Application of a novel lung protective drug formulated by silver nanoparticles containing Curcuma longa L leaf aqueous extract on α-Guttiferin-induced DNA fragmentation and apoptosis in lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines

Recently, scientists have understood that metallic nanoparticles green-synthesized by medicinal plants have signicant anti-cancer effects in the in vitro, in vivo, and clinical trial conditions. Also, the anti-lung cancer properties of metallic nanoparticles containing natural compounds have been indicated in many studies. In the recent research, we tried to investigate the application of a novel lung protective drug formulated by silver nanoparticles containing Curcuma longa L leaf aqueous extract on α-Guttiferin-induced DNA fragmentation and apoptosis in HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines. Also, we assessed the concentrations of inammatory cytokines, activity of caspase-3, and potential of mitochondrial membrane in the in vitro condition. Silver nanoparticles were characterized and analyzed by common physicochemical techniques including Transmission Electron Microscopy, Ultraviolet–Visible Spectroscopy, Fourier-Transform Infrared Spectroscopy, and Field Emission-Scanning Electron Microscopy. In the biological part of the present research, the cell viability of HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines was measured by trypan blue assay. Caspase-3 activity was assessed by the caspase activity colorimetric assay kit and mitochondrial membrane potential was studied by Rhodamine123 uorescence dye. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test was used to show DNA fragmentation and apoptosis. Also, the Rat inammatory cytokine assay kit was used to measure the concentrations of inammatory cytokines. Silver nanoparticles-treated cell cutlers signicantly (p ≤ 0.01) reduced the DNA fragmentation, caspase-3 activity, and inammatory cytokines concentrations, and raised the mitochondrial membrane potential and cell viability in the high concentration of α-Guttiferin-treated HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cells. The best result of lung protective and antioxidant properties of silver nanoparticles containing Curcuma longa L leaf aqueous extract was seen in the high dose of silver nanoparticles i.e., 4 µg. Assessment of the antioxidant properties of silver nanoparticles was done with the common free radical scavenging test i.e., DPPH in the presence of butylated hydroxytoluene as the positive control. The nanoparticles inhibited half of the DPPH molecules in the concentration of 149 µg/mL. According to the above results, silver nanoparticles containing Curcuma longa L leaf aqueous extract can be administrated as a lung protective drug for the treatment of lung diseases after approving in the clinical trial studies in humans.


Introduction
Metallic nanoparticles are one of main products of nanobiotechnology science. The recent evidences have indicated that metallic nanoparticles have signi cant therapeutic properties in the in vitro, in vivo, and clinical trial conditions. Different techniques and approaches were developed to synthesize metallic nanoparticles to make them one of the most applicable and widely used materials in science [1,2]. For instance, silver nanoparticles are used as antioxidants and biocides, and they are utilized either barely or integrated into other structures/substances. Additionally, silver nanoparticles have pharmaceutical and biomedical applications, such as cancer treatment and medical imaging, and not to mention other uses in engineering, cosmetics, and agriculture [2,3]. One crucial branch in nanotechnology is nano-medicine, which has evolved as an extension for the application of nanomaterials in treating several disorders [3].
Among all nanomaterials, the role of silver nanoparticles green synthesized by medicinal plants as protective and chemotherapeutic supplements in treating various diseases is unique [2].
Several studies have indicated signi cant antifungal effects of AgNPs green-synthesized by plants in the cure of candida diseases and their antibacterial properties in the treatment of Streptococcus, Staphylococcus, Pseudomonas, Salmonella, and Bacillus infectious. Also, silver nanoparticles synthesized by plants have been formulated due to the antiviral, antibacterial, antioxidant, anti-parasitic, anti-in ammatory, antifungal, wound healing, and anti-cancer properties [2,3]. In detail, it was reported that silver nanoparticles as useful metallic nanoparticles in medicine, can be used to diagnose several types of lung cancers [3a]. In this regard, Ahmeda et al. introduced a chemotherapeutic drug formulated by silver nanoparticles containing Melissa o cinalis for the treatment of several types of blood cancer [3e]. Another study was showed that silver nanoparticles green-synthesized by Spinacia oleracea L. had excellent anti-acute myeloid leukemia. In the previous research, silver nanoparticles signi cantly removed the leukemia cell lines and regulated the histopathological, immunological, biochemical, and hematological parameters in the animals [3f]. About lung protective properties of silver nanoparticles, Que et al. presented when silver nanoparticles are added to the lung cancer cell lines, ability of lung adenocarcinoma cells (A549) for migrating and metastasis reduce, the levels of reactive oxygen species (RPS) increase, and NF-κB lead to cellular apoptosis. In the previous study was reported the silver nanoparticles with size of more than 100 nm don't have lung protective effects against human lung cancer cell lines. There is a limit studies about the lung protective effects of medicinal plants greensynthesized silver nanoparticles [4]. He et al. reported the lung protective effects of green-synthesized silver nanoparticles on human lung cancer cell (H1299) in the in vitro condition. Green-synthesized silver nanoparticles inhibit NF-κB activity, reduce bcl-2 expression, raise survivin and caspase-3 expression and apply remarkable lung protective effects [5].
In the recent research, we decided to investigate the application of a novel lung protective drug formulated by silver nanoparticles containing Curcuma longa L on α-Guttiferin-induced cell death in HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines.

Preparation of Curcuma longa L leaf aqueous extract
Fully matured dark green leave of Curcuma longa L was collected in Kermanshah province, Iran in January 2020 (Fig. 1). After veri cation of the colored graphics, identi cation and characterization processes were performed.
Plant shells were washed with water for a while to remove them from their natural powders. After this procedure, the shells were dried in a sterile environment (botanical laboratory) for 20 days to dry in the natural environment. The next process was to grind these shells into powder.
The obtained powder samples were brought to the appropriate grain size for use and were used in the extraction. Then, 20 g of powder Curcuma longa L leaf was taken and added to 500 mL of boiling water and boiled for 5 minutes to obtain the extract. The resulting mixture was centrifuged for 15 minutes at 5000 rpm, then ltered and stored at 4 o C in the bottle for further experiments [2].

Synthesis of AgNPs
The AgNPs green synthesis was started with a reaction mixture of 100 mL of AgNO 3  After centrifuging the mixture at 12000 rpm for 20 min, the supernatant was discarded and the solid sample was washed with abundant pure water. After washing, it was centrifuged again at 1000 rpm for 15 minutes [3].
The characterizations of AgNPs were conducted using advanced spectroscopic techniques like FTIR and UV-Vis spectroscopy, TEM, and FE-SEM.
The presence of AgNPs was primarily con rmed by UV-Vis spectroscopy at 400-700 nm (Jasco V670 Spectrophotometer). The biomolecules that play an effective role in the reduction of AgNPs were investigated by the FT-IR spectrophotometer (Shimadzu IR a nity.1). Morphological properties of silver nanoparticles have been checked concerning structure, form, and thickness by applying FE-SEM and TEM microscopic techniques.

Cell culture
In this experiment, lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines were cultured according to protocol rules in the gibco RPMI1640 cell culture environment. In rst, 100 IU/mL penicillin (Sigma), 100 µg/mL streptomycin (Sigma) and 10%fetal bovine serum (FBS, Gibco) supported in cell cultures and standard case in T-25cm2 tissue culture bottles (5% incubated at 37ºC in CO 2 ). It led to changes in the morphology of α-Guttiferin cells, so apoptotic cells led to the cell being treated with diluted α-Guttiferin with 100 mM water.
For AgNO 3 × H 2 O, Curcuma longa L leaf aqueous extract, and silver nanoparticles solutions in the RPMI1640 water cell cultural environment were rst dissolved in DMSO and added to the cultural environment with a nal volume of 0.1%. Then, 12 h after the plating and washing, they were classi ed into several groups including [8]: For determining of cell death index in the various treatments of I-VII, TUNEL staining was used Eight random wells were selected to count TUNEL positive cells with an Olympus AX-70 uorescence microscope. The ratio of cell death index to apoptotic cells to total cells is equal [9].

Cell viability
Trypan blue was used for assessing cell viability. Vital Dye, whose membranes penetrate broken damaged or Dead Cells, is seen by penetrating dead cells in the Blue lobes in the Neubauer Lamela. For 12 hours, 96 wells with a 5 × 10 4 cell/mL density were coated on the cultural plate, then, they were cultured with various I-VII treatments and incubated at 37ºC at 5% CO 2 for 48 h. After trypinization of the cells, 200µL of the cell suspension was suspended by Neubauer Lamela 2-3 minutes after mixing with 0.4% 40µL of Trippan blue. The samples cell viability was measured by following the formula below [8].
Cell viability: Non-colored cells number/Total cells number

Caspase-3 activity
For plating the above cells, the well cell culture plate containing the PRMI1640 medium was used. After 12 h, the plate was washed by PBS. Then, the different treatments of I-VII were added to the cells. Trypsin was used for separating the cells from the ask. For removing the supernatant, all samples were centrifuged for 10 minutes. Then the centrifuging was done by adding lysate buffer and nally, they were transferred to the well cell culture plate. Later, 5µL N-acetyl-Asp-Glu-Val-Asp-p-nitroanilineDEVD-pNA was added to the well cell culture plate and incubated in 37ºC for 2 h. Then, releasing of pNA as a result of caspase-3 effect was recorded by Biotek (USA) Spectrophotometer [10].

Mitochondrial membrane potential (MMP)
For half an hour different treatments were exposed to 10 mg / mL rhodamine-123. The cell was then washed using PBS. Subsequently, 900µL triton X-100 was added to each well and held at 4 °C for 2 hours. The solutions were taken into microtubes to be centrifuged at 16000 rpm for 20 minutes. Fluorescent absorbing in cells was performed using a uorescent microplate reader (488 nm excitation and 520 nm emissions) [10].

Secretion of in ammatory cytokines
The pro-in ammatory cytokines concentrations including TNFα, IL-6, and IL-1β were measured using Rat V-Plex Kit.

Determination of the antioxidant property of silver nanoparticles
At the beginning of the study, 100 mL of methanol (50%) was added to the 39.4 g of DPPH. Also, several concentrations of AgNO 3 × H 2 O, Curcuma longa L leaf aqueous extract, and silver nanoparticles i.e., 0-1000 µg/mL, were considered. The DPPH was added to the various concentrations of AgNO 3 × H 2 O, Curcuma longa L, and AgNPs, and all samples were transferred to an incubator at the temperature of 37 °C. After 30 min incubating, the absorbances were determined at 517 nm. In this study, methanol (50%) and butylated hydroxytoluene (BHT) were negative and positive controls, respectively. Acceding to the following formula, the antioxidant properties of AgNO 3 × H 2 O, Curcuma longa L leaf aqueous extract, and silver nanoparticles were determined in detail [11]:

Statistical analysis
The data obtained as a result of experimental studies were evaluated by using the SPSS-22 program and in coordination with Tukeys post hoc process with ANOVA (p ≤ 0.01).

FT-IR analysis
Fourier-transform infrared spectroscopy (FTIR) is the technique used to acquire a solid, liquid or gas infrared spectrum of absorption or emission. Around the same time, an FTIR spectrometer gathers highspeci c data across a wide variety of the spectrum. This offers a signi cant advantage over a dispersion spectrometer, which measures the intensity at a time over a restricted range of wavelengths. Metaloxygen vibration of the FT-IR spectrum for metal nanoparticular biosynthesis induces peaks that are located at 400 and 700 cm − 1 [2,3]. The silver nanoparticles formation in the present study con rms by the existence of a peak at 643 cm − 1 belongs to the Ag-O bending vibration. In the eld of natural products, the IR spectroscopic approach is also an appropriate way to identify the bioactive components. Then the technique is a useful tool for identifying the presence of secondary metabolite over silver nanoparticles in the plants. The analysis has shown that in Curcuma longa L there are different IR bands related to the presence of different functional groups at 2039 cm − 1 band related to aliphatic "C-H" stretching; peaks of 3404 cm − 1 related to "O-H" stretching (For alcohols, carboxylic acids and phenols); peaks of 1127 cm − 1 could be compared to "-C-O" stretching and peaks of 1379 and 1612 cm − 1 correspond to "C = C" and "C = O" stretching found in phenolic and avonoid compounds, respectively (Fig. 2). The explanation for these peaks can be thought to be different compounds from previous research, such as the recorded avonoid, phenolic and carboxylic compounds [2,3].

FE-SEM analysis
An FE-SEM is an electron microscope (particles that have negative charge), rather than light, which works. Those electrons are emitted through a source of eld emissions. The object is scanned by electrons according to a zig-zag pattern and recorded the surface morphology and the size of materials [2,3]. The FE-SEM image of Curcuma longa L leaf aqueous extract mediated silver nanoparticles is shown in Fig. 4. The silver nanoparticles appeared as an agglomerated structure. The hydroxyl groups present in Curcuma longa L leaf aqueous extract could be responsible for agglomeration [2,3].
In the silver nanoparticles, the range size of 14

TEM analysis
TEM is the standard tool for calculating the size of nanoparticles, grain size, size distribution, and morphology directly [2,3]. Therefore, TEM analyses of silver nanoparticles were conducted to evaluate particle distributions and mean particle size (Fig. 5). The range size of the nanoparticles calculated through TEM images. Besides, the histogram plot in the TEM image uniformly revealed the particle size distribution of the biosynthesized silver nanoparticles between 14 and 27 nm.
In the previous studies, the size of silver nanoparticles formulated by aqueous extract of medicinal plants had been calculated in the ranges of 10-50 nm with the shape of spherical [2,3]. These literature studies and analyzes support the results of our study and experiment.

Lung protective potentials of green-synthesized silver nanoparticles
α-Guttiferin is prescribed as a chemical material and may be of concern because of its side effects [12,13]. Abuse of this chemical material may affect the upper respiratory system (including nasal cavity, pharynx, and larynx) and lower respiratory tract (Including trachea, primary bronchi, secondary bronchi, and lung); its metabolites may be harmful to the digestive and excretion system [14,15]. The pathway for the breakdown of α-Guttiferin passes through the liver and kidneys, and consequently, the potential of side effects is high in these organs [16,17]. In a study, the effects of α-Guttiferin on the lung and brain (cerebral cortex and hippocampus) are revealed. They emphasized the role of oxidative stress, neuronal, and pulmonary damage in the disruptive effects of α-Guttiferin abuse [16][17][18].
α-Guttiferin has direct effects on the upper and lower respiratory systems and increases the reactive oxygen species (ROS) by reducing the level of antioxidant activity in the plasma. Therefore, it seems necessary to evaluate the toxic effect of α-Guttiferin in upper and lower respiratory systems cells and study on compounds that can decline these toxic effects [16][17][18].
Different studies have indicated that α-Guttiferin decrease the anti-in ammatory cytokines such as IL13, IL10, IL5, IL4, and IL3 and increases the pro-in ammatory cytokines such as IL1α, IL1β, IL6, and TNFα. The cytotoxicity properties of α-Guttiferin increase cell death signi cantly and reduce cell proliferation in upper and lower respiratory system cells [15][16][17][18]. Also, a study showed that α-Guttiferin through apoptosis induction, caspase-3 and caspase-9 activation, and cell proliferation inhibition induced cell death in cells [17]. The ndings of our experiment also agreed with these results and revealed that α-Guttiferin at high concentrations (100 µM) reduced signi cantly (p ≤ 0.01) cell viability and increased in ammatory cytokines concentrations and caspase-3 activity. Treatment of these cells with both doses of silver nanopartivles synthesized using Curcuma longa L leaf aqueous extract increased the cell proliferation and cell viability potentials due to the cell cytotoxicity reduction (Fig. 6-9).
The previous researches presented that α-Guttiferin produced many free radicals, especially ROS in the body [16][17][18]. Free radicals with the degradation of DNA molecules cause cellular degradation and apoptosis in the cells [19]. ROS directly damages the DNA and increases the apoptosis in upper and lower respiratory systems cells through the production of free radicals [19][20][21]. In our study, the experiment of apoptosis by the TUNEL test indicated that α-Guttiferin caused DNA fragmentation and induced apoptosis in lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cells. Further experiments revealed that α-Guttiferin causes apoptosis in these cells by reducing the mitochondrial membrane potential. Our ndings also indicated that silver nanoparticles synthesized using Curcuma longa L leaf aqueous extract signi cantly (p ≤ 0.01) increased the mitochondrial membrane potential and reduced the rate of DNA fragmentation in lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cells treated with α-Guttiferin (Fig. 10,11).

Antioxidant potentials of green-synthesized silver nanoparticles
Traditional medicinal plants are well-known and essential natural antioxidant sources. Medicinal plantderived natural antioxidants are very e cient in blocking the process of oxidation by neutralizing free radicals. It is also commonly accepted that medicines taken from plant products are safer than their synthetic counterparts; however, the toxicity pro le of most medicinal plants have not been comprehensively assessed [22][23][24]. Combining them with metallic salts is a good option for increasing the antioxidant competencies of therapeutic plants.
In previous studies, a signi cant increase in antioxidant properties has been reported when produced by green synthesis with salts such as titanium, manganese, cobalt, palladium, gold, zinc, copper and iron [24][25][26]. So far, signi cant antioxidant potentials of silver nanoparticles synthesized by many plants such as Melissa o cinalis leaf, Thymus kotschyanus leaf, Phoenix dactylifera seed, Spinacia oleracea L leaf, and Pistacia atlantica leaf were proved [2].
It seems that silver nanoparticles green-synthesized by Curcuma longa L leaf, due to its antioxidant potential, signi cantly (p ≤ 0.01) raised cell viability and mitochondrial membrane potential, and reduced in ammatory cytokines concentrations, caspase-3 activity, and DNA fragmentation in the high concentration of α-Guttiferin-treated lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cells. Today, antioxidants are introduced as a reducer of cell cytotoxicity because they prevent ROS production and oxidative stresses in the cells [2,3].

Conclusion
Curcuma longa L leaf harvested from the Kermanshah city, Iran, was used for synthesizing silver nanoparticles as a suitable and safe material. The synthesized silver nanoparticles were characterized and analyzed by TEM, FE-SEM, and FT-IR and UV-Vis spectroscopy. In the UV-Vis, a clear peak with a wavelength of 439 nm indicated the formation of silver nanoparticles. The presence of many antioxidant compounds with related bonds caused the excellent condition to reduce silver in the silver nanoparticles.
FE-SEM and TEM analyses revealed a range size of 14-27 nm of the silver nanoparticles with a spherical shape.
In the biological experiments, we concluded that a high dose of α-Guttiferin causes cell death in lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines through the induction of cell in ammation and apoptosis. Silver nanoparticles enhanced cell viability in a high dose of α-Guttiferin-treated cells.
They repressed in ammatory cytokines (TNF-α, IL-6, IL1α, and IL-1β) production, mitochondrial membrane disruption, and caspase 3 activity. These events reveal that silver nanoparticles suppressed α-Guttiferin-induced cell death in a dose-dependent manner in lung HEL 299, MRC-5, IMR-90, CCD-19Lu, WI-38, and BEAS-2B cell lines. In the future, silver nanoparticles green-synthesized by Curcuma longa L leaf can be consumed for increasing the physiological activity of the respiratory system.

Con ict of Interest
The authors declare that there is no con ict of interest.