Panax ginseng leaf aqueous extract mediated green synthesis of AgNPs under ultrasound condition and investigation of its anti-lung adenocarcinoma

: Panax ginseng has many therapeutic uses in medicine. In the recent research, silver nanoparticles (AgNPs) were formulated by the Panax ginseng aqueous extract. The synthesized AgNPs ’ characterization was analyzed using UV-Vis spectrophotometry, energy dispersive X-ray spectroscopy, scanning electron microscopy, fourier transformed infrared spectroscopy, transmission electron microscopy, and elemental mapping. The AgNPs were analyzed for their surface morphology by SEM. The successful synthesis of AgNPs was evident with TEM images. The AgNPs had a uniform distribution and homogenous spherical shaped morphology with mean diameter in the range of 20 – 30 nm. The cytotoxic and anti-lung adenocarcinoma potentials of biologically formulated AgNPs against NCI-H1563, NCI-H1437, NCI-H1299, and NCI-H2126 cells were determined. The anti-lung adenocarcinoma properties of the AgNPs removed NCI-H1563, NCI-H1437, NCI-H1299, and NCI-H2126 cells. The AgNPs ’ IC 50 were 193, 156, 250, and 278 µg/mL against NCI-H1563, NCI-H1437, NCI-H1299, and NCI-H2126 cells, respectively. Also, AgNPs presented high antioxidant potential.


Introduction
Recently, nanotechnology has become an important and effective technology for science on a global scale [1][2][3].Engineering based nanotechnology is a powerful tool that has opened new ways of research and development in environmental science, agriculture, cosmetics, material science, bioscience, medicine, food, and information technology [4][5][6][7][8].It is applied as a tool to understand how the nano-sized materials change physicochemical properties [8][9][10][11].In fact, nanotechnology is a very practical knowledge that includes a wide range of sciences, and developing countries need investment and special attention to this science to improve the situation in the fields of treatment and health, environment, energy, and water.Nanomaterials main unit is placed in this range in three-dimensional space [12][13][14].Nanoparticles (NPs) have shown their effects in a broad spectrum against both groups of Gram-negative and Gram-positive bacteria and several cancers.For example, ZnO and AgNPs show concentration dependent antimicrobial properties against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, respectively.However, while different types of nanoparticles often have different effects [11][12][13][14][15], their anticancer mechanisms have not been fully determined.Anticancer mechanisms of silver nanoparticles (AgNPs) that have been accepted so far are generally described in one of these three models: (1) induction of oxidative stress, (2) release of metal ions, or (3) nonoxidative mechanisms [10][11][12][13][14].The modern formulation development over several types of AgNPs of different shapes and sizes have witnessed outstanding antimicrobial effects [13][14][15][16][17].
Natural molecules synthesized from herbs have helped in the formulation of modern anti-inflammatory supplements with significant remedial efficacy and less toxicity in the treatment of diseases [18][19][20].Metal NPs green-synthesized by plants have unique therapeutic effects.Currently, various physical and chemical processes are applied for the metal NPs formulation, which enables researchers to obtain NPs with their desired properties [21][22][23][24][25][26].However, these production methods are usually expensive and also seriously dangerous for the living organisms and environment, so there is a great need for an affordable and environmentally friendly alternative method to produce NPs [27][28][29][30].During the last decades, it has been shown that several biological systems including human cells, fungi, yeasts, bacteria, diatoms, algae, and plants can convert inorganic metal ions into metal NPs through reducing capacities.Today, the biological sources of NP synthesis follow more harmless protocols, and if NPs are used in fields related to human health, it is easy to make an aseptic environment during the NP biosynthesis process [28][29][30][31].The NPs production by plants has major advantages over other biological systems including yield on a higher scale, low cost in cultivation, short production time, safety, and compatibility with the environment [31][32][33][34].In the green method of producing metal NPs, some medicinal plants are used [33][34][35].
In this research, we are interested to spread a procedure for AgNPs fabrication by Panax ginseng leaf (Figure 1), without using any toxic regents.The poly hydroxy phytoorganic molecules of P. ginseng facilitated the green synthesis of AgNPs without aggregation and afforded spherical shape with very good dispersion.The bioformulated AgNPs were analyzed and their toxicity determined on lung cancer cells.

Experimental method 2.1 Materials
All materials were provided by Sigma Aldrich chemicals.P. ginseng leaf was obtained from the medical plant market.

Preparation of plant extract
The P. ginseng leaves aqueous extract was obtained by boiling 30 g dried plant part in deionized water for 30 min.A rotary evaporator system was applied to reduce the extract volume.Then, the concentrated extract was transferred to a freeze drier for 48 h.

AgNPs synthesis
An aqueous solution of freshly prepared AgNO 3 (10 mM, 10 mL) was mixed with the prepared extract (20 mL) and irradiated over ultrasonic conditions at 60°C for 1 h.The formation of AgNPs was indicated by the solution darkening to dark brown, due to the AgNPs' plasmon resonance band.The biosynthesized AgNPs were isolated from the medium by centrifugation.The synthesized AgNPs' characterization was analyzed by UV-Visible spectrophotometry, energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), fourier transformed infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and elemental mapping.

Cell culture and MTT assay
To assess the cytotoxic effect, several concentrations of NPs were prepared and used in the experiment.The cells' survival percentage was studied after 24, 48, and 72 h.Cells in 1640-RPMI culture medium were enriched with 10% FBS and streptomycin (50 µg/mL) and penicillin (50 IU/mL) antibiotics.The cultivation was carried out at 95% humidity saturation.After 5-6 passages, the cells were in the logarithmic phase of growth.After passaging the cells and counting them with a Marienfeld hemocytometer, 3 × 10 4 cells were poured into each well of the 96-well plate.For 24 h, it was placed in the CO 2 incubator of Mamrat Company with the culture medium containing serum, so that the cells stick to the well bottom.Then, the wells were treated with 20 µL of the desired concentrations and 100 µL of fresh medium, and each treatment was repeated three times.Then, the plates were transferred to a CO 2 incubator for 24, 48, and 72 h, after which, it was removed from the previous environment and washed with buffer.Then, 10 µL of MTT solution (Sigma) and 100 µL of fresh medium without serum was added to the wells and placed in a CO 2 incubator for 4 h.Then, the wells' supernatant was discarded and 150 mL of DMSO (Shikma Company) was added to each well for cell lysis.Then, the light absorption belonging to each well at a wavelength between 570 and 590 nm was read by the Diagnostic device and used to calculate the viability of the cells using the following formula: In this research, different concentrations of NPs were used as treatment groups and a group without NPs was used as a negative control.At the end, the treated cells were photographed using a Zeiss light microscope (Axiostar Plus) equipped with a Canon (Japan) camera [36].

Statistical analysis
The data statistical analysis was performed by SPSS24 software (ANOVA tests (p < 0.01)), according to the results of the Shapiro-Wilk test, all the data had a usual distribution.

Results and discussion
The AgNPs green synthesis was designed based on a bioinspired procedure by using aqueous extract of P. ginseng  The AgNPs' successful synthesis was visibly confirmed by the dark brown color of the solution.UV-Vis indicates a broad hump at ∼450 nm (λ max ), being recorded from 5 min of the reaction to 60 min (Figure 2).
TEM was applied to test the morphology, size, and shape of AgNPs synthesized by P. ginseng leaf extract (Figure 3).The TEM findings demonstrated the existence of nanoparticle as spherical shapes and well-dispersed with mean diameter within 20-30 nm.
The synthesized AgNPs' purity has been assessed by the EDX as reported in Figure 4.The results depict a sharp peak at 3 keV, confirmed the AgNPs as the maximum proportion.The carbon and oxygen small signals are detected in the lower energy region, assigned to the P. ginseng leaf component.
The EDX data were investigated by elemental mapping analysis.A segment of SEM image was scanned by X-ray (Figure 5).Today, plant natural products have provided a better model for designing the therapeutic agent's potential than synthetic drugs.Cancer is the uncontrolled proliferation and cells migration that has afflicted mankind since ancient times.Cancer treatment has always been a mystery [37][38][39].The largest cause of death in both women and men is reported to be cancer.Cancer is a general threat to the health of all humans.Every year, about 7,000,000 modern causes of cancer are identified and about 5,000,000 people die from it.Published research works indicate that around 14,000,000 people in the world are suffering from cancer.Recently, many research works have been made to combine drugs that have anti-cancer potential, and following that, hundreds of chemical medicinal agents that have anti-cancer properties have also been made [37,38].But an anti-cancer drug must first of all be able to kill or disable cancer cells without causing much usual cell damage [38,39].While chemical drugs have relatively improved a lot and modified forms of synthetic drugs have been researched and determined as an important aspect [39], natural products have the ability to act as models.Cancer cells are found six to ten times in a person's life.When human body immune system is strong, cancer cells are prevented and destroyed.When a person is diagnosed with cancer, it is a sign that the person has nutritional deficiencies.These deficiencies may be of lifestyle, food, environmental, and genetic factors [37][38][39].Chemotherapy, while causing fast-growing cancer cells' immediate poisoning, also removes fast-growing cancer cells in the intestines, bone marrow, etc., and can cause organ damage such as lungs, heart, liver, and kidneys [38,39].While radiation therapy destroys cancer cells, it also burns and destroys healthy cells.Initial treatment with radiotherapy and chemotherapy often reduces the size of the tumor.With this situation, long-term radiation therapy and chemotherapy do not lead to more destruction of the tumor.
When the body is affected by poisoning caused by radiation therapy and chemotherapy, the immune system is at risk and removed [37][38][39].Radiation therapy and chemotherapy can cause cancer cells genetic mutation, making them resistant and stable.Therefore, it will be hard to remove them.Failure to deliver nutrients needed by the cell prevents the increase and multiplication of the number of cells [38,39].Sugar feeds cancer cells.By stopping the consumption of sugar, an important factor of food supply for cancer cells is stopped.Medicinal plants provide health and energy and also cure various diseases such as cancers without causing poisoning.In the last Panax ginseng leaf aqueous extract mediated green synthesis of AgNPs  5 decade, a lot of studies has been done on natural products, which has led to the production of several important plantbased anticancer substances, the most important of which is paclitaxel (Taxol), from the small yew plant [37][38][39].
Oxidative stress is an imbalance of metabolic reactions and free radicals production that lead to the damage of nucleic acids, proteins, and lipids [25][26][27][28].These damages may occur because of the low level of antioxidants or extra production of radicals.Natural antioxidants and saturated production are necessary to inhibit oxidative stress effects [28][29][30][31].Antioxidants decrease the free radicals' harmful effect in the food and biological system in different ways and cause detoxification.It is possible to use NPs produced in a green way using plant substrates to formulate nanomaterials that are compatible with the environment and do not have any harmful materials [32][33][34][35][36]. Currently, the non-toxic materials used in the NPs synthesis is considered to inhibit biological hazards in the pharmaceutical and medical applications [32][33][34][35].Several researchers have considered plants bioactive substances or other sources such as fungi, yeast, and bacteria for the NPs synthesis [34][35][36].It is thought that the green formulation method will raise the performance and biocompatibility of metal NPs    because of the removal of harmful chemicals.During NPs biological production stages, it is more beneficial to produce them extracellularly by plants or their extracts [27][28][29][30].The recent research works have indicated that antioxidant properties of metallic NPs significantly increase their anticancer potential [34][35][36][37][38].

Conclusion
The study describes a simple and an ultrasound assisted AgNPs green synthesis using aqueous Panax ginseng acting as both the reducing and stabilizing agents.The characterization studies revealed the silver ions reduction as well as their saturation with P. ginseng component.As displayed from TEM analysis, size of the obtained AgNPs was in the range of 20-30 nm and had a spherical shape with suitable monodispersity, and no aggregation was seen.AgNPs were assessed in biological applications on lung cancer cells, i.e., NCI-H1563, NCI-H1437, NCI-H1299, and NCI-H2126.The viability of human cancer cells reduced in the presence of AgNPs.

Figure 2 :
Figure 2: UV-Vis spectrum of biosynthesis of AgNPs in the presence of P. ginseng leaf extract.

Figure 9 :
Figure 9: The anti-lung cancer potentials of NPs on NCI-H2126.Figure 7: The anti-lung cancer potentials of NPs on NCI-H1437.

Figure 7 :
Figure 9: The anti-lung cancer potentials of NPs on NCI-H2126.Figure 7: The anti-lung cancer potentials of NPs on NCI-H1437.

Figure 10 :
Figure 10: The cytotoxicity potentials of NPs on HUVEC.