The genotype complexity and poor prognosis of cancer are considered as the main barriers in its treatment. The use of nanotechnology as a practical field to develop effective methods in the diagnosis and treatment of cancer has been considered by many researchers today . Metal and metal oxides NPs such as Ag and ZnO-NPs play a prominent role in biomedical applications . In current study, Pecan smoke extract (PHSE) was used as a biological platform for the green synthesis of ZnO and Ag-NPs, and then the biological effects of nanoparticles were investigated and compared.
The use of biological systems such as plant extracts and metabolites as reducing and stabilizing agents for the fabrication of NPs is called the green synthesis method, which has been successfully used in cancer treatment approaches and is an effective step in eliminating the disadvantages of physical and chemical methods .
In this study, smoke from burning Peganum Harmala seeds was used to green synthesize of silver (ps: 25.99 nm and PDI: 0.25) and zinc oxide (ps: 55.72 nm and PDI: 0.25) nanoparticles. Harmala is a native plant of Iran and belongs to the Zygophyllaceae family [38, 39]. In the roots and seeds of this plant, alkaloid compounds, beta-carbolines such as harmin, Harman, quinazoline and etc. are found [40, 41] and its anti-cancer, antimicrobial, anti-inflammatory and antioxidant effects have been reported in previous studies . Due to the growing burden of cancer worldwide, different types of metal NPs have been fabricated through the green approach. For example, Zinc oxide-nanoparticles have been fabricated by various types of bio-platforms such as Laurus nobilis , Deverra tortuousa , Garcinia mangostana fruit , and Cucumis melo inodorus rough shell extracts . Also, there are several types of plant extracts that have been utilized for synthesizing Ag-NPs including Amphipterygium adstringens , Centella Asiatica , and Ficus benghalensis prop root extracts . In a study in 2015, Ag-NPs with a size of 5 to 25 nm from the aqueous extract of Caulerparacemosa were prepared by green method .. In another study, Ag-NPs were green synthesized using the sumac aqueous extract with an average size of 35nm  which is comparable to the Ag-NPs synthesized in the present study.
Similar to the present study, in a study conducted in 2021, globular nanoparticles with 55 nm were fabricated by the green method. In this study, Arthrospira platensis was used as a reducing agent for the formation of NPs .
In this study, the toxicity of Ag-NPs and ZnO-NPs against three cancer cell lines was compared with HFF cells. The most sensitive and resistant cell lines to treatment with Ag-NPs were HepG2 and PC3 with IC50 about1.95 and above of 40 µg/mL, respectively, while in the treatment with ZnO-NPs the highest and lowest resistance to treatment in HFF and A2780 cells with IC50 higher than 62 µg/mL and 11.65 µg/mL (respectively) was observed. Since ZnO-NPs did not show any toxic effect on normal cells at the studied concentrations, it can be said that ZnO-NPs are safer compared to Ag-NPs. Many studies similar to our study have shown the toxicity of various NPs including Ag-NPs and ZnO-NPs on cancer cells.
A study in 2018 showed higher cytotoxic effects of Ag-NPs synthesized from Artemisia tournefortiana extract against HT-29 (IC50=40.71 mg/mL) compared to the HEK293 (IC50=61.38 mg/mL) as a normal cells . In another study was reported the strongly cytotoxic effects of Ag-NPs synthesized by aqueous extract of Rubia tinctorum against HepG-2 cells (IC50: 6µg/mL) compared to the HFF (IC50: 100µg/mL) as a normal cells .
In a study was conducted in 2021 on the fabricated ZnO-NPs by Swertia chirayita leaf extract revealed the toxic effects against HCT-116 and Caco-2. The nanoparticles showed a strongly cytotoxic potency against HCT-116 and Caco-2 cell lines, but less so against the normal HEK-293 cell line . Also, in a study in 2019, Mahdizadeh et al. have reported the toxicity of ZnO-NPs from Cucumis melo on MCF7 (40 µg/mL) and Tubo (20 µg/mL) cells after 24 h .
ROS increasing, although under natural conditions causes diseases related to oxidative stress such as cancer, but in the treatment of cancer is used as one of the applied strategies in chemotherapy . Studies show that increasing the amount of ROS can lead to the release of cytochrome C and activation of apoptotic pathway by changing the permeability of the mitochondrial membrane . Our findings show the antioxidant effect of ZnO and Ag-NPs in vitro and its pro-oxidant effects inside the cancer cell. Our results exhibited a significant antioxidant capability for Ag-NPs for ABTS (IC50:150.07µg/mL) and DPPH (IC50: 707.33µg/mL) free radicals (Fig. 5). In other words, ZnO-NPs appeared as a weak radical scavenger compared with Ag-NPs and glutathione antioxidant potential.
In 2019, Ahmadi et al reported the inhibitory effect of ZnO-NPs against ABTS and DPPH free radicals with IC50 about of 31.2 and 60µg/mL , which shows a higher antioxidant power compared to our study. Similar to the present study, in a 2015 study, the inhibitory effects of Ag-NPs from Cassia roxburghii on ABTS free radicals (IC50: 140 µg/mL) and their reducing power on iron ions (FRAP of 2.64 (Mg−1)) were investigated and confirmed. Also, in a study in 2017, 23-95% of ABTS free radical scavenging capacity and 8.8 Mg−1 FRAP activities of Ag-NPs were reported . The results of many studies show that NPs increase ROS production in various cancer cells . Similar to the present study, in a 2020 study, the anti-proliferative and pro-oxidant effects of Ag-NPs from Beta vulgaris L were demonstrated in human hepatic cancer cells (HUH7) by ROS induction. Lee et al. reported the potential to stimulate ROS production in human keratinocytes treated with ZnO-NP. They showed that the mechanism of nanoparticle toxicity is related to ROS production, oxidative stress and apoptosis . In another study in 2017, Bai et al. have exhibited the increasing intracellular ROS in SKOV3 treated with ZnO-NPs .
The results of this investigation showed the wide range of safe toxic doses of ZnO and Ag-NPs indicate their pro-oxidant activity and thus approve their ROS-mediated cytotoxicity .
Angiogenesis is one of the most important features of tumor cells that help their survival and reducing angiogenesis plays the main role in preventing the growth of cancer cells and inflammation . The results of this survey confirmed the reduction of angiogenesis in the treatment with different concentrations of Ag-NPs in CAM and qPCR assay. Investigation of the effect of ZnO-NPs on angiogenesis showed that, they suppress angiogenesis by down-regulating VEGF and VEGFR gene expression at 0 to 500µg/mL concentrations and act as a pro-angiogenic compound at>500µg/mL doses. In many studies consist with the present investigation, the anti-angiogenesis effects of nanoparticles have been investigated and confirmed [64, 65] .
For example, a reduction in angiogenesis by the CAM method was reported in a 2015 study by examining the mean number and length of blood vessels in samples treated with Ag-NPs. Also, the inhibitory effect of Ag-NPs on VEGF expression in the mouse matrigel plug  and retinal endothelial cells  has been reported in previous studies.
Various investgations have shown the anti and pro angiogenesis potential of ZnO-NPs. In a study in 2019, the anti-angiogenesis effects of ZnO-NPs from Hyssops officinalis L were reported using the CAM assay . Ahtzaz et al reported the pro-angiogenic effects of ZnO-NPs in 2017 using the CAM method . In a study by Oikawa et al., The inhibitory effect of ZnO-NPs on the formation of blood vessels by reducing the expression of VEGF and VEGFR was reported  which is comparable to our results.