Calendula officinalis L. flower extract-mediated green synthesis of silver nanoparticles under LED light

Silver nanoparticles (AgNPs) are among the most known nanomaterials being used for several purposes, including medical applications. In this study, Calendula officinalis L. flower extract and silver nitrate were used for green synthesis of silver nanoparticles under red, green and blue light-emitting diodes. AgNPs were characterized by Ultraviolet-Visible Spectrophotometry, Field Emission Scanning Electron Microscopy, Dynamic Light Scattering, Electrophoretic Mobility, Fourier Transform Infrared Spectroscopy and X-ray Diffraction. Isotropic and anisotropic silver nanoparticles were obtained, presenting hydrodinamic diameters ranging 90 – 180 nm, polydispersity (PdI > 0.2) and moderate stability (zeta potential values around – 20 mV).

Green synthesis is considered a clean, nontoxic, simple and cost effective method to get nanoparticles. Different metal nanoparticles using silver, gold, zinc, copper and titanium can be synthesized by the reduction of metal precursor salts using plant extracts (Raj, Mali, Trivedi, 2018;Sengottaiyan et al., 2016;Muthusamy et al., 2015;Sone et al., 2015;Bindhu, Umadevi, 2015).
Calendula officinalis L. is a plant belonging to the Asteraceae family that present several medicinal properties and it is used in all over the world (Mishra et al., 2018) due to their anti-inflammatory, antitumor, antimicrobial and wound healing activities (Emre et al., 2018;López-Padilla et al., 2017). The main C. officinalis flower compounds are flavonoids, terpenoids, carotenoids, coumarines, quinones, amino acids and carbohydrates (Mishra et al., 2018;Nicolaus et al., 2017).
Asteraceae extracts-mediated green synthesis of metallic nanoparticles under different treatments are found in the literature (Francis et al., 2018;Vijayan et al., 2018), but photocatalytic reactions sensitized by light-emitting diodes (LEDs) has been used to get AgNPs with improved properties (Kumar et al., 2016;Lee et al., 2014;Stamplecoskie, Scaiano, 2010). This study describes the synthesis of AgNPs from C. officinalis flower extract and silver nitrate using red, green and blue LEDs.
Preparation of the C. officinalis flower extract 5 g of C. officinalis dried flower were weighed and washed several times with deionized water at room temperature. Then, the flowers were crushed and immersed in 100 mL of deionized water at 60-80 °C for 30 minutes. The C. officinalis flower extract was obtained by removing solids in simple filtration using qualitative filter paper (80 g, FITEC ® ) (Thema et al., 2016).

Preparation of AgNPs
For green synthesis of AgNPs, a solution of C. officinalis flower extract in AgNO 3 (1 mmol.L -1 ) was prepared in 1:20 ratio. This solution was exposed to red (630 nm), green (512 nm) and blue (455 nm) Light-Emitting Diodes (LEDs) light for 48 h.
The obtained AgNPs were named as follows: AgNPsR (obtained AgNPs under exposure to red LED), AgNPsG (obtained AgNPs under exposure to green LED) and AgNPsB (obtained AgNPs under exposure to blue LED).

Field Emission Scanning Electron Microscopy
Morphological analysis of AgNPs was performed on Field Emission Scanning Electron Microscopy (FE-SEM) (Mira3, TESCAN ® ) at 15 kV. Prior to the analysis, the AgNPs were placed on copper tapes in stubs, dried at room temperature and submitted to metallization with gold (SC7620, QUORUM ® ).

Hydrodynamic Diameter, Polydispersity Index and Zeta Potential analysis
Hydrodynamic Diameter and Polydispersity Index of the AgNPs were determined by Dynamic Light Scattering (DLS) and Zeta Potential was determined by electrophoretic mobility (Zetasizer Nano ZS90, MALVERN ® ) in three times (T = 0 day, T = 30 days and T = 60 days) to stability evaluation. Prior to the analysis, the samples were diluted 1:20, in deionized water.

Fourier Transform Infrared Spectroscopy
In order to confirm the presence of the C. officinalis flower extract in the coating of the AgNPs was used Fourier Transform Infrared (FTIR) Spectroscopy (IRPrestige-21, SHIMADZU ® ) in the range of 4000-400 cm -1 in 64 scans with 4 cm -1 resolution and potassium bromide pallet method (Kumar et al., 2016). Prior to the analysis, the AgNPs were centrifuged three times for 30 minutes at 18,000 rpm and then freeze-dried.
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RESULTS AND DISCUSSION
During preparation of the formulations there was a change of color from pale yellow to reddish brown, indicating that C. officinalis flower extract compounds were able to promote the reduction of silver from Ag +1 to Ag 0 , forming AgNPs (Baghizadeh et al., 2015).
Formation, size and shape of AgNPs can be characterized by UV-Vis spectroscopy (Baghizadeh et al., 2015) since AgNPs show optical absorption, named surface plasmon resonances, at wavelengths of 350-500 nm (Bindhu, Umadevi, 2015;Pal, Tak, Song, 2007). The larger is the nanoparticles, the greater is the wavelength of maximum absorbance and the bands intensity. The wavelength of maximum absorbance also varies according to the different AgNPs shapes (Khan et al., 2011;Bhui et al., 2009;Pal, Tak, Song, 2007).
According to Mie's theory, a single plasmon absorption band is expected in the spectra of spherical nanoparticles, whereas more than one plasmon absorption bands are expected in the spectra of anisotropic nanoparticles (Pal, Tak, Song, 2007). Figure 1 shows the plasmon absorption bands of AgNPs obtained using LEDs.   Table I shows that the use of red LED resulted in smaller nanoparticles than the use of green and blue LEDs. However, the hydrodynamic diameters values obtained for all AgNPs produced were higher than the hydrodynamic diameters values found in the literature for AgNPs obtained by green synthesis without LEDs (Baghizadeh et al., 2015;Bindhu, Umadevi, 2015;Bhui et al., 2009).
All dispersions showed polydispersity (PdI > 0.2) (Soema et al., 2015) and moderate stability once presented zeta potential values around -20 mV (Coviello et al., 2015). The negative zeta potential values obtained can be attributed to the C. officinalis flower extract compounds.
After 60 days, AgNPsR increased by 111,71% their hydrodynamic diameters, AgNPsG increased by 52,77% and AgNPsB increased by 35,49%. However, the zeta potential values remained very close to the initial values, evidencing the maintenance of stability.
The AgNPs FTIR spectra showed the same bands found in the C. officinalis flower extract spectrum (Figure 3 and Table II), confirming the presence of the C. officinalis flower extract in the coating of the AgNPs. However, a new C=O band of cetone groups appeared at 1722-1716 cm -1 in the AgNPs spectra, which may be result of a reduction reaction. Bands on ~1060 cm -1 suggest terpenoid or flavonoid compounds (Rad, Mokhtari, Abbasi, 2018;Hosseinkazemi et al., 2015).

CONCLUSION
Were obtained isotropic and anisotropic AgNPs with hydrodynamic diameters of 89 to 175 nm from C. officinalis flower extract and AgNO 3 under red, green and blue LED. AgNPs remained stable during the evaluated period with potential zeta values around -20 mV, but increased their hydrodynamic diameters considering that AgNPsB showed a smaller increase than AgNPsR and AgNPsG. AgNPs with required properties can be produced from the proposed method in order to be used as antimicrobial in health products.
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