Novel green synthesis of zinc oxide nanoparticles using Salvia rosmarinus extract for treatment of human lung cancer

: A green and low-cost approach was run to synthe-size zinc nanoparticles (NPs) using rosemary extract. The NPs were identi ﬁ ed by various methods, i.e., ultraviolet-visible and Fourier transform infrared spectroscopy, FE-scanning electron microscope imaging, X-ray di ﬀ raction, and energy-dispersive X-ray analysis. The radical scavenging activity and MTT assays were used to evaluate the biological activity of ZnO-NPs@Rosemary. The results revealed a spherical shape for ZnO-NPs@Rosemary with a crystal size of 30.74 nm. ZnO-NPs@Rosemary could scavenge the free radicals of DPPH with an IC 50 of 87.62 ± 0.47 μ g/mL. An MTT assay was run to investigate the anti-cancer activity of ZnO-NPs@Rosemary against PC-14, LC-2/ad, and HLC-1 as the selected lung cancer cell lines. The highest sensitivity of NPs was found against PC-14 with IC 50 of 178.84 ± 2.13. A dose-dependent activity was observed for ZnO-NPs@Rosemary against the chosen cell lines. The outcomes of the present study revealed an acceptable anti-lung cancer activity of ZnO-NPs@Rosemary.


Introduction
Cancer is known as one of the serious health troubles around the world.Oxidative stress is an important factor in cancer problems [1,2].According to many reports, lung cancer, with more than 1.7 million deaths in a year, is the most common type of illness [3,4].Cigarette smoking and airborne environmental pollution are the most critical risk factor for lung cancer, especially in developing countries [5,6].The therapeutic agents of tyrosine kinase inhibitors and immune checkpoint inhibitors are common remedies for lung cancer [7].Inducing apoptosis is one of the most effective methods to treat lung cancer [8].Nowadays, chemotherapy is the primary method to treat cancer problems; however, due to the side effects and drug resistance, the efficiency of chemotherapy seems to be decreased [9].Recently, the application of nanomedicine to treat different health problems has gained more attention of researchers worldwide.For cancer therapy, the limitation of the common chemical remedies, such as toxicity, poor plasma-solubility, and low bioavailability, may be considered as the essential factors that cause nanomedicine to have obtained more attention to control different cancers, including lung cancer [10].
Recently, nanotechnology as a new branch of science, which involves synthesis design, modification, and applications of particles with a size up to 100 nm, has gained a lot of attention from different research groups.In medicine, nanoparticles (NPs) are utilized to detect and treat the presence of diseases such as cancers [6,[11][12][13].The encapsulation of drugs or preparation of delivery systems by NPs are the new paths in targeted cancer treatment [14].Green synthesis of NPs, which comprises the green chemistry approaches to synthesize nanomaterials such as metallic NPs, is known to be a desired branch in nanotechnology.In this method, the NPs are synthesized using biological candidates such as fungi, bacteria, and plants [15].In comparison to chemical methods, which comprise toxic chemicals, eco-friendly, low cost, and easy handling are the benefits of green synthesis of metallic NPs, i.e., silver, gold, nickel, zinc, and others that have been synthesized and their biological activities have been investigated [15,16].The plants' metabolites, including proteins, enzymes, and other biomolecules, are the capping and reducing agents for synthesizing NPs [15].Among metallic NPs, zinc oxide (ZnO-NPs) is one of the metals that scientists are interested in for green synthesis by plants.ZnO-NPs are utilized in numerous applications, including cosmetics, biomedicine, detectors, and semiconductors [17].ZnO-NPs represent various therapeutic applications such as antibacterial, antifungal, antiviral, and anticancer properties [14].Furthermore, in our review of the literature, there was no report on toxic effect of ZnNPs, so these points promote us to green synthesize of ZnO-NPs using the aqueous extract of rosemary as the capping and reducing agent.
Salvia rosmarinus, commonly called rosemary, corresponds to the Lamiaceae family.The plant is an aromatic and evergreen, like the other species of the family, that grows in many regions of the world [18][19][20].Rosemary is used to cure renal colic, dysmenorrhea, and muscle spasms in different ethnomedicines [21].Rosemary can reduce the bilirubin level in the plasma and control hepatic glycogen content [22].So far, various pharmaceutical activities of rosemary, such as antitumor, antifungal, antiviral, antibacterial, anti-inflammatory, antiulcerogenic, and antioxidant activities, have been reported [21][22][23].Rosemary is a remedy for nervous, cardiovascular, gastrointestinal, and genitourinary problems [21].The plant is an additive candidate in diet and also in food industries [1].The existence of various classes of compounds as the plant secondary metabolites, such as polyphenols, flavonoids, and aromatic terpenoids, are responsible for the wide variety of rosemary biological properties.The plant is rich in phenols of carnosic, rosmarinic, and caffeic acid, and phenolic diterpenes of carnosol, betulinic acid, and ursolic acid [1,21,23,24].Rosemary essential oil, with a pleasant smell, is a popular additive in cosmetic industries; however, it is utilized in aromatherapy to cure skin, digestive and renal problems [1].Additionally, it has exhibited antibacterial, antioxidant, and anti-inflammatory activities [20].The essential oil is dominated by α-pinene, limonene, 1,8cineole, borneol, and camphor [23,25].
Since rosemary is an available plant in different climates which grows so vigorously under convenient conditions and due to the various applications of rosemary in traditional medicine, herein, we have reported the greensynthesis of ZnNPs using an aqueous extract of rosemary.The techniques of ultraviolet-visible (UV-Vis) and Fourier transform infrared (FT-IR) spectroscopy with Scanning Electron Microscope Imaging (SEM), X-Ray Diffraction analysis (XRD), and Energy-Dispersive X-ray (EDX) were run to characterize ZnNPs.Furthermore, the aim of the present research is to evaluate the anticancer activity of the ZnO-NPs@Rosemary against PC-14, LC-2/ad, and HLC-1 as the selected human lung cancer cell lines for the first time.Besides, the cytotoxicity of NPs was also studied against the HUVEC cell line.

Preparation of rosemary extract
20 g of fresh aerial parts of rosemary was boiled in 150 mL of deionized water (30 min) to prepare the plant extract.Next the obtained extract was cooled and filtered.Finally, the aqueous extract was kept in a cold place before use for the synthesize of ZnNPs.

Green synthesis of ZnO-NPs@Rosemary
A previous procedure was run for the green synthesis of ZnO-NPs [26].A 60 mL of rosemary extract was mixed with 25 mL of Zn (NO 3 ) 2 .6H 2 O (0.2 M) at an adjusted pH (8.5).The mixture was put in an ultrasonic bath with 75 W power and 60°C for 1 h.After that time, the formed precipitates were separated by a centrifuge with a speed of 10,000 rpm for 10 min.A solution of ethanol:water (50:50) was used to wash the NPs three times and interval centrifuging.The residue was placed in an oven and dried at 55°C for 6 h.The NPs, as a light brown powder, were named ZnO-NPs@Rosemary.

Antioxidant activity evaluation
The ability of ZnO-NPs@Rosemary to scavenge free radicals was examined using DPPH assay.The experiment was carried out according to a reported procedure [27].The methanol solution of DPPH (1 mL, 0.1 mM) was poured into 0.5 mL of ZnNPs solution with various concentrations (1-1,000 µg/mL).The mixture was left in the dark for 2 h.The absorbance was then read at 517 nm.A synthetic antioxidant butylated hydroxytoluene (BHT) was used as the positive control.Equation ( 1) was used to calculate the NPs' antioxidant activity.The IC 50 was obtained from the inhibition curve for the various concentrations of NPs.

Cytotoxicity and anti-human lung cancer activity of ZnO-NPs@Rosemary
The cytotoxicity and anti-lung cancer activity of ZnO-NPs were examined using the MTT method.The assay was run according to a previous report on the study of the anticancer activity of metallic NPs [28].In this study, the cytotoxicity activity of ZnO-NPs@Rosemary was studied against HUVEC cell line; furthermore, the anticancer activity was evaluated against human lung cancer cell lines of PC-14, LC-2/ad, and HLC-1.To carry out this research study, cancer cells were cultured in 10% complete DMEM and HUVEC cells in 10% complete culture medium (RPM) containing FBS and penicillin-streptomycin antibiotics in T25 flasks for cell culture.They were kept in a CO 2 incubator (temperature 37°C, 80% humidity, 5% CO 2 pressure).Whenever the cell line reached 80% density, the cells (-10 4 cells/cm 2 ) were cultured in a 96-well plate consisting of the medium.
Then, over a night, the ZnO-NPs with various concentrations were added to each well, and cell viability was estimated at day 1.Finally, the survival rate of cells treated with ZnO-NPs was analyzed using the MTT method.For this purpose, the culture medium was taken out from the wells containing treated cells and untreated cells (control group) and then 150 µL of culture medium containing 15 µL of MTT solution (5 mg/mL) was poured into each well.The plates were then placed in an incubator at 37°C for 4 h.Next the MTT was removed, and DMSO (150 µL) was added to the wells.Finally, the absorbance was measured at 570 nm using an ELISA reader (Stat Fax 2100, Germany).The percentage of cell viability was calculated using equation (2).

Statistical evaluation
One-way analysis of variance and Duncan post hoc using SPSS software was used for the analysis of the collected data.

Results and discussion
3.1 Chemical characterization of ZnO-NPs@Rosemary

UV-Vis analysis
The surface plasmon resonance of the ZnNPs is shown in Figure 1 as the UV-Vis spectrum.The band shifting is dependent on the particle size.The increase in the size cause shifts in the band to a higher wavelength.For ZnO-NPs@Rosemary, the bands at 265 and 350 nm indicate the synthesis of ZnNPs using rosemary.

FT-IR analysis
FT-IR analysis, as a qualitative method, is a sufficient technique to study nano metallic materials, especially for the metal oxide NPs that are synthesized using green approaches.The peaks between 400 and 700 cm −1 are attributed to M-O bond. Figure 2 presents the FT-IR spectrum of ZnNPs.The peaks at 453 and 518 cm −1 relate to the Zn-O bond.Similar peaks have been reported for ZnNPs [26].The other bands at different wavenumbers of 1,039, 1,492-1,695, 2,956, and 3,414 cm −1 belong to stretching vibration for different bonds of C-O, C]C and C]O, C-H, and O-H.The peaks pertain to the various functional groups of organic constituents, such as phenolics and flavonoids.The compounds can be found in rosemary extract [21,23,24].These compounds perform a reducing and capping role to synthesize ZnO-NPs@Rosemary.Zinc oxide@Rosemary against lung cancer cell line  3

XRD evaluation
The evaluation of the XRD diagram is a sufficient method for the crystallinity study of the green synthesis of metallic NPs.Furthermore, the crystal size of the NPs can be calculated by XRD details.  3).The calculation revealed a 30.74 nm for crystal size.This is less than the reported size for the green-synthesized ZnNPs using plant extract of Ziziphora clinopodioides [26].

FE-SEM imaging
There are many reports in the literature that have used FE-SEM imaging to examine the surface of NPs.The FE-SEM images of ZnO-NPs@Rosemary are presented in Figure 4(a and b).
The synthetic NPs are materialized with a spherical morphology and an average size smaller than 50 nm.ZnNPs  are aggregated, which is known to be a general property of green-synthesized metallic NPs, including zinc, nickel, titanium, and silver [26,[29][30][31].Previous studies have reported a size of 15-80 nm for zinc NPs that were synthesized using different plant extracts [8,26,32,33].
The EDX graph of ZnO-NPs@Rosemary is exhibited in Figure 5.The EDX is a beneficial approach for studying the elemental analysis of nanomaterials.The producing of ZnNPs is verified by the appearance of peaks below 1, after 8.5, and around 9.5 keV that belong to ZnLα, ZnKα, and ZnKβ.The linkage of rosemary extract to the surface of ZnO-NPs@Rosemary is approved by the peak around 0.5 keV, belonging to Okα.Similar peaks have been reported for ZnNPs previously [26,33].

Antioxidant activity of ZnO-NPs@Rosemary
Free radicals are one of the important factors that lead to most chronic diseases such as cancer, skin problems, and diabetes.The radicals cause damage to cells by reacting with biological molecules such as DNA, RNA, proteins, and other molecules.Free radicals are an effective agent to oxidize food.They can oxidize fats, oils, and vitamins, which causes a decrease in the quality of the food in our diet [34].Nowadays, it seems the consumption of antioxidants is necessary.The antioxidants react with radicals to neutralize them and prevent the damage of cells or biological molecules.Antioxidants can be obtained from natural  Zinc oxide@Rosemary against lung cancer cell line  5 or synthetic sources.Synthetic antioxidants such as BHT, TBHQ, and BHA have many disadvantages to human health.They have been identified as carcinogenic and adverse agents.Herbal products are known as the important natural origins of antioxidants.Phenolics, flavonoids, and terpenoids, as the secondary metabolites of the plants exhibited a vigorous antioxidant activity by neutralizing free radicals.These compounds also inhibit the growth of cancer cells [35,36].So far, many researchers have reported the antioxidant activity of metallic NPs, which are synthesized using plant extract [26,29,30,32].The green-synthesized ZnNPs using rosemary extract show a potent radical scavenging activity (RSA) with IC 50 of 87.62 ± 0.47 μg/mL, which is more than that of BHT, which is the selected positive control in this research.Figure 6 exhibits the RSA of ZnO-  NPs@Rosemary and BHT at different concentrations.The RSA of ZnNPs synthesized by other plants extract has been studied; according to a previous report, the ZnO-NPs scavenged the DPPH with an IC 50 of 250 μg/mL [26].

Cytotoxicity and anti-lung cancer activities of ZnO-NPs@Rosemary
In recent years, cancer has been introduced as a severe health trouble.Cancer is identified by uncontrolled increase in cellular division [6].The increase in abnormal cells leads to large masses and ground swell tumors [37][38][39].Tumors will be divided and surrounded the normal cells, which will be destroyed finally.Furthermore, the normal cells cannot access the primary nutrients and oxygen, which causes a decrease in health and results in death, unless the treatment is begun.The cancer cells are characterized by a high rate of proliferation, changes in morphology, and resistance to apoptosis [37,40].Lung cancer is responsible for the most cancer death, even more than colon, breast, and prostate cancer [4,41].Men are more prone to lung cancer than women [42,43].Chronic tobacco usage is the fundamental reason for lung cancer.The late diagnosis of the disease is the primary reason for the high mortality of lung cancer.According to the reports, more than 85% of patients exhibit non-small cell lung cancer, and around 15% have small cell lung cancer [42,44].Chemo-and radiotherapy are the common treatment methods for cancer, but the methods have shown side effects.Instead, paclitaxel, an herbal product, is the first choice to cure lung cancer and many other cancer types.The drugs affect on the mitochondria of cancer cells [9].In the past decade, nanomedicine has been introduced as an alternative to cure various diseases such as cancer.The NPs can be used as injectable drugs or other types of medicine for cancer therapy [45].The NPs can be manipulated to bind to specific receptors that are highly expressed in a tumor.The large surface with an ability for surface modifications is the benefit of NPs [46,47].So far, much research has been reported on the anticancer activity of different metallic NPs such as Ag, Au, Ti, Pd, Ni, and ZnNPs.The high antioxidant activity of the metallic NPs is responsible for their anti-cancer activity [48][49][50].Figure 7(a)-(c) shows the activity of ZnO-NPs@Rosemary and negative controls of rosemary extract, zinc nitrate, and commercial zinc oxide inhibiting the growth of PC-14, HLC-1, and LC-2/ad, respectively.The results reveal ZnO-NPs@Rosemary is more active compared to the controls against the selected lung cancer cell lines.According to the results, a dose-dependency is observed for mortality of the cell lines treated with ZnO-NPs.The highest activity was obtained against PC-14 with IC 50 of 178.84 ± 2.13 μg/mL followed by 285.50 ± 3.32 for LC-2/ad and 340.97 ± 5.71 for HLC-1.In contrast, the NPs showed low cytotoxicity against HUVEC as a normal cell line even at the high concentration of 1,000 μg/mL (Figure 7d), which reveals the potent activity of ZnO-NPs@Rosemary to cure lung cancer.

Conclusion
To summarize, the aqueous extract of rosemary, as the reducing agent, was used for the green synthesis of ZnNPs.The synthesized NPs were characterized by different techniques.The ZnNPs were materialized in spherical morphology.A 30.74 nm was calculated for the crystal size of synthesized Nps.The biological activity of NPs was evaluated using antioxidant and MTT assays.The results revealed a radical scavenging activity of ZnO-NPs@Rosemary with IC 50 of 87.62 ± 0.47 μg/mL.The NPs show an acceptable anti-lung cancer activity against cell lines of PC-14, HLC-1, and LC-2/ad.The highest activity was observed for PC-14 with IC 50 of 178.84 ± 2.13 μg/mL.On the other hand, the cytotoxicity analysis of ZnO-NPs@Rosemary showed low cell viability against HUVEC cell lines.

Figure 3
exhibits the XRD diagram of ZnNPs.The producing of ZnO is entirely approved by the appearance of the signals at 2 theta values of 31.45,34.20, 36.03,47.41, 56.37, and 67.45 that belong to planes of 100, 002, 101, 102, 110, and 311, respectively.These data are similar to those of green-synthetic zinc oxide using Alhagi maurorum extract.The crystal size of ZnNPs was calculated using Debye-Scherrer equation (