Greener pathway toward the synthesis of lichen‐based ZnO@TiO2@SiO2 and Fe3O4@SiO2 nanocomposites and investigation of their biological activities

Abstract A green way is introduced to biosynthesis of ZnO@TiO2@SiO2 and Fe3O4@SiO2 nanocomposites using the bioactive potential of Lecanora muralis (LM) lichen. UV‐Vis spectroscopy and GC–Mass analysis of the lichen show the presence of various bioactive constituents inside the lichen aqueous extract. The XRD, SEM, EDS, and elemental mapping techniques revealed the well fabrication of biosynthesized nanostructures. Also, investigation of antibacterial and antifungal activities of nanostructures demonstrated that green synthesized nanostructures have a very good antibacterial ability against Staphylococcus aureus, Escherichia coli, Pseudomonas spp. and Candida spp. pathogenic bacteria, and fungi but no antifungal activity toward the Aspergillus flavus, Aspergillus niger, and Aspergillus terrus fungi species.


| INTRODUC TI ON
Nowadays, synthetic nanomaterials are a great challenge for human life as they mostly produce using noneco-friendly and expensive processes through harsh conditions. In some cases, applications of them are limited due to the adsorption of chemicals and hazardous materials on their surfaces such as their employments in medicinal usages; thus, currently researchers trying to replace and improve the traditional methods of nanomaterial synthesis to the green, economic, and safe methods, Scheringer, 2008;Shi, Magaye, Castranova, & Zhao, 2013;Zhang, Guo, Li, Wang, & Liu, 2018). Green routes of science and technology cover a broad area mainly involves the monitoring and assessment, pollution prevention and control, and remediation and restoration. In fact, tracking those pollutions is essential to avoid the production of environmentally hazardous substances or alter human activities in ways that minimize damage to the environment.
In nanotechnology, both controlling the hazardous of nanomaterials before entering the environment and also improving the condition of ecosystem caused to introduce the concept of eco-nanotechnology as an important line of the environmentally friendly technologies, (Nasrollahzadeh, Sajadi, & Hatamifar, 2016;Oomen et al., 2015;Scott-Fordsmand et al., 2017;Sun et al., 2015;Vijayaraghavan & Ashokkumar, 2017).
Ever since the birth of mankind, human beings have been dependent on plants to fulfill their basic needs of life and even for the maintenance and restoration of health which among the plants, lichens are a wide range of habitats throughout the world, (Ismed et al., 2018;Ranković., Kosanić, & Stanojković, 2011;Walker., Lintott, 1997).
In fact, they are symbiotic organisms with no roots, leaves, or flowers and in a close symbiotic association consisting two unrelated organisms of fungal and photosynthetic partner such as green algae or cyanobacteria. Up to now, a lot of species of lichens and their products are known and directly or indirectly employed in medicinal and industrial usages. Among these compounds, secondary metabolites produced by lichens caused to their widely applications in folk and modern medicine due to their diverse bioactivities against various diseases such as usnic acid with a potential effect in cancer therapy due to its antimitotic and antiproliferative action. Lichen-forming fungi produce antibiotic secondary metabolites that protect many animals from pathogenic microorganisms; thus, in this way they demonstrated a very good antibacterial activity. Screening the lichens has revealed the frequent occurrence of metabolites with antioxidant properties. In fact, they act as bioreducer to prevent the side effects of free radicals inside the human bodies. Beside the medicinal effects of antioxidant phytochemicals inside the lichens, they have a very good potential to reduce and stabilize metallic salts and convert them to the metal nanoparticles through an electron transfer mechanism, (Kumar, Kumar, & Kumar, 2016;Agbanelo, Adesalu, 2016;Nugraha et al. 2019;Jayanthi, Priya, Monica Devi, & Benila Smily, 2012;Santiago, Sangvichien, Boonprago, & Dela Cruz, 2013;Rankovic, Kosanic, 2015;Boustie, & Grube, 2005).
Therefore, during this study we used Lecanora muralis (LM) collected from Iraqi Kurdistan as the source of bioactive antioxidant phytochemicals to green synthesis of ZnO@TiO 2 @SiO 2 and Fe 3 O 4 @ SiO 2 nanocomposites and evaluation of their antibacterial and antifungi activities.

| Instrumentations
All chemicals used in current study were purchased from Merck and Aldrich companies. XRD analysis was carried out for both lichen thalli and nanostructures to determine the types and formation of organic biominerals and the mineralogy of prepared nanostructures. The instrument used was PANalytical X'Pert 3 Powder using Cu Kα radiation equipped with a diffractometer system XPERT-PRO. The diffraction pattern was recorded for 2θ from 5° to 70° and a 2θ step scan of 0.010° was used, counting for 0.5 s at every step. The voltage and current of the generator were set at 45 kV and 40 mA, respectively. The prepared nanomaterials coated with gold using coater machine; then, particle morphology was investigated using FEI (Quanta 450) scanning electron microscopy equipped with Quantax EDS-Xflash 6/10 microanalyzer for detecting chemical composition of the prepared nanomaterials. The GC-MS analysis of bioactive compounds in both species

| Study area
The lichen was collected from Grdmandil mountain, Choman district, Erbil Provinces, located between 36°39.60 Latitude and 44°57.40 Longitude at the elevation 1,381 m above sea level. The main structure component of the of the rock-inhabiting lichens in the studied area is Quartz, Hematite, Magnetite, and Maghemite Q (subcell) as shown in Figure 1.

| Specimen (lichen) collection and identification
Specimen of lichen Lecanora muralis (LM) was collected with its rock sample. The species of lichen was identified previously in this area by Aziz (2005). Possible dust was carefully removed with soft brush on the lichen thalli then carefully detached from substratum with a sharp blade. Pretreated lichen was air-dried then ground in a porcelain mortar to obtain the required amount for the analysis,

| GC-Mass analysis of Lecanora muralis lichen species
The pulverized of lichen thalli Lecanora muralis submerged in methanol, incubated overnight with stirring on a magnetic stirrer, and filtered through Whatmann No. 41 filter paper; then, 1 μl of the sample of the solutions was employed in GC-MS for analysis of different compounds, Figure 3. The GC-Mass analysis of the lichens showed the presence of various types of bioactive phytochemicals responsible for green synthesis of nanocomposites, Table 1.

| Preparation of lichen extracts
The Lecanora muralis (LM) lichen was used to green synthesis of nanocomposites. 2 g Lecanora muralis (LM) was mixed to 30 ml distilled water at 80°C for 1 hr then filtered. The filtrate was used as lichen extract to biosynthesis of nanoparticles.

| Green synthesis of Lecanora muralis (LM) based ZnO@TiO 2 @SiO 2 and Fe 3 O 4 @SiO 2 NCs
One pot green synthesis procedure was used during the current study. Initially, 0.5 g of ZnCl 2 , 1.5 g of titanyl hydroxide TiO(OH) 2 and 2.5 g Na 2 SiO 3 were mixed with 20 ml LM extract at pH 9 (as adjusted using Na 2 CO 3 ) and 80°C under stirring for 5 hr until the formation of a slightly light precipitate of ZnO@TiO 2 @SiO 2 NCs. The precipitate was separated using filtration, washed with hot distillate water to remove the impurities then dried, and kept to further investigations.
In case of fabrication of Fe 3 O 4 @SiO 2 nanocomposites, 0.7 g FeCl 2 and 1.2 g FeCl 3 were mixed with 20 ml LM extract containing 2 g Na 2 SiO 3 at pH 9 (as adjusted using Na 2 CO 3 ) while stirring at 80°C for 5 hr. In the next step, the precipitate was separated using filtration, washed with hot distillate water to remove the impurities then dried, and kept to further investigations, Scheme 1.

| Bioactivity of lichen extract and prepared nanomaterials
For more accuracy, both disk diffusion and well diffusion methods were used to determine antibacterial activity. The antifungal (Candida spp.) activity of the lichen extract and nanostructures was monitored using the poisoned food method.

| Antibacterial activity
Disk diffusion method inoculated bacterial suspension 10 5 CFU/ml of Mueller-Hinton agar. Sterile filter paper disks loaded separately with lichen extract and prepared nanomaterials were placed on the top of Mueller-Hinton agar plates.
The plates were incubated at 37°C for 24 hr. The diameter of The diameter of inhibition zone was measured after 24 hr incubation using a caliper and recorded in millimeters.

| Antifungal activity (Aspergillus spp.)
The poisoned food method was used in the preliminary screening of lichen aqueous extracts and prepared nanomaterials for their antifungal properties' evaluation. First, the mycelia growths were evaluated in 60 mm Petri dishes filled with PDA solid medium amended with lichen extracts and prepared nanomaterials. Next, the center of each Petri dish was inoculated with 5 mm diameter disk of fungal mycelium, taken from pure culture (7 days old). Then, all inoculated dishes were incubated at 25°C for 6 days. After that, the radial mycelial growth was measured 6 days after inoculation. Finally, the antifungal activity of each extract was calculated in terms of inhibition percentage of mycelia growth by using the formula of: where dc is the average increase in mycelia growth in control, and dt is the average increase in mycelia growth in treated, (Hale, 1974;Singh, Tripathi, 1999).

| RE SULTS AND D ISCUSS I ON
Lichens contain various secondary bioactive metabolites within the thalli and typically form crystals on the surface of the fungal hyphae. Their secondary metabolites have many pharmaceutical properties such as antimicrobial, antioxidant, antiviral, anticancer, antigenotoxic, anti-inflammatory, analgesic, and antipyretic activities, Table 1. Hence, the present study is undertaken the potential ability of lichen extract as the source of antioxidants to biosynthesis of some bioactive nanostructures.

| Identification of the green synthesized lichenbased Fe 3 O 4 @SiO 2 NCs
According the XRD shown in Figure 4, it clearly reveals the pres-    Thus, all analysis showed the fabrication of the Fe3O4@SiO2 NCs.

| Identification of the green synthesized lichenbased ZnO@TiO 2 @SiO 2 and Fe 3 O 4 @SiO 2 NCs
Besides the application of LM lichen to biosynthesis of Fe3O4@ SiO2 NCs, it was also used to synthesis of bioactive ZnO@TiO 2 @  Figures 8 and 9. Therefore, all analysis confirmed the fabrication of ZnO@TiO 2 @SiO 2 NCs using the bioactive potential of the lichen.

| CON CLUS IONS
During this research, the ability of Lecanora muralis (LM) lichen aqueous extract was examined to biosynthesis of Fe 3 O 4 /SiO 2 and ZnO/ TiO 2 /SiO 2 nanocomposites through a simple, eco-friendly, economic, and rapid method as characterized using micrograph and diffractogram techniques. Analysis of the lichen extract using GC-Mass technique revealed the presence of valuable bioactive phytochemicals inside that. Also, the green synthesized nanocomposites showed a very good bioactivity against some common pathogenic bacteria and fungi due to the accumulation of lichen phytochemicals on the surface of them. F I G U R E 1 0 Antibacterial (disk diffusion method) and antifungal activities of LM lichen extract and green nanomaterials

ACK N OWLED G M ENT
The authors are grateful to Soran University for partial support of this work.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing. F I G U R E 11 Antibacterial (Well diffusion method) and antifungal activities of LM lichen extract and green nanomaterials