, Waste Biomass Valorizat. (2018), 10.1007/s12649-0180540-2 [12] T. Mosmann, J. Immunol. Methods. 65 (1983) 55–63 [13] S. Jeyarani, N.M. Vinitha, P. Puja, S. Senthamilselvi, U. Devan, A.J. Velangani, M. biruntha, A. Pugazhendhi, P. Kumar, J. Photoch. Photobio B. 202 (2020) 111715. [14] R. Manikandan, B. Manikandan, T. Raman, K. Arunagirinathan, N.M. Prabhu, M. Jothi Basu, M. Perumal, S. Palanisamy, A. Munusamy, Spectrochim Acta A., 138 (2015), 120-129 [15] R. Manikandan, R. Anjali, M. Beulaja, N.M. Prabhu, A. Koodalingam, G. Saiprasad, P. Chitra, M. Arumugam, Process Biochem., 79 (2019) 135-141. [16] S. Rajeshkumar, C. Kannan, G. Annadurai, Drug. Invent. Today, 4 (10) (2012), 511-513 [17] J. Devi, B. Saraniya Bhimba, D.M. Valentin Peter, Indian J. Geo-Mar. Sci., 42 (2012), 125130 [18] B. Song, C. Zhang, G. Zeng, J. Gong, Y. Chang, Y. Jiang, Arch. Biochem. Biophys. 604 (2016) 167-176 [19] Vivek R, Thangam R, Muthuchelian K, Gunasekaran P, Kaveri K, Kannan S, Process Biochem. (2012), 47, 2405–2410 Figure legends Fig.1 (a) TEM imgae of AgNPs synthesized from S. filamentosa, (b) SEM-EDS of AgNPs. Fig.2. FT-IR Spectrum of AgNPs Fig.3. XRD pattern of AgNPs Fig.4. Antibacterial activity of AgNPs (a) Staphylococcus sp., (b) Klebsiella sp. Fig.5. Fluorescence based staining (a & b) and dual staining (c & d)
Highlights Silver nanoparticles (AgNPs) were successfully synthesized from the marine seaweed Spyridia filamentosa The adopted synthesis method was eco-friendly, and rapid. The AgNPs are well dispersed and spherical in shape 20-30 nm in size. AgNPs showed extensive antibacterial activity against both Gram-positive and Gramnegative bacteria AgNPs exhibited potential cytotoxic activity against MCF-7 cells.
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Author contributions All authors conceived the Study, N. Valarmathi and S. Arunprakash designed and performed the experiment, collected data, analysed data and wrote the draft manuscript. Fuad Ameen and Almansob contributed data analysis and revisions. P. Kumar contributed FT-IR analysis cytotoxicity experiments and revision. M. Goarthanan antibacterial activity experiments, data analysis, and revision. All authors commented on the manuscript.
Conflict of interest
The authors declare that they don’t have any conflict of interest in this manuscript
Declaration Statement The authors declared that they dont have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. any conflict of interest in this manuscript.
Supplementary Figures Fig.1. FT-IR spectrum of S. filamentosa extract