Facile Synthesis of Silver Nanoparticles Using Euphorbia Antiquorum L

Journal of Saudi Chemical Society (2016) xxx, xxx–xxx

King Saud University Journal of Saudi Chemical Society

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Facile synthesis of silver nanoparticles using Euphorbia antiquorum L. latex extract and evaluation of their biomedical perspectives as anticancer agents

Chandrasekaran Rajkuberan, Seetharaman Prabukumar, Gnansekar Sathishkumar, Arockiasamy Wilson, Keepanan Ravindran, Sivaperumal Sivaramakrishnan *

Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathidasan University, Tiruchirappalli, 620 024 Tamil Nadu, India

Received 27 June 2015; revised 7 January 2016; accepted 8 January 2016

KEYWORDS Abstract This study reveals the rapid biosynthesis of silver nanoparticles (EAAgNPs) using aque- Crystal structure; ous latex extract of Euphorbia antiquorum L as a potential bioreductant. Synthesized EAAgNPs Euphorbia antiquorum L.; generate the surface plasmonic resonance peak at 438 nm in UV–Vis spectrophotometer. Size Silver nanoparticles; and shape of EAAgNPs were further characterized through transmission electron microscope Anticancer; (TEM) which shows well-dispersed spherical nanoparticles with size ranging from 10 to 50 nm. Human pathogens Energy dispersive X-ray spectroscopic analysis (EDAX) conﬁrms the presence of silver (Ag) as the major constituent element. X-ray diffraction (XRD) pattern of EAAgNPs corresponding to (111), (200), (220) and (311) planes, reveals that the generated nanoparticles were face centered cubic crystalline in nature. Interestingly, fourier-transform infrared spectroscopy (FTIR) analysis shows the major role of active phenolic constituents in reduction and stabilization of EAAgNPs. Phyto-fabricated EAAgNPs exhibits signiﬁcant antimicrobial and larvicidal activity against bacterial human pathogens as well as disease transmitting blood sucking parasites such as Culex quinquefasciatus and Aedes aegypti (IIIrd instar larvae). On the other hand, in vitro cytotoxicity assessment of bioformulated EAAgNPs has shown potential anticancer activity against human cervical carcinoma cells (HeLa). The preliminary biochemical (MTT assay) and microscopic studies depict

* Corresponding author. Tel.: +0431 2407086: fax: +0431 2407045. E-mail address: [email protected] (S. Sivaramakrishnan). Peer review under responsibility of King Saud University.

Production and hosting by Elsevier http://dx.doi.org/10.1016/j.jscs.2016.01.002 1319-6103 Ó 2016 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

that the synthesized EAAgNPs at minimal dosage (IC50 =28lg) triggers cellular toxicity response. Hence, the EAAgNPs can be considered as an environmentally benign and non-toxic nanobioma- terial for biomedical applications. Ó 2016 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction tion. The latex was collected from the plant by a small incision near the end of the branches and collected into a small tube Nano-scale materials with a size range of 1–100 nm find a containing an equal volume of double distilled water (1:1 niche in materials science owing to their defined size, shape, ratio). After collection, the mixture (water and latex) was optical, mechanical, biological properties and significant use brought immediately to the laboratory and centrifuged at ° in various applications such as space, electronics, chemicals, 10,000 RPM for 20 min at 4 C. The resulting supernatant biomedical imaging, sensors, agriculture, tissue engineering, was used further in all experiments as a source of latex extract. drug delivery and targeting [1,2]. In particular, synthesis of noble metal nanoparticles (silver, gold, platinum, and palla- 2.2. Synthesis of EAAgNPs dium) is fascinating among researchers around the world [3]. Of all the metals, silver nanoparticles (AgNPs) have gained For synthesis, 3% aqueous latex extract and 1 mM AgNO3 tremendous interest due to its increasing commercial demand was used. Briefly, the reaction mixture was prepared by adding day by day. [4]. Conventional methodologies of AgNPs syn- 8 mL of AgNO3 and 2 mL of 3% aqueous latex extract. This thesis posses many disadvantages and are not eco-friendly stoichiometric proportion was fixed based on the initial test [5]. To switch over to these technical hitches a clean, stable with UV–Vis spectroscopy. Further, the reaction mixture and non toxic synthesis procedure for synthesis of AgNPs was kept in dark condition at room temperature for 24 h for should be adopted. The use of green chemistry route (plants) the development of a deep brownish color which indicates in AgNP synthesis is an imperative due to its ease of develop- the formation of EAAgNPs. ment and is devoid of hazardous by products. The concept of AgNPs green synthesis was first developed 2.3. Characterization of silver nanoparticles by Raveendran et al. [6] where b-D-glucose and starch were used as a reducing and capping agent to prepare AgNPs. As Synthesized nanoparticles were monitored by measuring the of today, various biological entities such as bacteria, fungi, absorbance spectra at regular intervals using UV–Vis spec- yeast, algae, actinomycetes, and plants were employed for troscopy in the wavelength of 300–700 nm in JASCO V-650 AgNP synthesis. The use of plant materials in nanoparticle spectrophotometer. After preliminary confirmation of AgNPs synthesis could be more advantageous than microbes because by UV–Vis spectroscopy, the synthesized nanoparticle solution it does not need intra or extra cellular synthesis, purification was centrifuged at 15000 RPM for 20 min and the resulting and maintenance of microbial culture [7]. pellet was dissolved in double distilled water and filtered Latex is a milky white fluid secreted by laticifer cells in which through a Whatman filter paper (0.45 lm). The resulting sus- sub-cellular organelles intensively synthesize secondary metabo- pension was used for further characterization studies. For lites and exhibit antibacterial, antifungal, insecticidal, molluscici- TEM analysis, a drop of aqueous solution containing the dal, antimutagenic, antitumor and inflammatory activities [8,9]. AgNPs was sampled onto carbon coated copper grids and Traditionally, Euphorbiaceae family plants bear latex, dried under an infrared lamp. TEM observations were made which is widely used for therapeutic purposes since it contains with TECNAI-10 operated at an accelerating voltage at highly active constituents. Euphorbia antiquorum belonging to 100 kv. In addition the presence of metals in the sample Euphorbiaceae, a succulent plant characterized by having 3 (AgNPs) was analyzed by EDS. The functional groups present winged branchlets and stipular spines on sinuate repent wings in the AgNPs and aqueous latex extract were analyzed by [10]. E. antiquorum latex is a white, thick fluid that flows using FTIR spectra using PerkinElmer [Model RX1] with a throughout the plant and in ancient times this plant was used wavelength range between 4000 and 400 cm1. For FTIR anal- for insect pest control and herbivorous attacks [11]. Moreover, ysis, the samples were mixed with KBr powder and pelletized, the latex was used as an antibacterial, antifungal, anticancer, after drying the spectrum data were recorded. X-ray diffrac- antiviral, treatment of ulcer and menstrual disorders in tradi- tion measurements of aqueous latex reduced AgNPs was car- tional folk medicine [12]. Hitherto, only a limited number of ried on XRD 600, Shimadzu, Japan instruments operating at articles were available for latex mediated synthesis of AgNPs voltage of 40 kv and current of 30 mA with CuKa radiation. and its biological applications. Hence, an attempt was made Concentration of synthesized AgNPs was measured with using the latex of E. antiquorum to synthesize EAAgNPs with- inductively coupled plasma atomic emission spectroscopy out additional chemicals and for different biological activities. (ICP-OES Perkin Elmer Optima 5300 DV model).

2. Experimental section 2.4. Antimicrobial activity of EAAgNPs 2.1. Preparation of E. antiquorum aqueous extract The antimicrobial activity of EAAgNPs was evaluated using E. antiquorum growing in the vicinity of Bharathidasan an agar well diffusion method [13] against human pathogenic University campus was used as a source for the latex collec- bacteria in the Mueller Hinton agar media (MHA) with

Please cite this article in press as: C. Rajkuberan et al., Facile synthesis of silver nanoparticles using Euphorbia antiquorum L. latex extract and evaluation of their biomedical perspectives as anticancer agents, Journal of Saudi Chemical Society (2016), http://dx.doi.org/10.1016/j.jscs.2016.01.002 Synthesis of silver nanoparticles and evaluation of their biomedical perspectives 3 different concentrations of EAAgNPs (50 and 100 lg/mL). aegypti and C. quinquefasciatus. Different concentrations of Streptomycin and crude aqueous latex extract was used as a latex (1000–200 ppm) and EAAgNPs (10–2 ppm) were assayed control. After a 24 h incubation at 37 °C, a zone of inhibition for larvicidal activity. After 24 h, the numbers of dead larvae was measured. were counted and percentage of mortality rate was calculated from the average of three replicates. 2.5. Cytotoxicity of EAAgNPs 2.8. Statistical analysis Cytotoxicity study of EAAgNPs was assayed in HeLa cell lines using 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bro- The mortality data were subjected to probit regression analy- mide] MTT assay [14]. Briefly, the human cervical cancer cell sis. All other statistical calculations for determining lethal con- line (HeLa) was obtained from National Centre for Cell centrations LC50,LC90 and their 95% confidence interval Science (NCCS), Pune and grown in Eagles Minimum Essen- (upper confidence limit) and (lower confidence limit) values tial Medium (EMEM). The cells were seeded in 96 well plates were determined using statistical software (version 16). at a concentration of 1.5 104 cells/well, allowed to attach for 24 h and treated with different concentrations 3.125, 6.25, 12.5, 25 and 50 lg of EAAgNPs. Following drug addition, the plates were incubated for an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative humidity. After 48 h of incubation, 15 ll of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100 ll of DMSO and then measured the absorbance at 570 NM using a micro plate reader. The medium containing without EAAgNPs was served as control and triplicate was maintained for all concentrations. The % cell inhibition was determined using the following formula: % Cell Inhibition ¼ 100 AbsðsampleÞ=AbsðcontrolÞ100

2.6. Mosquito larvicidal bioassay

Mosquito larvae (Aedes Aegypti and Culex quinquefasciatus) were collected from the stagnant water area and a pond in the local vicinity of the university as per the protocol [15,16]. The collected larvae were kept in enamel trays containing tap water and maintained as per the method of Kamaraj [17]. For bioassay, twenty-ﬁve numbers of IIIrd instar mosquito larvae (A. aegypti and C. quinquefasciatus) were introduced into 500 mL glass beaker containing 249 mL of dechlorinated tap water and 1 mL of desired concentrations of aqueous latex extract (1000, 800, 600, 400 and 200 ppm/l) and synthesized AgNPs (10,8,6,4 and 2 ppm/l) was added sep- arately and kept in an environmental chamber at 25 °C with a 16:8 light/dark cycle. Three replicates were set up for each concentration and larvae placed in de-chlorinated water alone considered as negative control. Control mortality was cor- rected by using Abbotts formula and percentage mortality was calculated as follows; Number of dead larvae Percentage mortality ¼ 100 Number of larvae introduced Mortality was assessed after a 24 h exposure to ascertain the acute toxicity for IIIrd instar larvae of A. aegypti and C. Figure 1 (a) Color change pattern, formation of brown color quinquefasciatus. indicates the synthesis of EAAgNPs, (b) UV–Vis spectroscopy produces intense SPR spectra at 438 nm, absorbance maxima was 2.7. Dose–response bioassay noticed as the incubation time rose upto 24 h. (c) FTIR transmit- tance of aqueous latex extract and synthesized EAAgNPs shows Synthesized EAAgNPs and crude plant latex were subjected to the stretches of major functional biocompounds in reduction and dose–response bioassay against IIIrd instar larvae of A. stabilization of EAAgNPs.

Figure 2 (a) TEM micrograph of EAAgNPs at different magniﬁcation displays spherical particles with a thin biomolecule coating on the surface. (b) Particle size distribution graph of EAAgNPs, the mean size was calculated as 21.39 nm. (c) EDAX analysis produces a strong peak for silver that conﬁrms the stability and purity of synthesized EAAgNPs.

3. Results and discussion

Natural products have a profound therapeutic value and there is a very high demand for plant bioactive compounds in com- bination with nanomaterials for various applications in the medical ﬁeld. Therefore, in the study aqueous latex extracts of E. antiquorum are used for fabrication of silver nanoparticles.

3.1. Biosynthesis of EAAgNPs

On adding, 2 mL of 3% aqueous latex extract in 8 mL of 1 mM AgNO3 solution, the reaction mixture turned into yel- lowish brown color (Fig. 1a) due to the excitation of surface plasmon resonance (SPR) [18]. The optimal conditions for rapid generation of EAAgNPs were ﬁxed to be (incubation Figure 3 X-ray diffractogram intensities were interpreted with time: 24 h, room temperature, metal ion concentration JCPDS which shows the diffraction planes at (111), (200), (220) 1 mM, pH 8, the stoichiometric proportion of 1 mM AgNO3 and (311) which depicts that synthesized EAAgNPs were cubic and aqueous latex extract 8:2). In our study, we have observed crystalline in nature. the complete reduction of silver ions after 24 h of incubation at

Figure 4a Bactericidal effects of EAAgNPs against human pathogens A – E. coli,B–K. Pneumoniae,C–P.mirabilis,D–V.cholerae,E – E. faecalis. Concentrations: (1) latex extract; (2) antibiotic; (3) 50 lg/ml; (4) 100 lg/ml. room temperature (Fig. 1b). Incubation time has a direct cor- stretch, C–C stretch, N–H bend and OH stretch. Therefore, relation with synthesis rate of EAAgNPs, as incubation time it clearly shows the presence of phenolic compounds and pro- increases from 0 to 24 h intensity of SPR raised and yield of tein moieties present in the latex extract act as stabilizing/ EAAgNPs was also found to be higher due to augmented con- reducing agent. Moreover, the E. antiquorum latex contains tact time [19]. For better synthesis the reaction medium was Euphorbia, euphol, isohelianol and camelliol C euphol 3-Ocin- tuned to alkaline pH to upsurge the availability of the plant namate, antiquol A, antiquol B, 24-methylenecycloartanol and active biocompounds [20]. cycloeucalenol [21]. Hence, we speculate that the above biomolecules play a vital role in the synthesis of EAAgNPs. TEM analysis was carried out to determine the surface morphology 3.2. Characterization of EAAgNPs of the AgNPs. Our TEM micrograph, (Fig. 2a) clearly display the spherical shape of the EAAgNPs with a size distribution FTIR analysis was carried out to know the biocomponents from 10 to 50 nm having the mean value of 21.39 nm with their corresponding spectrum in the given sample(s). (Fig. 2b). Analysis of the EDAX spectrum at 3 KeV conﬁrms (Fig. 1c) illustrates the possible biomolecules involved in the the presence of silver as the major constituent element reduction of Ag+ and capping of the EAAgNPs. The obtained (Fig. 2c). A thin layer of some capping organic biomaterials spectrum reveals intense bands of EAAgNPs located at 652.69, from aqueous latex extracts provides stability and prevents 695.77, 762.31, 829.46, 1124.94, 1402.35, 1570.75, 2078.56, aggregation of AgNPs. Depending upon the part of the plant 3431.78 corresponding to C–Br stretch, C–Cl stretch, C–N material used, the phytochemical constituents will differ;

3.4. Anticancer activity of EAAgNPs

As observed from (Fig. 5a), as concentration of EAAgNPs increases (3, 6.25, 12.5, 25 and 50 lg) cytotoxicity in HeLa cell lines was increased. The IC50 value of EAAgNPs was 28 lg respectively. The morphological changes of cells due to varying concentrations of EAAgNPS were observed in a microscope, captured and recorded. The control cells were viable, healthy, whereas HeLa cell proliferation was significantly inhibited by EAAgNP treatment. These cells showed increased apoptosis and morphological changes with clearly visible debris due to cell death. A graph was constructed with % inhibition versus concentration of EAAgNPs (Fig. 5b). These results show that EAAgNPs exhibit cytotoxicity against HeLa cell lines in a dose dependent manner. In our earlier study, biosynthesized AgNPs using the latex of Calotropis gigantea has shown a dose dependent anticancer- ous effect against HeLa cell lines with IC50 value of 91.3 lg Figure 4b Antimicrobial activity of EAAgNPs against bacterial [30]. A similar observation was also noted in EAAgNPs and pathogens. this might be due to the capping of plant compounds that upsurge the activity of EAAgNPs [31]. The molecular mechanism of EAAgNP cytotoxicity is due to the induction of Reac- tive Oxygen Species (ROS) causing damage to cellular material similarly the size, shape and composition of AgNPs will vary such as proteins, lipids, DNA eventually leading to death [32] accordingly [22]. ICP-OES analysis of silver in EAAgNPs or increase of intracellular oxidative stress leading to cell death was 45.53 mg/L. XRD pattern of EAAgNPs shows intense process via. apoptosis or necrosis [33]. AgNPs have antiangio- peaks at 2h values of 38.13°, 44.26°, 64.46°, 77.51° correspond- genic properties that block the activity of abnormally express- ing to the (111), (200), (220) and (311) face centered cubic ing signaling proteins [34]. Moreover, the latex of E. structure of EAAgNPs (Fig. 3). X-ray diffraction pattern of antiquorum is safe, non toxic and does not pose any toxicity pure silver ions is known to display peaks at 2h values at in the normal cell line as reported by Sumathi et al. [35]. Fur- 38.0°, 44.2°, 64.4°, 77.3°, 81.5°. [23]. In EAAgNPs, XRD pat- ther studies are needed to analyze the molecular mechanism tern exhibits a peak corresponding to silver ions which clearly and effect of EAAgNPs on the mammalian immune system indicates that the particles are in the nano regime while to explore as a possible source of novel anticancer drugs. another unidentified peak is due to biomolecules present in the sample. Therefore, XRD results suggest that the crystal- 3.5. Mosquitocidal activity of EAAgNPs lization of the bioorganic phase occurs on the surface of EAAgNPs. A similar XRD peak was observed, where AgNPs IIIrd instar larvae of A. aegypti and C. quinquefasciatus were are synthesized using the latex of Jatropha curcas [24]. assessed for larvicidal efficacy of EAAgNPs. The LC50 and LC90 of the EAAgNPs and aqueous latex extract are reported 3.3. Antimicrobial activity of EAAgNPs in Tables 1 and 2. The tested concentrations (2, 4, 6, 8 and 10 ppm) of EAAgNPs against IIIrd instar larvae showed AgNPs is a very good antimicrobial agent due to its non-toxic prominent activity with 100% mortality observed after 24 h effect on human cells and a weaker ability of bacteria to treatment. The aqueous latex extract showed activity at con- develop resistance against silver ions [25]. In this study, after centrations ranging from 200 to 1000 ppm against both the lar- a 24 h incubation, it was noticed that EAAgNPs show mild vae mosquitoes. Therefore, the mortality rate is directly antimicrobial activity against all tested human pathogens while proportional to the concentration of the sample, while the the crude latex extract does not show any inhibitory action sample concentration was increased the mortality rate also (Figs. 4a and b). This may be due to the capping of phytocon- increases. The mechanism behind the mosquito death is due stituents from the latex on the surface of nanoparticles [26]. to the penetration of AgNPs orally or through breaching of The reasons behind the antimicrobial activity of AgNPs were the cuticle membrane, thereby entering into the body cavity. not clearly known, but several hypotheses are put forward In the body it binds to the sulfur containing proteins and phos- for the antimicrobial activity of AgNPs. Firstly, Ag+ ions phorous containing DNA leads to inhibition of protein and interact with bacterial cell membrane, causing plasmolysis DNA synthesis. Therefore, the cellular membrane permeability resulting in inhibition of cell membrane synthesis [27]. Sec- is decreased and disturbance in proton motive force causes ondly, AgNPs interfere with the thiol group of bacterial cells morphological deformation and leads to death [36] leading to ceasure of respiratory chain reaction, cell division Several studies have reported the latex has anti insecticidal and death [28]. Finally, AgNPs intrude into the bacterial mem- properties like inhibition of egg hatching, affecting larval brane, causing condensation of DNA damage and also by growth and mortality [37]. Recent studies have shown that affecting protein synthesis [29]. Therefore, these mechanisms latex mediated synthesis of AgNPs is a very effective larvicidal might be involved in the inhibitory action of EAAgNPs agent. Patil et al. [38] synthesizes AgNPs from the latex of against the tested pathogens. Plumeria rubra and evaluated for its bioefficacy against IInd

Figure 5 (a) Graphical plot between % inhibition Vs concentration (b) Microscopic images of against HeLa cell at different concentrations (A) control, (B) 3 lg, (C) 6.25 lg, (D) 12.5 lg, (E) 25 lg and (F) 50 lg.

Table 1 Larvicidal efﬁcacy of aqueous latex extract of E. antiquorum against IIIrd instar larvae of A. aegypti and C. quinquefasciatus

after 24 h of exposure. Distilled water (control) shows nil mortality, significant at P < 0.05 level; LC50 lethal concentration that kills 50% of the exposed adults and larvae, UCL upper confidence limit, LCL lower confidence limit.

Aqueous latex extract Concentration (ppm/l) and % mortality LC50 (ppm) LFL–UFL LC90 (ppm) LFL–UFL Aedes aegypti (IIIrd instar larvae) 1000 – 96.66 ± 2.88 221.91 797.70 800 – 93.33 ± 2.88 (156.87–275.59) (649–1100) 600 – 78.33 ± 2.88 400 – 65.00 ± 5.00 200 – 51.66 ± 7.63 Culex quinquefasciatus (IIIrd instar larvae) 1000 – 85.00 ± 5.00 440.70 1540 800 – 71.66 ± 2.88 (370–510) (1178–2397) 600 – 56.66 ± 2.88 400 – 45.00 ± 5.00 200 – 23.33 ± 2.88

and IVth instar larvae of A. aegypti and Anopheles stephensi 3 ppm against IIIrd instar larvae stages of mosquitoes suggest- (LC 50 1.10, 1.74, 1.49, 1.82 ppm and LC90 2.16, 3.77, 3.34, ing the possibility of use as a biolarvicidal agent. AgNPs have 3.86 ppm). Patil et al. [39] reported AgNPs synthesized from good larvicidal property, even at very low concentrations. [40] Pergularia daemia latex and assayed against the larval instars Rahuman et al. [41] reported the petroleum ether extract of of A. aegypti and A. stephensi. AgNPs-treated ﬁrst, second, the stem bark of Euphorbia tirucalli and tested against A. third, and fourth instars of A. aegypti (LC50 04.39, 5.12, aegypti (LC50 4.25; LC90 13.14 ppm) and C. quinquefasciatus 5.66, 6.18; LC90, 9.90, 11.13, 12.40, 12.95 ppm) and A. ste- (LC50 5.52; LC90 25.67 ppm) respectively. Borase et al. [42] phensi (LC50 4.41, 5.35, 5.91, 6.47; LC90 10.10, 12.04, 13.05, reported the phytosynthesis of silver AgNPs from leaves of 14.08 ppm). In the study, EAAgNPs show LC50 less than E. tirucalli and tested against IInd and IVth instar larvae of

Table 2 Larvicidal efﬁcacy of EAAgNPs against IIIrd instar larvae of A. aegypti and C. quinquefasciatus after 24 h of exposure.

Distilled water (control) shows nil mortality, significant at P < 0.05 level; LC50 lethal concentration that kills 50% of the exposed adults and larvae, UCL upper confidence limit, LCL lower confidence limit.

Silver nanoparticles EAAgNPs Concentration (ppm/l) and % mortality LC50 (ppm) LC90 (ppm) LFL–UFL LFL–UFL Aedes aegypti (IIIrd instar larvae) 10 – 100 ± 0.00 2.4 7.8 8 – 91.66 ± 2.88 (1.87–2.96) (6.51–10.44) 6 – 78.33 ± 2.88 4 – 63.33 ± 2.88 2 – 46.66 ± 2.88 Culex quinquefasciatus (IIIrd instar larvae) 10 – 98.33 ± 2.88 2.8 12.06 8 –85.66 ± 5.00 (2.05–3.45) (9.20–16.31) 6 – 70.00 ± 5.00 4 – 55.00 ± 5.00 2 – 43.33 ± 2.88

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