nanomaterials Article
Antimicrobial, Antioxidant, and Anticancer Activities of Biosynthesized Silver Nanoparticles Using Marine Algae Ecklonia cava Jayachandran Venkatesan 1 , Se-Kwon Kim 2 and Min Suk Shim 1, * 1 2
*
Division of Bioengineering, Incheon National University, Incheon 406-772, Korea; [email protected] Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Korea; [email protected] Correspondence: [email protected]; Tel.: +82-32-835-8268
Academic Editor: Guogang Ren Received: 14 September 2016; Accepted: 25 November 2016; Published: 6 December 2016
Abstract: Green synthesis of silver nanoparticles (AgNPs) has gained great interest as a simple and eco-friendly alternative to conventional chemical methods. In this study, AgNPs were synthesized by using extracts of marine algae Ecklonia cava as reducing and capping agents. The formation of AgNPs using aqueous extract of Ecklonia cava was confirmed visually by color change and their surface plasmon resonance peak at 418 nm, measured by UV-visible spectroscopy. The size, shape, and morphology of the biosynthesized AgNPs were observed by transmission electron microscopy and dynamic light scattering analysis. The biosynthesized AgNPs were nearly spherical in shape with an average size around 43 nm. Fourier transform-infrared spectroscopy (FTIR) analysis confirmed the presence of phenolic compounds in the aqueous extract of Ecklonia cava as reducing and capping agents. X-ray diffraction (XRD) analysis was also carried out to demonstrate the crystalline nature of the biosynthesized AgNPs. Antimicrobial results determined by an agar well diffusion assay demonstrated a significant antibacterial activity of the AgNPs against Escherichia coli and Staphylococcus aureus. Antioxidant results determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay revealed an efficient antioxidant activity of the biosynthesized AgNPs. The biosynthesized AgNPs also exhibited a strong apoptotic anticancer activity against human cervical cancer cells. Our findings demonstrate that aqueous extract of Ecklonia cava is an effective reducing agent for green synthesis of AgNPs with efficient antimicrobial, antioxidant, and anticancer activities. Keywords: anticancer; antimicrobial; antioxidant; biosynthesis; Ecklonia cava; nanoparticle
1. Introduction In recent years, noble metal nanoparticles (NPs) have been intensively utilized for biomedical applications, such as diagnostics, drug delivery, and tissue engineering, due to their unique physicochemical and optoelectronic properties [1–4]. Among various noble metal nanoparticles, silver nanoparticles (AgNPs) have received great attention in a variety of applications, including nanoelectronic devices, sensors, imaging contrast agents, filters, and antimicrobial agents due to their good electrical conductivity, stability, optical property, and antimicrobial activity [5,6]. AgNPs have also extended their applications to cancer therapy. Several in vitro studies using AgNPs have demonstrated their potential as effective anticancer agents [7–10]. They have exhibited apoptosis-mediated, strong anticancer efficacies in a variety of cancer cells, including human cervical cancer [8], lung cancer [9], and breast cancer cells [10]. It has been well-documented that performance and applicability of AgNPs critically depend on their size, shape, composition, and surface chemistry [11–13]. Synthesis of noble
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metal NPs with controlled shape and size can be achieved through many different synthetic methods: evaporation-condensation, gamma irradiation, electron irradiation, microwave processing, microemulsion, sonochemical, electrochemical, photochemical, etc. [14–17]. One of the popular methods for the synthesis of defined noble metal NPs is chemical reduction [14]. Reduction of silver complexes in dilute solution with a proper reductant can lead to the formation of colloidal AgNPs [14]. Although this method offers significant advantages of simple equipment and convenient operation, it involves a use of hazardous chemicals and high temperature conditions, which are rather environmentally unfriendly and energetically inefficient [14,18]. To overcome the drawbacks of the chemical reduction method, biogenic synthesis that employs microorganisms and naturally occurring products has emerged as an environmentally friendly synthetic method (i.e., green chemistry) [17,19–21]. This method offers a facile and convenient entry to producing various noble metal NPs. Biogenic synthesis of noble metal NPs mainly relies on various extracts (e.g., nucleic acids, enzymes, proteins, peptides, vitamins, and polysaccharides) of microorganisms. For example, extracts of fungi, bacteria, and algae were extensively utilized to synthesize AgNPs, as shown in Table 1. Table 1. Biosynthesized AgNPs using various microorganisms. No.
Species
Reaction Time
Size
Applications
Ref.
1 2 3 4 5 6 7 8 9 10 11
Trichoderma viride Chaetoceros calcitrans Aspergillus clavatus Porphyra vietnamensis Gelidiella acerosa Sargassum tenerrimum Ulva fasciata Padina tetrastromatica Turbinaria conoides Padina pavonica Cryphonectria Pterocladia capillacae, Jania rubins, Ulva faciata, Colpmenia sinusa Gracilaria corticata Sargassum cinereum Padina gymnospora Sargassum plagiophyllum Sargassum longifolium Pithophora oedogonia Sesbania grandiflora Stenotrophomonas maltophilia Croton bonplandianum Chenopodium murale Alternanthera sessilis Pseudomonas putida Citrullus colocynthis Chlorella vulgaris Lovoa trichilioïdes Ficus elastica Ficus religiosa Morinda pubescens Saccharomyces boulardii Alternanthera sessilis (Linn.) Citrus reticulata juice Caulerpa racemosa Spirogyra varians Enteromorpha flexuos Cyanobacteria and Microalgae Dracocephalum moldavica Dimocarpus longan Lour. Curculigo orchioides rhizome Rhus chinensis
48 h 2 weeks N.A. 15 min 48 h 20 min N.A 24 h N.A 24 h 24 h
5–40 nm N.A. 10–25 nm 13 ± 3 nm 22 nm 20 nm 28–41 nm 14 nm 96 nm 46.8 nm 30–70nm
Antimicrobial Antimicrobial Antifungal Antibacterial Antifungal Anti-bacterial Antimicrobial Antibacterial Antibacterial Antimicrobial Antimicrobial
[13] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
3h
20 nm
Antimicrobial
[32]
20 min 3h N.A. 24 h 64 h N.A. 15 min N.A. N.A. N.A. N.A. 20 min 24 h 24 h 2h 30 min 10 min N.A. 4h N.A. N.A. 24 h N.A. N.A. 72 h 1h 5h N.A. 12 h
18–46 nm 45–76 nm 25–40 nm 21–48 nm N.A. 34.03 nm 12 nm 93 nm 32 nm 30–50 nm 10–30 nm 6–16 nm 31 nm 7 nm 37–43 nm 50–60 nm 5–35 nm N.A. 3–10 nm 20–30 nm N.A. 5–25 nm 17.6 nm 2–32 nm 13–31 nm 31 ± 6 nm 9–32 nm 15–18 nm 150 nm.
Antifungal Antimicrobial Antimicrobial Antimicrobial Antifungal Antibacterial Antimicrobial Antimicrobial Antimicrobial and anticancer Antimicrobial Anticancer Antibacterial and anticancer Anticancer Anticancer and antimicrobial Anti-bacterial Air pollution control In vivo antitumor Antioxidant and anticancer Anticancer Antimicrobial and antioxidant Antimicrobial Antibacterial Antibacterial Antimicrobial Antibacterial Antimicrobial Anticancer Larvicidal and Anticancer Antibacterial
[33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61]
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
N.A. = Not Available.
Nanomaterials 2016, 6, 235 40 Curculigo orchioides rhizome Nanomaterials 41 2016, 6, 235Rhus chinensis
3 of 17 N.A. 12 h
15–18 nm 150 nm.
Larvicidal and Anticancer Antibacterial
[60] [61] 3 of 18
N.A. = Not Available.
Seaweeds from fromthe theocean oceanare are considered “sea vegetables”, commonly as in food in Seaweeds considered as as “sea vegetables”, commonly usedused as food AsiaAsia-Pacific as Korea, China, Japan. Seaweeds oftenused usedtotoproduce produce hydrocolloids hydrocolloids Pacific areasareas suchsuch as Korea, China, andand Japan. Seaweeds areare often such as alginate, agar, and carrageenan. carrageenan. Ecklonia cava is an edible brown alga, mainly present in Korea, Previous studies studies reported reported that that phlorotannins phlorotannins such such as as phloroglucinol phloroglucinol are main Japan, and China. Previous responsible components in Ecklonia cava [62–65]. These compounds in Ecklonia cava have proved to be responsible bioactivities such suchas asantioxidant antioxidant[62,66], [62,66],anticancer anticancer [67–69], and antimicrobial activities for bioactivities [67–69], and antimicrobial activities [70].[70]. In In addition, antioxidant compounds from Eckloniacava cavaextracts, extracts,including including(a) (a)phloroglucinol; phloroglucinol;(b) (b) eckol; eckol; addition, antioxidant compounds from Ecklonia fucodiphlorethol G; G;(d) (d)phlorofucofuroeckol phlorofucofuroeckolA;A;(e)(e) 7-phloroeckol; dieckol; 6,60 -bieckol; (c) fucodiphlorethol 7-phloroeckol; (f) (f) dieckol; (g) (g) 6,6′-bieckol; (h) 0 0 (h) triphlorethol-A; and 2,7 -phloroglucinol-6,6 -bieckol (Figure1)1)[42,62,67–71], [42,62,67–71],could couldact actas as effective effective triphlorethol-A; and (i) (i) 2,7′-phloroglucinol-6,6′-bieckol (Figure reductants to synthesize noble metal NPs.
Figure phloroglucinol; (b) (b) eckol; (c) fucodiphlorethol G; (d) Figure 1. 1. Chemical Chemicalstructures structuresof:of:(a) (a) phloroglucinol; eckol; (c) fucodiphlorethol G; 0 phlorofucofuroeckol A; (e) 7-phloroeckol; (f) dieckol; (g) 6,6′-bieckol; (h) triphlorethol-A; and (i) (d) phlorofucofuroeckol A; (e) 7-phloroeckol; (f) dieckol; (g) 6,6 -bieckol; (h) triphlorethol-A; 2,7′and phloroglucinol-6,6′-bieckol. (i) 2,70 -phloroglucinol-6,60 -bieckol.
In this study, AgNPs were synthesized via reduction of silver ions using aqueous extracts of In this study, AgNPs were synthesized via reduction of silver ions using aqueous extracts Ecklonia cava as an alternative to conventional chemical reduction methods. To the best of our of Ecklonia cava as an alternative to conventional chemical reduction methods. To the best of our
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knowledge, use of Ecklonia cava extracts for the biosynthesis of AgNPs has not been attempted yet. knowledge, use of Ecklonia cava extracts for the biosynthesis of AgNPs has not been attempted yet. We synthesize AgNPs and subsequently characterize their morphologies and compositions. We also We synthesize AgNPs and subsequently characterize their morphologies and compositions. We also investigate whether phloroglucinol and their derivatives obtained from Ecklonia cava contribute to investigate whether phloroglucinol and their derivatives obtained from Ecklonia cava contribute to the reduction of silver ions required for the formation of AgNPs. Antimicrobial, antioxidant, and the reduction of silver ions required for the formation of AgNPs. Antimicrobial, antioxidant, and anticancer activities of the biosynthesized AgNPs are also investigated. anticancer activities of the biosynthesized AgNPs are also investigated. 2. Results Results and and Discussion Discussions 2. Spectroscopy 2.1. Biosynthesis of AgNPs and Characterization by UV-Vis Spectroscopy Ecklonia cava cava (1% (1% w/w) w/w) was silver nitrate nitrate (AgNO (AgNO33) Aqueous extract of Ecklonia was mixed with 1 mM of silver temperature. The formation of AgNPs was confirmed by the appearance of dark solution at room temperature. yellow color, color, which which is is aa typical typical color color of of AgNPs AgNPs in in solution solution due due to to their their surface surface plasmon plasmon resonance resonance (SPR) [72]. [72]. The The color colorof ofreaction reactionsolution solutionwas wasyellowish yellowishbrown brownupon upon1 h 1 of h of reaction, it changed (SPR) reaction, butbut it changed to to dark brown color when reduction of silver ions completed at h72(Figure h (Figure dark brown color when fullfull reduction of silver ions waswas completed at 72 2). 2).
Figure 2. Schematic representation representation of green green synthesis synthesis of of AgNPs. AgNPs. Ecklonia cava is collected from the sea and then ground ground into into aa fine fine powder. powder. The The aqueous aqueous extract extract of of Ecklonia Ecklonia cava cava is is mixed mixed with with 11 mM mM AgNO AgNO33 solution and stirred stirred for for 72 72 hh to to synthesize synthesize AgNPs. AgNPs.
UV-Vis spectroscopy spectroscopy was was used used to to confirm confirm the the synthesis synthesis of of AgNPs AgNPs with with aqueous aqueous extract extract of of UV-Vis Ecklonia cava. UV-Vis spectra scanned after time intervals of 0.5 h, 1 h, 18 h, and 24 h from the initiation Ecklonia cava. UV-Vis spectra scanned after time intervals of 0.5 h, 1 h, 18 h, and 24 h from the of reactionofare represented in Figure in 3. Strong SPR peak SPR of AgNPs atAgNPs 418 nmatwas observed initiation reaction are represented Figure 3. Strong peak of 418clearly nm was clearly upon 18 h of reaction, indicating the formation of AgNPs. It was also found that intensity of the SPR observed upon 18 h of reaction, indicating the formation of AgNPs. It was also found that intensity of peak increased with reaction time (Figure demonstrating the increased concentration of AgNPs. the SPR peak increased with reaction time 3), (Figure 3), demonstrating the increased concentration of The UV-Vis spectraspectra and visual observation demonstrate thatthat formation ofofAgNPs almost AgNPs. The UV-Vis and visual observation demonstrate formation AgNPswas was almost completed within 24 h. We also investigated the effect of temperature on the formation of AgNPs. completed within 24 h. We also investigated the effect of temperature on the formation of AgNPs. When the the reaction reaction temperature temperatureincreased increasedto to200 200 ◦°C, the formation formation of of AgNPs AgNPs was was accelerated accelerated and and thus thus When C, the completed within 5 h (data not shown). completed within 5 h (data not shown).
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Figure AgNPs at different time intervals. Figure 3. 3. UV-Vis UV-Vis absorption absorption spectra of biosynthesized UV-Vis absorption spectra spectra of of biosynthesized biosynthesized AgNPs AgNPs at at different differenttime timeintervals. intervals.
2.2. 2.2. Thermogravimetric Thermogravimetric Analysis Analysis (TGA) (TGA) 2.2. Thermogravimetric Analysis (TGA) Thermal extracts biosynthesized Thermal properties properties of of Ecklonia Ecklonia cava cava extracts extracts and and biosynthesized biosynthesized AgNPs AgNPs were were confirmed confirmed by by Thermal properties of Ecklonia cava and AgNPs were confirmed by thermogravimetric analysis (TGA) using a Pyris 1 TGA analyzer (Perkin-Elmer, Waltham, thermogravimetric analysis analysis (TGA) (TGA) using using a Pyris 1 TGA analyzer (Perkin-Elmer, USA), thermogravimetric (Perkin-Elmer, Waltham, MA, MA, USA), as shown Figure 4. TGA result exhibits the strong deflection point at 230 for Ecklonia cava as shown shown in in Figure Figure 4. 4. The The TGA TGA result result exhibits exhibits the the strong strong deflection deflection point point at at 230 230 ◦°C °C for Ecklonia Ecklonia cava cava as in The C for extracts, indicating their decomposition temperature. No significant difference has been observed extracts, indicating indicating their their decomposition decomposition temperature. temperature. No significant significant difference difference has has been been observed observed in in extracts, in TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of organic materials (i.e., Ecklonia cava) in the biosynthesized AgNPs. organic materials materials (i.e., (i.e., Ecklonia Ecklonia cava) cava)in inthe thebiosynthesized biosynthesizedAgNPs. AgNPs. organic
Figure 4. Thermogravimetric analysis ofof aqueous aqueous extract of of Ecklonia cava cava (black curve) curve) and Figure Figure 4. 4. Thermogravimetric Thermogravimetric analysis analysis of aqueous extract extract of Ecklonia Ecklonia cava (black (black curve) and and biosynthesized AgNPs (red curve). biosynthesized biosynthesized AgNPs AgNPs (red (red curve). curve).
2.3. Fourier Transform-Infrared (FT-IR) Spectroscopy 2.3. Fourier Transform-Infrared (FT-IR) (FT-IR) Spectroscopy Spectroscopy To determine the possible biomolecules and functional groups involved in the formation of To determine the possible biomolecules and functional groups involved in the formation of AgNPs, FT-IR spectroscopy was employed. biosynthesized AgNPs and aqueous AgNPs, FT-IR spectroscopy was employed. employed. FT-IR FT-IR spectra spectra of of biosynthesized AgNPs and aqueous in Figure 5. The aqueous extract of Ecklonia cava showed the peaks extract of Ecklonia cava were shown extract of Ecklonia cava were shown in Figure − 5.1The aqueous extract of Ecklonia cava showed the peaks 1 , 1412 cm −1 , and 3341−1cm−1 . The broad peak around −11 −1,−1412 −1, 1600 −1 at 871 cm− ,1027 1027cm cm−1−1−, 11231 , 1231cm cm , 1600 cm , cm cm , and 3341 −1 −1 −1 −1 at 871 cm , 1027 cm , 1231 cm , 1412 cm , 1600 cm , and 3341 cm cm−1.. The The broad broad peak peak around around 3341 3341 −1 in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. 3341 cm −1 cm in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. −1 cm in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. The The The absorption band observed at 1600 cm−1 can be assigned to the N–H bending vibration of amine or −1 can be assigned to the N–H bending vibration of amine or absorption band observed at 1600 cm −1 −1 is attributed absorption band observed at observed 1600 cm atcan be cm assigned to the N–H bending vibration vibration of amine of or amide groups [32]. The band 1412 to the C–N stretching −1 is attributed to the C–N stretching vibration of amide groups [32]. The band observed at 1412 cm − 1 − 1 −1 amide or groups The [73]. bandThe observed at 1412 cm atis1231 attributed to the C–N vibration of amine amide[32]. groups absorption bands cm and 1027 cmstretching correspond to C–O −1 −1 amine amine or or amide amide groups groups [73]. [73]. The The absorption absorption bands bands at at 1231 1231 cm cm−1and and 1027 1027 cm cm−1 correspond correspond to to C–O C–O or or −1 −1 C–O–C C–O–C stretching stretching vibrations vibrations [74]. [74]. Similar Similar kinds kinds of of peaks peaks were were observed observed at at 823 823 cm cm−1,, 1030 1030 cm cm−1,, 1243 1243
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2016, 6, 235 vibrations [74]. Similar kinds of peaks were observed at 823 cm−1 , 10306cm of 17 −1 , orNanomaterials C–O–C stretching − 1 − 1 − 1 − 1 1243 cm , 1370−1−1cm , 1609−1−1cm , and 3347−1 cm for biosynthesized AgNPs (Figure 5A). Similar FT-IR −1,−1 cm AgNPs (Figure (Figure5A). 5A).Similar SimilarFT-IR FT-IR cm ,1370 1370cm cm , ,1609 1609 cm cm , , and and 3347 3347 cm cm−1 for biosynthesized biosynthesized AgNPs absorption bands from the AgNPs implies that aqueous extract of Ecklonia cava could act as capping absorption extract of of Ecklonia Eckloniacava cavacould couldact actasascapping capping absorptionbands bandsfrom fromthe theAgNPs AgNPs implies implies that aqueous extract agents as well as reducing agents for the formation of stable AgNPs. agents stable AgNPs. AgNPs. agentsasaswell wellasasreducing reducingagents agentsfor for the the formation formation of stable
Figure 5. Fourier transform-infrared spectra of: (A) biosynthesized AgNPs; and (B) aqueous extract 5. Fourier Fourier transform-infrared transform-infraredspectra spectraof: of:(A) (A)biosynthesized biosynthesizedAgNPs; AgNPs; and aqueous extract Figure 5. and (B)(B) aqueous extract of of Ecklonia cava. of Ecklonia cava. Ecklonia cava.
2.4. X-Ray Diffraction (XRD) Analysis 2.4. X-ray X-RayDiffraction Diffraction(XRD) (XRD)Analysis Analysis 2.4. An XRD spectrum of biosynthesized AgNPs is shown in Figure 6. Distinct XRD patterns were An XRD XRD spectrum spectrum of biosynthesized biosynthesized AgNPs AgNPs is is shown in in Figure 6. 6. Distinct XRD XRD patterns patterns were were An observed at 28.0°, 32.5°,of 38.2°, 44.3°, 46.4°, 55.1°, 57.5°,shown 64.6°, andFigure 77.2°. TheDistinct peaks at 2θ values of 38.2°, ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ observed at 28.0°, 32.5°, 38.2°, 44.3°, 46.4°, 55.1°, 57.5°, 64.6°, and 77.2°. The peaks at 2θ values of 38.2°, observed at 28.0 32.5 corresponds , 38.2 , 44.3 ,to 46.4 , 64.6 and (3 77.2 peaks at 2θ values ofcubic 38.2◦ , 44.3°, 64.6° and ,77.2° (1 1, 55.1 1), (2, 057.5 0), (2 2 0),, and 1 1). The planes of face-centered ◦ ◦ ◦ 44.3°,, 64.6 64.6°and and77.2 77.2°corresponds correspondstoto(1(1 1 1), 0 (2 0),2(2 0), and (3 planes 1 1) planes of face-centered cubic 44.3 1(Joint 1), (2Committee 0(20), 0),2on and (3 1 1) of face-centered cubic (FCC) (FCC) structure of silver, respectively Powder Diffraction Standard (JCPDS) file: (FCC) structure of silver, respectively (Joint Committee on Powder Diffraction Standard (JCPDS) file: structure silver, on Powder Diffraction Standard (JCPDS) 04-0783). 04-0783).of It is inrespectively agreement(Joint with Committee several studies that have reported similar XRD file: patterns of 04-0783). It is in agreement with several studies that have reported similar XRD patterns It biosynthesized is in agreement with several studies that have reported similar XRD patterns of biosynthesized AgNPs [59]. It was found that other co-existing peaks at 2θ values of 28.0°, 32.5°, 46.4°,of ◦ , 32.5 ◦ , 46.4 biosynthesized AgNPs [59]. was peaks at planes 2θ values of ◦28.0°, 46.4°, AgNPs It was found thatItto other co-existing at values of228.0 , 55.1◦32.5°, , andcubic 57.5◦ 55.1°, [59]. and 57.5° correspond (1 1found 1), (2 0that 0), other (2peaks 2 0),co-existing (3 2θ 1 1), and (2 2) of face-centered 55.1°, and 57.5° correspond to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes of face-centered cubic correspond (1 1 1), 0 0), (2 2 0), (3 1respectively 1), and (2 2 2) planesfile: of face-centered cubicThis crystalline crystallinetophase of(2silver chloride, (JCPDS 31-1238) [75–80]. result phase clearlyof crystalline phase of silverof(JCPDS chloride, (JCPDS file: 31-1238) [75–80]. This resultextract clearly silver chloride, respectively file:respectively 31-1238) [75–80]. This result clearly indicates the production of indicates the production Ag/AgCl composite nanoparticles (Ag/AgCl NPs) using aqueous indicates the production of Ag/AgCl composite nanoparticles (Ag/AgCl NPs) using aqueous extract Ag/AgCl composite nanoparticles (Ag/AgCl using aqueous extractextract of Ecklonia cava. The chloride of Ecklonia cava. The chloride ions might beNPs) originated from aqueous of Ecklonia cava. The offormation Ecklonia cava. TheNPs chloride ions might be from ofAgCl Ecklonia The ions might be from aqueous extract of Ecklonia cava. The formation NPs might be of originated AgCl might be attributed to originated the interaction of aqueous silver ionsextract withof chloride ionscava. present formation of the AgCl NPs becava. attributed to chloride the interaction of silver ions with chloride ions present in aqueous extract of might Ecklonia Similar results have previously reported regarding the attributed to interaction of silver ions with ions been present in aqueous extract of Ecklonia cava. inbiosynthesis aqueous extract of Ecklonia cava. reported Similar results have previously reported regarding the of AgNPs [75,78]. Similar results have been previously regarding thebeen biosynthesis of AgNPs [75,78]. biosynthesis of AgNPs [75,78].
Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk). Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk).
Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk).
2.5. Size and Morphology Analysis of Biosynthesized AgNPs
2.5. Size and Morphology Analysis of Biosynthesized AgNPs
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Transmission images of of biosynthesized biosynthesized AgNPs AgNPsinindifferent different Transmission electron electron microscopy microscopy (TEM) (TEM) images magnifications Figure7.7.The The AgNPs were polydispersed, magnificationswere wereshown shown in Figure AgNPs were polydispersed, and and theirtheir sizes sizes were were in thein the range 15–30nm nm(Figure (Figure7A,B). 7A,B). addition, most themwere wereofofspherical sphericalshape. shape.The The mean range ofof 15–30 InInaddition, most ofofthem mean hydrodynamicdiameter diameterofofthe theAgNPs AgNPs dispersed dispersed in deionized 4343 nm hydrodynamic deionizedwater, water,determined determinedby byDLS, DLS,was was nm with PDI 0.27(Figure (Figure7C). 7C). with PDI ofof0.27
Figure 7. (A,B) Transmission electron microscopy images of biosynthesized AgNPs at different Figure 7. (A,B) Transmission electron microscopy images of biosynthesized AgNPs at different magnifications; and (C) particle size distribution of AgNPs determined by DLS. magnifications; and (C) particle size distribution of AgNPs determined by DLS.
2.6. Antimicrobial 2.6. AntimicrobialActivity Activityby byBiosynthesized Biosynthesized AgNPs AgNPs AgNPs activities[6]. [6].Antimicrobial Antimicrobialactivity activity AgNPswere werewell-known well-knownto tohave have strong strong antimicrobial antimicrobial activities ofof biosynthesized AgNPs was shown in Figure 8. Antimicrobial activity of the biosynthesized AgNPs was biosynthesized AgNPs was shown in Figure 8. Antimicrobial activity of the biosynthesized AgNPs investigated by growing Escherichia coli (E.coli coli) 1053610536 and Staphylococcus aureus (S. aureus) ATCC was investigated by growing Escherichia (E.ATCC coli) ATCC and Staphylococcus aureus (S. aureus) 6538 colonies Luria–Bertani (LB) broth agar plates. shown Figurein8A, significant inhibition ATCC 6538 on colonies on Luria–Bertani (LB) broth agarAs plates. Asinshown Figure 8A, significant zone of E. coli wasofobserved with the colonies treated withtreated AgNPs, as compared those treated with inhibition zone E. coli was observed with the colonies with AgNPs, as to compared to those treatedextract with aqueous extract Ecklonia cavaalso alone. It was also clearly observed that biosynthesized aqueous of Ecklonia cavaof alone. It was clearly observed that biosynthesized AgNPs revealed AgNPs revealed a concentration-dependent antibacterial activity. E. coli colonies withexhibited 40 µ g a concentration-dependent antibacterial activity. E. coli colonies treated with 40 µgtreated of AgNPs of AgNPs exhibited a larger diameter of zone of inhibition (12 ± 1 mm) (Figure 8(Ad)), as compared a larger diameter of zone of inhibition (12 ± 1 mm) (Figure 8(Ad)), as compared to those treated with those treated(10 with µ g of(Figure AgNPs 8(Ac)). (10 ± 1 mm) (Figure 8(Ac)). Efficient antimicrobial activity of 20toµg of AgNPs ± 20 1 mm) Efficient antimicrobial activity of the biosynthesized the biosynthesized AgNPs was observed S. colonies aureus. The S. aureus treated with 40 AgNPs was also observed with S.also aureus. The S. with aureus treated with colonies 40 µg of AgNPs exhibited µ g of AgNPs exhibited a larger diameter of zone of inhibition (Figure 8(Bd)), as compared to those a larger diameter of zone of inhibition (Figure 8(Bd)), as compared to those treated with aqueous treated aqueous extract of Ecklonia alone (Figurethat 8(Bb)). It was reported that synthesized extract of with Ecklonia cava alone (Figure 8(Bb)).cava It was reported synthesized AgNPs with Rhus chinensis AgNPs with Rhus chinensis extracts exhibited efficient antimicrobial activity against S. aureus, extracts exhibited efficient antimicrobial activity against S. aureus, Staphylococcus saprophyticus, E. coli, Staphylococcus E. coli, Pseudomonas This to study usedgood 50 µantibacterial g to 70 µ g and Pseudomonassaprophyticus, aeruginosa [61]. Thisand study used 50 µgaeruginosa to 70 µg of[61]. AgNPs achieve of AgNPs to achieve good antibacterial activity against all of the tested bacteria. In this study, activity against all of the tested bacteria. In this study, effective antimicrobial activity was achieved effective antimicrobial activity was achieved by using only 40 µ g of biosynthesized AgNPs, by using only 40 µg of biosynthesized AgNPs, demonstrating a higher antimicrobial activity of the demonstrating a higher antimicrobial activity of the biosynthesized AgNPs using Ecklonia cava biosynthesized AgNPs using Ecklonia cava extracts. Half maximal effective concentration (EC50 ) of extracts. Half maximal effective concentration (EC50) of AgNPs against E. coli was found to be 15.2 AgNPs against E. coli was found to be 15.2 µg/mL, whereas a slightly higher EC50 (i.e., 16.2 µg/mL) µ g/mL, whereas a slightly higher EC50 (i.e., 16.2 µ g/mL) was required for S. aureus. was required for S. aureus. Although AgNPs have demonstrated effective antimicrobial activities, the mechanism of action Although AgNPs have demonstrated effective antimicrobial activities, the mechanism of action on microorganisms has not been clearly elucidated yet. It has been proposed that silver ions released onfrom microorganisms has not with been thiol clearly elucidated yet. has been proposed that silver ions released AgNPs can interact groups present in Itrespiratory enzymes of bacterial cells, thus disrupting their respiration process [81]. Another possible mechanism of cell death is the interaction
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from AgNPs can interact with thiol groups present in respiratory enzymes of bacterial cells, thus Nanomaterials 2016, 6, 235 8 of 17 disrupting their respiration process [81]. Another possible mechanism of cell death is the interaction of silver ions with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication of silver ions2016, with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication Nanomaterials 6, 235 8 of 17 andand thus cellcell death [82]. thus death [82]. of silver ions with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication and thus cell death [82].
Figure 8. Antimicrobial activity of biosynthesized AgNPs, determined by an agar well diffusion assay.
Figure 8. Antimicrobial activity of biosynthesized AgNPs, determined by an agar well diffusion assay. Pictures show inhibition zones produced by the biosynthesized AgNPs against E. coli and S. aureus. Pictures show inhibition zones produced by the biosynthesized AgNPs against E. coli and S. aureus. (A) E. coli colonies treated with:of(a) 20 µ g of aqueous extract of Ecklonia cava; (b)well 40 µdiffusion g of aqueous Figure 8. Antimicrobial activity biosynthesized AgNPs, determined by an agar assay. (A) E. coli colonies treated with: (a) 20 µg of aqueous extract of Ecklonia cava; (b) 40 µg of aqueous extract ofshow Ecklonia cava; (c) zones 20 µ g of AgNPs; and (d)biosynthesized 40 µ g of AgNPs.AgNPs (B) S. aureus colonies with: Pictures inhibition produced by the against E. coli treated and S. aureus. extract of Ecklonia cava;extract (c) 20 µg of AgNPs; and (d) 40 µgaqueous of AgNPs. (B) S. Ecklonia aureus colonies treated with: (a) g ofcolonies aqueoustreated of Ecklonia (b)aqueous 40 µ g ofextract cava; µ g of (A)20 E.µcoli with: (a) 20cava; µ g of of extract Eckloniaofcava; (b) 40 µ g(c) of20 aqueous (a) 20 µg of aqueous extract of Ecklonia cava; (b) 40 µg of aqueous extract of Ecklonia cava; (c) 20 µg of AgNPs; and (d) 40cava; µ g of(c) AgNPs. extract of Ecklonia 20 µ g of AgNPs; and (d) 40 µ g of AgNPs. (B) S. aureus colonies treated with: AgNPs; and (d) 40 µg of AgNPs. (a) 20 µ g of aqueous extract of Ecklonia cava; (b) 40 µ g of aqueous extract of Ecklonia cava; (c) 20 µ g of
2.7. Antioxidant AgNPs AgNPs; andActivity (d) 40 µ gbyofBiosynthesized AgNPs.
2.7. Antioxidant Activity by Biosynthesized AgNPs Antioxidant activity of biosynthesized AgNPs was evaluated by 1,1-diphenyl-2-picrylhydrazyl
2.7. Antioxidant Activity by Biosynthesized AgNPs (DPPH) radicalactivity scavenging assay (Figure 9). Freewas radical scavenging activity of AgNPs was Antioxidant of biosynthesized AgNPs evaluated by 1,1-diphenyl-2-picrylhydrazyl determined by a decrease in absorbance of DPPH solution at 517 nm. When was activity assay of biosynthesized AgNPs wasscavenging evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) Antioxidant radical scavenging (Figure 9). Free radical activity of DPPH AgNPssolution was determined mixed with 250 µ g/mL of Ecklonia cava extract or biosynthesized AgNPs, ca. 50% of scavenging radical scavengingofassay (Figure 9). at Free scavenging AgNPs was by a(DPPH) decrease in absorbance DPPH solution 517radical nm. When DPPHactivity solutionofwas mixed with activity was by achieved (Figure 9). DPPH radical scavenging activities of When Ecklonia cava solution extract and determined a decrease in absorbance of DPPH solution at 517 nm. DPPH was 250 µg/mL of Ecklonia cava extract or biosynthesized AgNPs, ca. 50% of scavenging activity was biosynthesized AgNPs wereEcklonia similar at the extract same concentrations (e.g., 100, 250, and 500 µof g/mL). High mixed (Figure with 2509). µ g/mL cava or biosynthesized ca. 50% scavenging achieved DPPHofradical scavenging activities of EckloniaAgNPs, cava extract and biosynthesized antioxidant of Ecklonia possibly due to polyphenolic as previously activity wasactivity achieved (Figurecava 9). extract DPPH is radical scavenging activities ofcompounds, Ecklonia cava extract and AgNPs were similarresult at theindicates same concentrations (e.g., 100, 250, of and 500 µg/mL). Highisantioxidant reported [67]. This that antioxidant activity(e.g., biosynthesized AgNPs highly biosynthesized AgNPs were similar atstrong the same concentrations 100, 250, and 500 µ g/mL). High activity of Ecklonia cava extract is possibly due toon polyphenolic compounds, as previously reported related to the Ecklonia cava extract remained thedue surface of the AgNPs. Due to as thepreviously efficient [67]. antioxidant activity of Ecklonia cava extract is possibly to polyphenolic compounds, Thisantioxidant result indicates that antioxidant activityand of AgNPs, biosynthesized AgNPs is highly to the activities ofstrong both Ecklonia extracts combination of AgNPs andrelated Ecklonia reported [67]. This result indicates thatcava strong antioxidant activity of biosynthesized AgNPs is highly Ecklonia cava extract remained on thea good surface of the AgNPs. Due theand efficient antioxidant activities cava with effects be candidate as pharmaceutical nutraceutical products. related tosynergistic the Ecklonia cavacan extract remained on the surface ofto the AgNPs. Due to the efficient
of both Eckloniaactivities cava extracts AgNPs, of AgNPs and Ecklonia cava with antioxidant of bothand Ecklonia cavacombination extracts and AgNPs, combination of AgNPs and synergistic Ecklonia effects can be a good candidate as pharmaceutical and nutraceutical products. cava with synergistic effects can be a good candidate as pharmaceutical and nutraceutical products.
Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of Ecklonia cava extract and biosynthesized AgNPs. (ns: non-significant; * p < 0.05).
Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH)radical radicalscavenging scavengingactivity activityofofEcklonia Eckloniacava cavaextract extractand Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH) 2.8. Anticancer Activity by Biosynthesized AgNPs and biosynthesized AgNPs. (ns: non-significant; * p < 0.05). biosynthesized AgNPs. (ns: non-significant; * p < 0.05).
2.8. Anticancer Activity by Biosynthesized AgNPs
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In In recent recent years, years, searching searching anticancer anticancer drug drug candidates candidates from from marine marine resources resources is is increasing increasing due due to their lower side effects [83,84]. Anticancer activity of biosynthesized AgNPs using to their lower side effects [83,84]. Anticancer activity of biosynthesized AgNPs using Ecklonia Ecklonia cava cava extracts extracts was was investigated investigated by by using using human human cervical cervical cancer cancer cells cells (HeLa (HeLa cells). cells). Figure Figure 10A 10A shows shows the the cytotoxicity of AgNPs at different concentrations. IC value of the AgNPs was found to be 50 cytotoxicity of AgNPs at different concentrations. IC50 value of the AgNPs was found to be around around 59 The cells cells treated treated with with aqueous aqueous extract extract of of Ecklonia 59 µg/mL. µ g/mL. The Ecklonia cava cava alone alone did did not not show show any any noticeable noticeable cytotoxicity concentrations suchsuch as 250 (data not shown). A similarAanticancer capability cytotoxicityatathigh high concentrations asµg/mL 250 µ g/mL (data not shown). similar anticancer of biosynthesized AgNPs against HeLa cells was found in other recent studies [85]. Biosynthesized capability of biosynthesized AgNPs against HeLa cells was found in other recent studies [85]. AgNPs using Podophyllum hexandrum exhibited an efficient anticancer activity against HeLa cells with Biosynthesized AgNPs using Podophyllum hexandrum exhibited an efficient anticancer activity against an IC50cells value of 20 High effect of biosynthesized using Cymodocea serrulata HeLa with anµg/mL. IC50 value ofcytotoxic 20 µ g/mL. High cytotoxic effectAgNPs of biosynthesized AgNPs using was also reported [86]. Their IC value against HeLa cells was 34.5 µg/mL. 50 Cymodocea serrulata was also reported [86]. Their IC50 value against HeLa cells was 34.5 µ g/mL.
Figure Figure 10. 10. (A) (A) Anticancer Anticancer activity activity of of biosynthesized biosynthesized AgNPs AgNPs against against HeLa HeLa cells cells (ns: (ns: non-significant; *** *** pp
Antimicrobial, Antioxidant, and Anticancer Activities of Biosynthesized Silver Nanoparticles Using Marine Algae Ecklonia cava Jayachandran Venkatesan 1 , Se-Kwon Kim 2 and Min Suk Shim 1, * 1 2
*
Division of Bioengineering, Incheon National University, Incheon 406-772, Korea; [email protected] Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Korea; [email protected] Correspondence: [email protected]; Tel.: +82-32-835-8268
Academic Editor: Guogang Ren Received: 14 September 2016; Accepted: 25 November 2016; Published: 6 December 2016
Abstract: Green synthesis of silver nanoparticles (AgNPs) has gained great interest as a simple and eco-friendly alternative to conventional chemical methods. In this study, AgNPs were synthesized by using extracts of marine algae Ecklonia cava as reducing and capping agents. The formation of AgNPs using aqueous extract of Ecklonia cava was confirmed visually by color change and their surface plasmon resonance peak at 418 nm, measured by UV-visible spectroscopy. The size, shape, and morphology of the biosynthesized AgNPs were observed by transmission electron microscopy and dynamic light scattering analysis. The biosynthesized AgNPs were nearly spherical in shape with an average size around 43 nm. Fourier transform-infrared spectroscopy (FTIR) analysis confirmed the presence of phenolic compounds in the aqueous extract of Ecklonia cava as reducing and capping agents. X-ray diffraction (XRD) analysis was also carried out to demonstrate the crystalline nature of the biosynthesized AgNPs. Antimicrobial results determined by an agar well diffusion assay demonstrated a significant antibacterial activity of the AgNPs against Escherichia coli and Staphylococcus aureus. Antioxidant results determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay revealed an efficient antioxidant activity of the biosynthesized AgNPs. The biosynthesized AgNPs also exhibited a strong apoptotic anticancer activity against human cervical cancer cells. Our findings demonstrate that aqueous extract of Ecklonia cava is an effective reducing agent for green synthesis of AgNPs with efficient antimicrobial, antioxidant, and anticancer activities. Keywords: anticancer; antimicrobial; antioxidant; biosynthesis; Ecklonia cava; nanoparticle
1. Introduction In recent years, noble metal nanoparticles (NPs) have been intensively utilized for biomedical applications, such as diagnostics, drug delivery, and tissue engineering, due to their unique physicochemical and optoelectronic properties [1–4]. Among various noble metal nanoparticles, silver nanoparticles (AgNPs) have received great attention in a variety of applications, including nanoelectronic devices, sensors, imaging contrast agents, filters, and antimicrobial agents due to their good electrical conductivity, stability, optical property, and antimicrobial activity [5,6]. AgNPs have also extended their applications to cancer therapy. Several in vitro studies using AgNPs have demonstrated their potential as effective anticancer agents [7–10]. They have exhibited apoptosis-mediated, strong anticancer efficacies in a variety of cancer cells, including human cervical cancer [8], lung cancer [9], and breast cancer cells [10]. It has been well-documented that performance and applicability of AgNPs critically depend on their size, shape, composition, and surface chemistry [11–13]. Synthesis of noble
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metal NPs with controlled shape and size can be achieved through many different synthetic methods: evaporation-condensation, gamma irradiation, electron irradiation, microwave processing, microemulsion, sonochemical, electrochemical, photochemical, etc. [14–17]. One of the popular methods for the synthesis of defined noble metal NPs is chemical reduction [14]. Reduction of silver complexes in dilute solution with a proper reductant can lead to the formation of colloidal AgNPs [14]. Although this method offers significant advantages of simple equipment and convenient operation, it involves a use of hazardous chemicals and high temperature conditions, which are rather environmentally unfriendly and energetically inefficient [14,18]. To overcome the drawbacks of the chemical reduction method, biogenic synthesis that employs microorganisms and naturally occurring products has emerged as an environmentally friendly synthetic method (i.e., green chemistry) [17,19–21]. This method offers a facile and convenient entry to producing various noble metal NPs. Biogenic synthesis of noble metal NPs mainly relies on various extracts (e.g., nucleic acids, enzymes, proteins, peptides, vitamins, and polysaccharides) of microorganisms. For example, extracts of fungi, bacteria, and algae were extensively utilized to synthesize AgNPs, as shown in Table 1. Table 1. Biosynthesized AgNPs using various microorganisms. No.
Species
Reaction Time
Size
Applications
Ref.
1 2 3 4 5 6 7 8 9 10 11
Trichoderma viride Chaetoceros calcitrans Aspergillus clavatus Porphyra vietnamensis Gelidiella acerosa Sargassum tenerrimum Ulva fasciata Padina tetrastromatica Turbinaria conoides Padina pavonica Cryphonectria Pterocladia capillacae, Jania rubins, Ulva faciata, Colpmenia sinusa Gracilaria corticata Sargassum cinereum Padina gymnospora Sargassum plagiophyllum Sargassum longifolium Pithophora oedogonia Sesbania grandiflora Stenotrophomonas maltophilia Croton bonplandianum Chenopodium murale Alternanthera sessilis Pseudomonas putida Citrullus colocynthis Chlorella vulgaris Lovoa trichilioïdes Ficus elastica Ficus religiosa Morinda pubescens Saccharomyces boulardii Alternanthera sessilis (Linn.) Citrus reticulata juice Caulerpa racemosa Spirogyra varians Enteromorpha flexuos Cyanobacteria and Microalgae Dracocephalum moldavica Dimocarpus longan Lour. Curculigo orchioides rhizome Rhus chinensis
48 h 2 weeks N.A. 15 min 48 h 20 min N.A 24 h N.A 24 h 24 h
5–40 nm N.A. 10–25 nm 13 ± 3 nm 22 nm 20 nm 28–41 nm 14 nm 96 nm 46.8 nm 30–70nm
Antimicrobial Antimicrobial Antifungal Antibacterial Antifungal Anti-bacterial Antimicrobial Antibacterial Antibacterial Antimicrobial Antimicrobial
[13] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
3h
20 nm
Antimicrobial
[32]
20 min 3h N.A. 24 h 64 h N.A. 15 min N.A. N.A. N.A. N.A. 20 min 24 h 24 h 2h 30 min 10 min N.A. 4h N.A. N.A. 24 h N.A. N.A. 72 h 1h 5h N.A. 12 h
18–46 nm 45–76 nm 25–40 nm 21–48 nm N.A. 34.03 nm 12 nm 93 nm 32 nm 30–50 nm 10–30 nm 6–16 nm 31 nm 7 nm 37–43 nm 50–60 nm 5–35 nm N.A. 3–10 nm 20–30 nm N.A. 5–25 nm 17.6 nm 2–32 nm 13–31 nm 31 ± 6 nm 9–32 nm 15–18 nm 150 nm.
Antifungal Antimicrobial Antimicrobial Antimicrobial Antifungal Antibacterial Antimicrobial Antimicrobial Antimicrobial and anticancer Antimicrobial Anticancer Antibacterial and anticancer Anticancer Anticancer and antimicrobial Anti-bacterial Air pollution control In vivo antitumor Antioxidant and anticancer Anticancer Antimicrobial and antioxidant Antimicrobial Antibacterial Antibacterial Antimicrobial Antibacterial Antimicrobial Anticancer Larvicidal and Anticancer Antibacterial
[33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61]
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
N.A. = Not Available.
Nanomaterials 2016, 6, 235 40 Curculigo orchioides rhizome Nanomaterials 41 2016, 6, 235Rhus chinensis
3 of 17 N.A. 12 h
15–18 nm 150 nm.
Larvicidal and Anticancer Antibacterial
[60] [61] 3 of 18
N.A. = Not Available.
Seaweeds from fromthe theocean oceanare are considered “sea vegetables”, commonly as in food in Seaweeds considered as as “sea vegetables”, commonly usedused as food AsiaAsia-Pacific as Korea, China, Japan. Seaweeds oftenused usedtotoproduce produce hydrocolloids hydrocolloids Pacific areasareas suchsuch as Korea, China, andand Japan. Seaweeds areare often such as alginate, agar, and carrageenan. carrageenan. Ecklonia cava is an edible brown alga, mainly present in Korea, Previous studies studies reported reported that that phlorotannins phlorotannins such such as as phloroglucinol phloroglucinol are main Japan, and China. Previous responsible components in Ecklonia cava [62–65]. These compounds in Ecklonia cava have proved to be responsible bioactivities such suchas asantioxidant antioxidant[62,66], [62,66],anticancer anticancer [67–69], and antimicrobial activities for bioactivities [67–69], and antimicrobial activities [70].[70]. In In addition, antioxidant compounds from Eckloniacava cavaextracts, extracts,including including(a) (a)phloroglucinol; phloroglucinol;(b) (b) eckol; eckol; addition, antioxidant compounds from Ecklonia fucodiphlorethol G; G;(d) (d)phlorofucofuroeckol phlorofucofuroeckolA;A;(e)(e) 7-phloroeckol; dieckol; 6,60 -bieckol; (c) fucodiphlorethol 7-phloroeckol; (f) (f) dieckol; (g) (g) 6,6′-bieckol; (h) 0 0 (h) triphlorethol-A; and 2,7 -phloroglucinol-6,6 -bieckol (Figure1)1)[42,62,67–71], [42,62,67–71],could couldact actas as effective effective triphlorethol-A; and (i) (i) 2,7′-phloroglucinol-6,6′-bieckol (Figure reductants to synthesize noble metal NPs.
Figure phloroglucinol; (b) (b) eckol; (c) fucodiphlorethol G; (d) Figure 1. 1. Chemical Chemicalstructures structuresof:of:(a) (a) phloroglucinol; eckol; (c) fucodiphlorethol G; 0 phlorofucofuroeckol A; (e) 7-phloroeckol; (f) dieckol; (g) 6,6′-bieckol; (h) triphlorethol-A; and (i) (d) phlorofucofuroeckol A; (e) 7-phloroeckol; (f) dieckol; (g) 6,6 -bieckol; (h) triphlorethol-A; 2,7′and phloroglucinol-6,6′-bieckol. (i) 2,70 -phloroglucinol-6,60 -bieckol.
In this study, AgNPs were synthesized via reduction of silver ions using aqueous extracts of In this study, AgNPs were synthesized via reduction of silver ions using aqueous extracts Ecklonia cava as an alternative to conventional chemical reduction methods. To the best of our of Ecklonia cava as an alternative to conventional chemical reduction methods. To the best of our
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knowledge, use of Ecklonia cava extracts for the biosynthesis of AgNPs has not been attempted yet. knowledge, use of Ecklonia cava extracts for the biosynthesis of AgNPs has not been attempted yet. We synthesize AgNPs and subsequently characterize their morphologies and compositions. We also We synthesize AgNPs and subsequently characterize their morphologies and compositions. We also investigate whether phloroglucinol and their derivatives obtained from Ecklonia cava contribute to investigate whether phloroglucinol and their derivatives obtained from Ecklonia cava contribute to the reduction of silver ions required for the formation of AgNPs. Antimicrobial, antioxidant, and the reduction of silver ions required for the formation of AgNPs. Antimicrobial, antioxidant, and anticancer activities of the biosynthesized AgNPs are also investigated. anticancer activities of the biosynthesized AgNPs are also investigated. 2. Results Results and and Discussion Discussions 2. Spectroscopy 2.1. Biosynthesis of AgNPs and Characterization by UV-Vis Spectroscopy Ecklonia cava cava (1% (1% w/w) w/w) was silver nitrate nitrate (AgNO (AgNO33) Aqueous extract of Ecklonia was mixed with 1 mM of silver temperature. The formation of AgNPs was confirmed by the appearance of dark solution at room temperature. yellow color, color, which which is is aa typical typical color color of of AgNPs AgNPs in in solution solution due due to to their their surface surface plasmon plasmon resonance resonance (SPR) [72]. [72]. The The color colorof ofreaction reactionsolution solutionwas wasyellowish yellowishbrown brownupon upon1 h 1 of h of reaction, it changed (SPR) reaction, butbut it changed to to dark brown color when reduction of silver ions completed at h72(Figure h (Figure dark brown color when fullfull reduction of silver ions waswas completed at 72 2). 2).
Figure 2. Schematic representation representation of green green synthesis synthesis of of AgNPs. AgNPs. Ecklonia cava is collected from the sea and then ground ground into into aa fine fine powder. powder. The The aqueous aqueous extract extract of of Ecklonia Ecklonia cava cava is is mixed mixed with with 11 mM mM AgNO AgNO33 solution and stirred stirred for for 72 72 hh to to synthesize synthesize AgNPs. AgNPs.
UV-Vis spectroscopy spectroscopy was was used used to to confirm confirm the the synthesis synthesis of of AgNPs AgNPs with with aqueous aqueous extract extract of of UV-Vis Ecklonia cava. UV-Vis spectra scanned after time intervals of 0.5 h, 1 h, 18 h, and 24 h from the initiation Ecklonia cava. UV-Vis spectra scanned after time intervals of 0.5 h, 1 h, 18 h, and 24 h from the of reactionofare represented in Figure in 3. Strong SPR peak SPR of AgNPs atAgNPs 418 nmatwas observed initiation reaction are represented Figure 3. Strong peak of 418clearly nm was clearly upon 18 h of reaction, indicating the formation of AgNPs. It was also found that intensity of the SPR observed upon 18 h of reaction, indicating the formation of AgNPs. It was also found that intensity of peak increased with reaction time (Figure demonstrating the increased concentration of AgNPs. the SPR peak increased with reaction time 3), (Figure 3), demonstrating the increased concentration of The UV-Vis spectraspectra and visual observation demonstrate thatthat formation ofofAgNPs almost AgNPs. The UV-Vis and visual observation demonstrate formation AgNPswas was almost completed within 24 h. We also investigated the effect of temperature on the formation of AgNPs. completed within 24 h. We also investigated the effect of temperature on the formation of AgNPs. When the the reaction reaction temperature temperatureincreased increasedto to200 200 ◦°C, the formation formation of of AgNPs AgNPs was was accelerated accelerated and and thus thus When C, the completed within 5 h (data not shown). completed within 5 h (data not shown).
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Figure AgNPs at different time intervals. Figure 3. 3. UV-Vis UV-Vis absorption absorption spectra of biosynthesized UV-Vis absorption spectra spectra of of biosynthesized biosynthesized AgNPs AgNPs at at different differenttime timeintervals. intervals.
2.2. 2.2. Thermogravimetric Thermogravimetric Analysis Analysis (TGA) (TGA) 2.2. Thermogravimetric Analysis (TGA) Thermal extracts biosynthesized Thermal properties properties of of Ecklonia Ecklonia cava cava extracts extracts and and biosynthesized biosynthesized AgNPs AgNPs were were confirmed confirmed by by Thermal properties of Ecklonia cava and AgNPs were confirmed by thermogravimetric analysis (TGA) using a Pyris 1 TGA analyzer (Perkin-Elmer, Waltham, thermogravimetric analysis analysis (TGA) (TGA) using using a Pyris 1 TGA analyzer (Perkin-Elmer, USA), thermogravimetric (Perkin-Elmer, Waltham, MA, MA, USA), as shown Figure 4. TGA result exhibits the strong deflection point at 230 for Ecklonia cava as shown shown in in Figure Figure 4. 4. The The TGA TGA result result exhibits exhibits the the strong strong deflection deflection point point at at 230 230 ◦°C °C for Ecklonia Ecklonia cava cava as in The C for extracts, indicating their decomposition temperature. No significant difference has been observed extracts, indicating indicating their their decomposition decomposition temperature. temperature. No significant significant difference difference has has been been observed observed in in extracts, in TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of TGA curves between Ecklonia cava extracts and AgNPs. This result clearly indicates the presence of organic materials (i.e., Ecklonia cava) in the biosynthesized AgNPs. organic materials materials (i.e., (i.e., Ecklonia Ecklonia cava) cava)in inthe thebiosynthesized biosynthesizedAgNPs. AgNPs. organic
Figure 4. Thermogravimetric analysis ofof aqueous aqueous extract of of Ecklonia cava cava (black curve) curve) and Figure Figure 4. 4. Thermogravimetric Thermogravimetric analysis analysis of aqueous extract extract of Ecklonia Ecklonia cava (black (black curve) and and biosynthesized AgNPs (red curve). biosynthesized biosynthesized AgNPs AgNPs (red (red curve). curve).
2.3. Fourier Transform-Infrared (FT-IR) Spectroscopy 2.3. Fourier Transform-Infrared (FT-IR) (FT-IR) Spectroscopy Spectroscopy To determine the possible biomolecules and functional groups involved in the formation of To determine the possible biomolecules and functional groups involved in the formation of AgNPs, FT-IR spectroscopy was employed. biosynthesized AgNPs and aqueous AgNPs, FT-IR spectroscopy was employed. employed. FT-IR FT-IR spectra spectra of of biosynthesized AgNPs and aqueous in Figure 5. The aqueous extract of Ecklonia cava showed the peaks extract of Ecklonia cava were shown extract of Ecklonia cava were shown in Figure − 5.1The aqueous extract of Ecklonia cava showed the peaks 1 , 1412 cm −1 , and 3341−1cm−1 . The broad peak around −11 −1,−1412 −1, 1600 −1 at 871 cm− ,1027 1027cm cm−1−1−, 11231 , 1231cm cm , 1600 cm , cm cm , and 3341 −1 −1 −1 −1 at 871 cm , 1027 cm , 1231 cm , 1412 cm , 1600 cm , and 3341 cm cm−1.. The The broad broad peak peak around around 3341 3341 −1 in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. 3341 cm −1 cm in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. −1 cm in the spectra indicates the existence of O–H group of polyphenols or polysaccharides. The The The absorption band observed at 1600 cm−1 can be assigned to the N–H bending vibration of amine or −1 can be assigned to the N–H bending vibration of amine or absorption band observed at 1600 cm −1 −1 is attributed absorption band observed at observed 1600 cm atcan be cm assigned to the N–H bending vibration vibration of amine of or amide groups [32]. The band 1412 to the C–N stretching −1 is attributed to the C–N stretching vibration of amide groups [32]. The band observed at 1412 cm − 1 − 1 −1 amide or groups The [73]. bandThe observed at 1412 cm atis1231 attributed to the C–N vibration of amine amide[32]. groups absorption bands cm and 1027 cmstretching correspond to C–O −1 −1 amine amine or or amide amide groups groups [73]. [73]. The The absorption absorption bands bands at at 1231 1231 cm cm−1and and 1027 1027 cm cm−1 correspond correspond to to C–O C–O or or −1 −1 C–O–C C–O–C stretching stretching vibrations vibrations [74]. [74]. Similar Similar kinds kinds of of peaks peaks were were observed observed at at 823 823 cm cm−1,, 1030 1030 cm cm−1,, 1243 1243
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2016, 6, 235 vibrations [74]. Similar kinds of peaks were observed at 823 cm−1 , 10306cm of 17 −1 , orNanomaterials C–O–C stretching − 1 − 1 − 1 − 1 1243 cm , 1370−1−1cm , 1609−1−1cm , and 3347−1 cm for biosynthesized AgNPs (Figure 5A). Similar FT-IR −1,−1 cm AgNPs (Figure (Figure5A). 5A).Similar SimilarFT-IR FT-IR cm ,1370 1370cm cm , ,1609 1609 cm cm , , and and 3347 3347 cm cm−1 for biosynthesized biosynthesized AgNPs absorption bands from the AgNPs implies that aqueous extract of Ecklonia cava could act as capping absorption extract of of Ecklonia Eckloniacava cavacould couldact actasascapping capping absorptionbands bandsfrom fromthe theAgNPs AgNPs implies implies that aqueous extract agents as well as reducing agents for the formation of stable AgNPs. agents stable AgNPs. AgNPs. agentsasaswell wellasasreducing reducingagents agentsfor for the the formation formation of stable
Figure 5. Fourier transform-infrared spectra of: (A) biosynthesized AgNPs; and (B) aqueous extract 5. Fourier Fourier transform-infrared transform-infraredspectra spectraof: of:(A) (A)biosynthesized biosynthesizedAgNPs; AgNPs; and aqueous extract Figure 5. and (B)(B) aqueous extract of of Ecklonia cava. of Ecklonia cava. Ecklonia cava.
2.4. X-Ray Diffraction (XRD) Analysis 2.4. X-ray X-RayDiffraction Diffraction(XRD) (XRD)Analysis Analysis 2.4. An XRD spectrum of biosynthesized AgNPs is shown in Figure 6. Distinct XRD patterns were An XRD XRD spectrum spectrum of biosynthesized biosynthesized AgNPs AgNPs is is shown in in Figure 6. 6. Distinct XRD XRD patterns patterns were were An observed at 28.0°, 32.5°,of 38.2°, 44.3°, 46.4°, 55.1°, 57.5°,shown 64.6°, andFigure 77.2°. TheDistinct peaks at 2θ values of 38.2°, ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ observed at 28.0°, 32.5°, 38.2°, 44.3°, 46.4°, 55.1°, 57.5°, 64.6°, and 77.2°. The peaks at 2θ values of 38.2°, observed at 28.0 32.5 corresponds , 38.2 , 44.3 ,to 46.4 , 64.6 and (3 77.2 peaks at 2θ values ofcubic 38.2◦ , 44.3°, 64.6° and ,77.2° (1 1, 55.1 1), (2, 057.5 0), (2 2 0),, and 1 1). The planes of face-centered ◦ ◦ ◦ 44.3°,, 64.6 64.6°and and77.2 77.2°corresponds correspondstoto(1(1 1 1), 0 (2 0),2(2 0), and (3 planes 1 1) planes of face-centered cubic 44.3 1(Joint 1), (2Committee 0(20), 0),2on and (3 1 1) of face-centered cubic (FCC) (FCC) structure of silver, respectively Powder Diffraction Standard (JCPDS) file: (FCC) structure of silver, respectively (Joint Committee on Powder Diffraction Standard (JCPDS) file: structure silver, on Powder Diffraction Standard (JCPDS) 04-0783). 04-0783).of It is inrespectively agreement(Joint with Committee several studies that have reported similar XRD file: patterns of 04-0783). It is in agreement with several studies that have reported similar XRD patterns It biosynthesized is in agreement with several studies that have reported similar XRD patterns of biosynthesized AgNPs [59]. It was found that other co-existing peaks at 2θ values of 28.0°, 32.5°, 46.4°,of ◦ , 32.5 ◦ , 46.4 biosynthesized AgNPs [59]. was peaks at planes 2θ values of ◦28.0°, 46.4°, AgNPs It was found thatItto other co-existing at values of228.0 , 55.1◦32.5°, , andcubic 57.5◦ 55.1°, [59]. and 57.5° correspond (1 1found 1), (2 0that 0), other (2peaks 2 0),co-existing (3 2θ 1 1), and (2 2) of face-centered 55.1°, and 57.5° correspond to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes of face-centered cubic correspond (1 1 1), 0 0), (2 2 0), (3 1respectively 1), and (2 2 2) planesfile: of face-centered cubicThis crystalline crystallinetophase of(2silver chloride, (JCPDS 31-1238) [75–80]. result phase clearlyof crystalline phase of silverof(JCPDS chloride, (JCPDS file: 31-1238) [75–80]. This resultextract clearly silver chloride, respectively file:respectively 31-1238) [75–80]. This result clearly indicates the production of indicates the production Ag/AgCl composite nanoparticles (Ag/AgCl NPs) using aqueous indicates the production of Ag/AgCl composite nanoparticles (Ag/AgCl NPs) using aqueous extract Ag/AgCl composite nanoparticles (Ag/AgCl using aqueous extractextract of Ecklonia cava. The chloride of Ecklonia cava. The chloride ions might beNPs) originated from aqueous of Ecklonia cava. The offormation Ecklonia cava. TheNPs chloride ions might be from ofAgCl Ecklonia The ions might be from aqueous extract of Ecklonia cava. The formation NPs might be of originated AgCl might be attributed to originated the interaction of aqueous silver ionsextract withof chloride ionscava. present formation of the AgCl NPs becava. attributed to chloride the interaction of silver ions with chloride ions present in aqueous extract of might Ecklonia Similar results have previously reported regarding the attributed to interaction of silver ions with ions been present in aqueous extract of Ecklonia cava. inbiosynthesis aqueous extract of Ecklonia cava. reported Similar results have previously reported regarding the of AgNPs [75,78]. Similar results have been previously regarding thebeen biosynthesis of AgNPs [75,78]. biosynthesis of AgNPs [75,78].
Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk). Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk).
Figure 6. X-ray diffraction patterns of biosynthesized AgNPs (dot circle) and AgCl NPs (asterisk).
2.5. Size and Morphology Analysis of Biosynthesized AgNPs
2.5. Size and Morphology Analysis of Biosynthesized AgNPs
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Transmission images of of biosynthesized biosynthesized AgNPs AgNPsinindifferent different Transmission electron electron microscopy microscopy (TEM) (TEM) images magnifications Figure7.7.The The AgNPs were polydispersed, magnificationswere wereshown shown in Figure AgNPs were polydispersed, and and theirtheir sizes sizes were were in thein the range 15–30nm nm(Figure (Figure7A,B). 7A,B). addition, most themwere wereofofspherical sphericalshape. shape.The The mean range ofof 15–30 InInaddition, most ofofthem mean hydrodynamicdiameter diameterofofthe theAgNPs AgNPs dispersed dispersed in deionized 4343 nm hydrodynamic deionizedwater, water,determined determinedby byDLS, DLS,was was nm with PDI 0.27(Figure (Figure7C). 7C). with PDI ofof0.27
Figure 7. (A,B) Transmission electron microscopy images of biosynthesized AgNPs at different Figure 7. (A,B) Transmission electron microscopy images of biosynthesized AgNPs at different magnifications; and (C) particle size distribution of AgNPs determined by DLS. magnifications; and (C) particle size distribution of AgNPs determined by DLS.
2.6. Antimicrobial 2.6. AntimicrobialActivity Activityby byBiosynthesized Biosynthesized AgNPs AgNPs AgNPs activities[6]. [6].Antimicrobial Antimicrobialactivity activity AgNPswere werewell-known well-knownto tohave have strong strong antimicrobial antimicrobial activities ofof biosynthesized AgNPs was shown in Figure 8. Antimicrobial activity of the biosynthesized AgNPs was biosynthesized AgNPs was shown in Figure 8. Antimicrobial activity of the biosynthesized AgNPs investigated by growing Escherichia coli (E.coli coli) 1053610536 and Staphylococcus aureus (S. aureus) ATCC was investigated by growing Escherichia (E.ATCC coli) ATCC and Staphylococcus aureus (S. aureus) 6538 colonies Luria–Bertani (LB) broth agar plates. shown Figurein8A, significant inhibition ATCC 6538 on colonies on Luria–Bertani (LB) broth agarAs plates. Asinshown Figure 8A, significant zone of E. coli wasofobserved with the colonies treated withtreated AgNPs, as compared those treated with inhibition zone E. coli was observed with the colonies with AgNPs, as to compared to those treatedextract with aqueous extract Ecklonia cavaalso alone. It was also clearly observed that biosynthesized aqueous of Ecklonia cavaof alone. It was clearly observed that biosynthesized AgNPs revealed AgNPs revealed a concentration-dependent antibacterial activity. E. coli colonies withexhibited 40 µ g a concentration-dependent antibacterial activity. E. coli colonies treated with 40 µgtreated of AgNPs of AgNPs exhibited a larger diameter of zone of inhibition (12 ± 1 mm) (Figure 8(Ad)), as compared a larger diameter of zone of inhibition (12 ± 1 mm) (Figure 8(Ad)), as compared to those treated with those treated(10 with µ g of(Figure AgNPs 8(Ac)). (10 ± 1 mm) (Figure 8(Ac)). Efficient antimicrobial activity of 20toµg of AgNPs ± 20 1 mm) Efficient antimicrobial activity of the biosynthesized the biosynthesized AgNPs was observed S. colonies aureus. The S. aureus treated with 40 AgNPs was also observed with S.also aureus. The S. with aureus treated with colonies 40 µg of AgNPs exhibited µ g of AgNPs exhibited a larger diameter of zone of inhibition (Figure 8(Bd)), as compared to those a larger diameter of zone of inhibition (Figure 8(Bd)), as compared to those treated with aqueous treated aqueous extract of Ecklonia alone (Figurethat 8(Bb)). It was reported that synthesized extract of with Ecklonia cava alone (Figure 8(Bb)).cava It was reported synthesized AgNPs with Rhus chinensis AgNPs with Rhus chinensis extracts exhibited efficient antimicrobial activity against S. aureus, extracts exhibited efficient antimicrobial activity against S. aureus, Staphylococcus saprophyticus, E. coli, Staphylococcus E. coli, Pseudomonas This to study usedgood 50 µantibacterial g to 70 µ g and Pseudomonassaprophyticus, aeruginosa [61]. Thisand study used 50 µgaeruginosa to 70 µg of[61]. AgNPs achieve of AgNPs to achieve good antibacterial activity against all of the tested bacteria. In this study, activity against all of the tested bacteria. In this study, effective antimicrobial activity was achieved effective antimicrobial activity was achieved by using only 40 µ g of biosynthesized AgNPs, by using only 40 µg of biosynthesized AgNPs, demonstrating a higher antimicrobial activity of the demonstrating a higher antimicrobial activity of the biosynthesized AgNPs using Ecklonia cava biosynthesized AgNPs using Ecklonia cava extracts. Half maximal effective concentration (EC50 ) of extracts. Half maximal effective concentration (EC50) of AgNPs against E. coli was found to be 15.2 AgNPs against E. coli was found to be 15.2 µg/mL, whereas a slightly higher EC50 (i.e., 16.2 µg/mL) µ g/mL, whereas a slightly higher EC50 (i.e., 16.2 µ g/mL) was required for S. aureus. was required for S. aureus. Although AgNPs have demonstrated effective antimicrobial activities, the mechanism of action Although AgNPs have demonstrated effective antimicrobial activities, the mechanism of action on microorganisms has not been clearly elucidated yet. It has been proposed that silver ions released onfrom microorganisms has not with been thiol clearly elucidated yet. has been proposed that silver ions released AgNPs can interact groups present in Itrespiratory enzymes of bacterial cells, thus disrupting their respiration process [81]. Another possible mechanism of cell death is the interaction
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from AgNPs can interact with thiol groups present in respiratory enzymes of bacterial cells, thus Nanomaterials 2016, 6, 235 8 of 17 disrupting their respiration process [81]. Another possible mechanism of cell death is the interaction of silver ions with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication of silver ions2016, with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication Nanomaterials 6, 235 8 of 17 andand thus cellcell death [82]. thus death [82]. of silver ions with bases and phosphorus groups of DNA, leading to the inhibition of DNA replication and thus cell death [82].
Figure 8. Antimicrobial activity of biosynthesized AgNPs, determined by an agar well diffusion assay.
Figure 8. Antimicrobial activity of biosynthesized AgNPs, determined by an agar well diffusion assay. Pictures show inhibition zones produced by the biosynthesized AgNPs against E. coli and S. aureus. Pictures show inhibition zones produced by the biosynthesized AgNPs against E. coli and S. aureus. (A) E. coli colonies treated with:of(a) 20 µ g of aqueous extract of Ecklonia cava; (b)well 40 µdiffusion g of aqueous Figure 8. Antimicrobial activity biosynthesized AgNPs, determined by an agar assay. (A) E. coli colonies treated with: (a) 20 µg of aqueous extract of Ecklonia cava; (b) 40 µg of aqueous extract ofshow Ecklonia cava; (c) zones 20 µ g of AgNPs; and (d)biosynthesized 40 µ g of AgNPs.AgNPs (B) S. aureus colonies with: Pictures inhibition produced by the against E. coli treated and S. aureus. extract of Ecklonia cava;extract (c) 20 µg of AgNPs; and (d) 40 µgaqueous of AgNPs. (B) S. Ecklonia aureus colonies treated with: (a) g ofcolonies aqueoustreated of Ecklonia (b)aqueous 40 µ g ofextract cava; µ g of (A)20 E.µcoli with: (a) 20cava; µ g of of extract Eckloniaofcava; (b) 40 µ g(c) of20 aqueous (a) 20 µg of aqueous extract of Ecklonia cava; (b) 40 µg of aqueous extract of Ecklonia cava; (c) 20 µg of AgNPs; and (d) 40cava; µ g of(c) AgNPs. extract of Ecklonia 20 µ g of AgNPs; and (d) 40 µ g of AgNPs. (B) S. aureus colonies treated with: AgNPs; and (d) 40 µg of AgNPs. (a) 20 µ g of aqueous extract of Ecklonia cava; (b) 40 µ g of aqueous extract of Ecklonia cava; (c) 20 µ g of
2.7. Antioxidant AgNPs AgNPs; andActivity (d) 40 µ gbyofBiosynthesized AgNPs.
2.7. Antioxidant Activity by Biosynthesized AgNPs Antioxidant activity of biosynthesized AgNPs was evaluated by 1,1-diphenyl-2-picrylhydrazyl
2.7. Antioxidant Activity by Biosynthesized AgNPs (DPPH) radicalactivity scavenging assay (Figure 9). Freewas radical scavenging activity of AgNPs was Antioxidant of biosynthesized AgNPs evaluated by 1,1-diphenyl-2-picrylhydrazyl determined by a decrease in absorbance of DPPH solution at 517 nm. When was activity assay of biosynthesized AgNPs wasscavenging evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) Antioxidant radical scavenging (Figure 9). Free radical activity of DPPH AgNPssolution was determined mixed with 250 µ g/mL of Ecklonia cava extract or biosynthesized AgNPs, ca. 50% of scavenging radical scavengingofassay (Figure 9). at Free scavenging AgNPs was by a(DPPH) decrease in absorbance DPPH solution 517radical nm. When DPPHactivity solutionofwas mixed with activity was by achieved (Figure 9). DPPH radical scavenging activities of When Ecklonia cava solution extract and determined a decrease in absorbance of DPPH solution at 517 nm. DPPH was 250 µg/mL of Ecklonia cava extract or biosynthesized AgNPs, ca. 50% of scavenging activity was biosynthesized AgNPs wereEcklonia similar at the extract same concentrations (e.g., 100, 250, and 500 µof g/mL). High mixed (Figure with 2509). µ g/mL cava or biosynthesized ca. 50% scavenging achieved DPPHofradical scavenging activities of EckloniaAgNPs, cava extract and biosynthesized antioxidant of Ecklonia possibly due to polyphenolic as previously activity wasactivity achieved (Figurecava 9). extract DPPH is radical scavenging activities ofcompounds, Ecklonia cava extract and AgNPs were similarresult at theindicates same concentrations (e.g., 100, 250, of and 500 µg/mL). Highisantioxidant reported [67]. This that antioxidant activity(e.g., biosynthesized AgNPs highly biosynthesized AgNPs were similar atstrong the same concentrations 100, 250, and 500 µ g/mL). High activity of Ecklonia cava extract is possibly due toon polyphenolic compounds, as previously reported related to the Ecklonia cava extract remained thedue surface of the AgNPs. Due to as thepreviously efficient [67]. antioxidant activity of Ecklonia cava extract is possibly to polyphenolic compounds, Thisantioxidant result indicates that antioxidant activityand of AgNPs, biosynthesized AgNPs is highly to the activities ofstrong both Ecklonia extracts combination of AgNPs andrelated Ecklonia reported [67]. This result indicates thatcava strong antioxidant activity of biosynthesized AgNPs is highly Ecklonia cava extract remained on thea good surface of the AgNPs. Due theand efficient antioxidant activities cava with effects be candidate as pharmaceutical nutraceutical products. related tosynergistic the Ecklonia cavacan extract remained on the surface ofto the AgNPs. Due to the efficient
of both Eckloniaactivities cava extracts AgNPs, of AgNPs and Ecklonia cava with antioxidant of bothand Ecklonia cavacombination extracts and AgNPs, combination of AgNPs and synergistic Ecklonia effects can be a good candidate as pharmaceutical and nutraceutical products. cava with synergistic effects can be a good candidate as pharmaceutical and nutraceutical products.
Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of Ecklonia cava extract and biosynthesized AgNPs. (ns: non-significant; * p < 0.05).
Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH)radical radicalscavenging scavengingactivity activityofofEcklonia Eckloniacava cavaextract extractand Figure 9. 1,1-diphenyl-2-picrylhydrazyl (DPPH) 2.8. Anticancer Activity by Biosynthesized AgNPs and biosynthesized AgNPs. (ns: non-significant; * p < 0.05). biosynthesized AgNPs. (ns: non-significant; * p < 0.05).
2.8. Anticancer Activity by Biosynthesized AgNPs
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Nanomaterials 2016,Activity 6, 235 by Biosynthesized AgNPs 2.8. Anticancer
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In In recent recent years, years, searching searching anticancer anticancer drug drug candidates candidates from from marine marine resources resources is is increasing increasing due due to their lower side effects [83,84]. Anticancer activity of biosynthesized AgNPs using to their lower side effects [83,84]. Anticancer activity of biosynthesized AgNPs using Ecklonia Ecklonia cava cava extracts extracts was was investigated investigated by by using using human human cervical cervical cancer cancer cells cells (HeLa (HeLa cells). cells). Figure Figure 10A 10A shows shows the the cytotoxicity of AgNPs at different concentrations. IC value of the AgNPs was found to be 50 cytotoxicity of AgNPs at different concentrations. IC50 value of the AgNPs was found to be around around 59 The cells cells treated treated with with aqueous aqueous extract extract of of Ecklonia 59 µg/mL. µ g/mL. The Ecklonia cava cava alone alone did did not not show show any any noticeable noticeable cytotoxicity concentrations suchsuch as 250 (data not shown). A similarAanticancer capability cytotoxicityatathigh high concentrations asµg/mL 250 µ g/mL (data not shown). similar anticancer of biosynthesized AgNPs against HeLa cells was found in other recent studies [85]. Biosynthesized capability of biosynthesized AgNPs against HeLa cells was found in other recent studies [85]. AgNPs using Podophyllum hexandrum exhibited an efficient anticancer activity against HeLa cells with Biosynthesized AgNPs using Podophyllum hexandrum exhibited an efficient anticancer activity against an IC50cells value of 20 High effect of biosynthesized using Cymodocea serrulata HeLa with anµg/mL. IC50 value ofcytotoxic 20 µ g/mL. High cytotoxic effectAgNPs of biosynthesized AgNPs using was also reported [86]. Their IC value against HeLa cells was 34.5 µg/mL. 50 Cymodocea serrulata was also reported [86]. Their IC50 value against HeLa cells was 34.5 µ g/mL.
Figure Figure 10. 10. (A) (A) Anticancer Anticancer activity activity of of biosynthesized biosynthesized AgNPs AgNPs against against HeLa HeLa cells cells (ns: (ns: non-significant; *** *** pp
