Comparative studies on chemically synthesized and biosynthesized zinc oxide nanoparticles using Desmodium sp. and their potential as UV filters

Plant-based synthesis of nanoparticles has been a great interest topic due to reducing the use of toxic materials and the presence of bioactive compounds. This study investigated the chemically synthesized and biosynthesized zinc oxide (ZnO) nanoparticles using Desmodium triquetrum leaf extract and their potential as UV filters. These ZnO were evaluated using FE-SEM, EDX, XRD, and FTIR. The in-vitro Sun Protector Factor (SPF) was determined using a spectrophotometer. SEM image revealed the flower shape morphology of biosynthesized ZnO and chemically synthesized ZnO with different particle sizes. The biosynthesized ZnO nanoparticles exhibited smaller particle sizes than chemically synthesized ZnO. XRD analysis demonstrated that the average crystallite sizes of biosynthesized and chemically synthesized ZnO were 10.34 nm and 15.08 nm, respectively. The biosynthesized ZnO showed an SPF value of 25.12, indicating stronger UV protection ability than chemically synthesized ZnO (SPF=9.72) at a concentration of 1 mg/mL. These results indicate that the biosynthesized ZnO nanoparticles could be a great candidate as a UV filter for further sunscreen formulations.


Introduction
Nanoparticles possess nanoscale size and high surface areas that provide opportunities to utilize in various applications, including imaging, biotechnology, energy-based research, catalysis, agriculture, environment, food, and medical applications [1,2].Three primary methods, including chemical, physical and biological, have been applied to synthesize nanoparticles.However, chemical and physical methods have drawbacks related to toxic chemical release, high energy consumption, and complicated synthesis circumstances and equipment [3].To counter those disadvantages, the biological methods, especially plant-based synthesis, have gained growing interest in recent years due to their cost-effective, simple process, non-toxic waste products, biocompatible, and no surfactant/capping agent needed as bioactive compounds in plant extract could act as stabilizing and capping agents [4,5].
Among metal oxide nanoparticles, ZnO nanoparticles have a wide band gap and high binding energy that provide opportunities for scientific and industrial applications [6].ZnO nanoparticles show potential use in many fields, such as catalysis, piezoelectric devices, anticancer agents, antimicrobial agents, chemical sensors, anti-corrosives, and nanogenerators [7].Zinc oxide nanoparticles also have unique properties, including low toxicity, whitening properties, and a strong capability to absorb UV which make them good for personal cosmetics such as sunscreen and skin creams [8,9].
Plant-based synthesis of ZnO nanoparticles has been conducted not only using leaf extract but also other parts of plants including flowers, fruit, bark and seed.According to previous studies, ZnO nanoparticles have been synthesized successfully using aqueous fruit extracts of Myristica fragrans [10] , leaf extracts of Elaeagnus angustifolia L [11], bark extract of Cinnamomum verum [12], seed extract of Eriobutria japonica [13], and flower extract of Nyctanthes arbor-tristis [14].The presence of bioactive compounds in plant extract, such as flavonoids, phenolic compounds, alkaloids, terpenoids, and saponins could act as reducing agents as well as stabilizing and capping agents for the synthesis of zinc oxide nanoparticles.
In this study, we investigated chemically and biosynthesized ZnO using Desmodium triquetrum leaf extract and their application as a UV filter.Desmodium triquetrum extract has been studied as an effective reducing and capping agent to synthesize silver nanoparticles and their application as antibacterial, antibiofilm and anticancer [15].However, using Desmodium triquetrum to synthesize zinc oxide nanoparticles and their application as potential UV filters has not been reported yet.

Materials
Desmodium triquetrum (DT) was procured from the Research Center for Traditional Medicine, Tawangmangu, Indonesia.Zinc nitrate tetrahydrate and sodium hydroxide were obtained from Sigma Aldrich.

Preparation of Extract
The dried leaves of DT were ground using an electric grinder to acquire fine powder.Leaf powder (4 gr) in distilled water (200 mL) was stirred at 65°C for 30 minutes.The resultant extract was filtered and stored at 4°C for further use.

Chemical synthesis of ZnO nanoparticles using reduction-precipitation method (c-ZnO)
Sodium hydroxide (0.5M) was added dropwise to 100 mL of zinc nitrate tetrahydrate (0.05M) until the pH reached 10.The mixture was heated at 55°C for 20 minutes with continuous stirring.The white precipitation was obtained on the bottom of the beaker glass, indicating the formation of ZnO.The separation of zinc oxide was conducted using centrifugation at 8000 rpm for 15 minutes.The distilled water was used to rinse the precipitation.This process was repeated twice to get rid of the residues.The final zinc oxide was dried in an oven (65°C) for 24 hours.The white powder was stored at 4°C.

Biosynthesis of ZnO nanoparticles using Desmodium triquetrum extract (b-ZnO)
The plant extract of 15 mL was mixed with 100 mL of zinc nitrate tetrahydrate (0.05M) under constant stirring.Sodium hydroxide was added dropwise in the mixture until pH reached 10.Continuous stirring was conducted for 20 minutes at 55°C.The precipitation was collected by centrifugation at 8000 rpm for 15 minutes.Distilled water was used to rinse the precipitation.The final product was dried in an oven at 65°C for 24 hours and then stored at 4°C.

Characterization of ZnO nanoparticles
The morphology of ZnO nanoparticles was analysed by FE-SEM (Jeol JIB-4610F).EDX detector attached to the FE-SEM was applied to demonstrate elemental analysis.The crystalline structure of the zinc oxide was observed by XRD (PANalytical AERIS).FTIR (Thermo Scientific Nicolet iS-10) was utilized to identify functional groups bonded with the zinc oxide.

Evaluation of Sun Protection Factor (SPF)
Four milligrams of ZnO were prepared in ethanol (4 mL).The ultrasonication was applied to the sample for 10 minutes.Then, the absorption spectra were evaluated by a spectrophotometer in the range of 290 to 320 nm and analysed by the Mansur equation [16] as below: SPFspectrophotometric = CF x EE (λ) x I (λ) x Abs (λ) Where CF= correction factor (=10); EE = spectrum of erythermal effect; I = spectrum of solar intensity.The constant value was applied for EE x I as presented in Table 1 [17].

FE-SEM and EDX Analysis
FE-SEM images show the morphology of c-ZnO and b-ZnO nanoparticles as presented in Figure 1a and Figure 1b.The flower-like structures which consist of petals and spherical shapes are observed.Image J software was applied to calculate the particle size of c-ZnO and b-Zno.The diameter of spherical and length of petal shape from c-ZnO was found to be nm -666.21nm and 101.73 nm -796.32 nm respectively and b-ZnO possessed particle sizes of 30.10 nm-100.33nm (spherical shape) and 139.4-505.27nm (petal shape).The b-ZnO image (Figure 1b) exhibits smaller particle sizes than the c-ZnO (Figure 1a) which might be due to the presence of a capping agent derived from the plant extract.Aqueous extract of Desmodium triquetrum contains polyphenols that could act not only as reducing agents but also as capping agents [15].Capping agents can prevent the over-growth of nanoparticles and agglomeration in the synthesis process [18].Furthermore, the elemental analysis determined by EDX is presented in Figure 1c and Figure 1d.The result exhibits the characteristic of zinc and oxygen with signal peaks at ∼1.0, 8.6, and 9.5 KeV which is related to Zn and ∼ 0.5 KeV which is assigned to O.The quantitative analysis of c-ZnO demonstrates that the atomic of Zn and O was found to be 46.6% and 53.4% respectively (Figure 1c).Meanwhile, b-ZnO nanoparticles show 45.4% for Zn and 54.6% for O.

Evaluation of Sun Protection Factor (SPF)
Spectrophotometric analysis was conducted to obtain absorption characteristics of c-ZnO and b-ZnO.
According to the result of Mansur's mathematical equation which was applied to calculate SPF values of ZnO nanoparticles based on their absorption in the wavelength range of 290 nm and 320 nm, c-ZnO showed a lower SPF value, which was found to be 9.72 at a concentration of 1 mg/mL and b-ZnO demonstrated an SPF value of 25.12.This result indicates that b-ZnO nanoparticles are more effective in shielding skin from UV radiation than c-ZnO nanoparticles.The b-ZnO nanoparticles in this study also show higher SPF value than another report studying biosynthesized ZnO using Solanum lycopersicum fruit extract with an SPF value of 16.8 at a concentration of 10 mg/mL [28].US Food and Drug Administration (FDA) suggest that the formulation with SPF > 2 could be recognized as sunscreen and an SPF value of 15 or higher has good protection for the skin and minimizes the harmful effect of the UV rays [29].The b-ZnO nanoparticles in this study possess good potential as a UV filter for further sunscreen formulation.

Conclusion
Zinc oxide nanoparticles were successfully synthesized by chemical and biological method using Desmodium triquetrum extract in this study.Flower-like structures of ZnO nanoparticles are presented in the FE-SEM image.The b-ZnO nanoparticles have smaller particle sizes and crystalline sizes than c-ZnO nanoparticles.The FTIR spectrum shows the functional groups of bioactive compounds derived from the plant extract.The b-ZnO nanoparticles possess a higher SPF value than the c-ZnO, suggesting the b-ZnO nanoparticles have a stronger capability to absorb UV light and are more effective in protecting skin from damage due to UV irradiation.Therefore, the biosynthesized ZnO nanoparticles in this study could be a good ingredient in sunscreen formulation.

Figure 1 .
Figure 1.FE-SEM images of c-ZnO (a) and b-ZnO (b) and EDX analysis of c-ZnO (c) and b-ZnO (d).
FTIR spectra of c-ZnO and b-ZnO nanoparticles are presented in Figure3.In the c-ZnO and b-ZnO spectrum, the presence of ZnO vibration is demonstrated in the range of 410-500 cm -1[21].The c-

Table 1 .
Normalized function for SPF calculation.