An in-vitro Cytotoxic and Genotoxic Properties of Allamanda Cathartica L. Latex Green NPs on Human Peripheral Blood Mononuclear Cells

Green synthesis of silver nanoparticles (NPs) by green route approaches has advantages over conventional methods. In green synthesis, we use eco-friendly plant extracts contain secondary metabolites and bioactive components, proteins that act as both reducing and capping agents, form stable and shape-controlled green silver nanoparticles. The current study deals with the synthesis of silver nanoparticles using the aqueous latex extract of Allamanda cathartica. The green silver nanoparticles are characterized by using different spectroscopic methods like ultra violet-visible spectroscopy (UV-Vis), Fourier transform-infrared spectroscopy (FTIR), transmission electron microscope (TEM), scanning electron microscope (SEM) and X-ray diffraction (XRD). Results indicated that the crystalline natured particles were spherical shaped with an average of 35 nm in size, and that the stability of silver nanoparticles was due to its high negative zeta potential of –27.6 mV. The current study also revealed that green silver nanoparticles had very good genotoxic and cytotoxic activity in peripheral blood mononuclear cells (PBMCs). Leukemia leads to the development of high numbers of white blood cells, which is one of the major types of cancers that affect children. Many of the chemicals used for the treatment produce remarkable side effects. To overcome this problem, we made an attempt to see the efficacy of latex green silver nanoparticle on peripheral blood mononuclear cells and deoxyribonucleic acid fragmentation, which leads to the development of future therapeutic drugs.


Introduction
The basic concept of nanotechnology is the engineering of functional systems at the molecular scale. Nanoparticle (NP), a most popular term and the field of nanotechnology, is one the most active research area. NPs are the essential measure of nanotechnology. The most noteworthy attribute of NPs is that they exhibit large surface area to volume ratio [1]. In general, particles with a size less than 100 nm are referred to as NPs. Metal NPs have gained importance because of their unique physicochemical characteristics, including catalytic, optical, electronic, magnetic properties and antimicrobial activities [2,3]. Many processes are currently available for synthesizing metal NPs. There is an obvious necessity for an alternative, cost-effective ecofriendly method for NP production, since these productions are expensive, labor intensive and bio-hazardous [4]. Due to their withstanding ability to harsh process conditions, inorganic metal oxides have been paid much attention to by researchers [5]. Silver is one of the important members of the metals used for synthesizing NPs, and many plant extracts have shown to be useful in biosynthesis of silver NPs [6,12,13].
Plants have proven to be one of the richest sources of natural products, most of which are therapeutically useful. Plant extracts reduce the metal ions in a shorter time as compared to any other source. When compared to others, NPs synthesized from plants takes a few minutes to hours, depending on the plant type and the concentration of phytochemicals [14]. Several factors together are responsible for the formation of NPs such as plant source pigments, alkaloids, polyphenols and proteins present in the plant extract [15]. Several reports suggested that the phytochemicals, that is, pectins, flavonoids, terpenoids, ascorbic acid, sugars, carotenoids and myriad present in plant materials act as reducing agents as well as a capping agent in the conversion of metal ions to form stable NPs [16]. Of about 10% of flowering plants of Angiosperms contain milky fluid called latex [17]. Latex contains alkaloids, resins, proteins, starches, sugars, oils, tannins, and gums that coagulate on exposure to air. It is usually exuded after tissue injury. In most plants, latex is white, but some have yellow, orange or scarlet latex [18].
Allamanda belongs to Apocynaceae. This plant is well known in traditional medicine to treat various ailments. The whole plant, including leaves, roots and flowers, contains cathartic. Some species of Allamanda are active against carcinoma cells, the human immunodeficiency virus (HIV) and pathogenic organisms [19]. Keeping in view the medicinal properties of latex from Allamanda and by using green technology in the present study, we tried synthesizing and characterizing green NPs. We characterized green Ag NPs, and the synthesized particles were tested against human peripheral blood mononuclear cells (PBMCs) to find out their antiproliferative activity. Leukemia leads to the development of high numbers of white blood cells [20]. It is one of the major types of cancers that effect children. Many of the chemicals used for the treatment produce remarkable side effects. To overcome this problem, we made an attempt to see the efficacy of latex silver NPs on PBMCs and DNA fragmentation. The overall process of the present study was represented in schematic diagram as given below (Schematic 1). All the aqueous solutions and buffers were prepared by distilled water wherever necessary.
Plant material: The plant material, Allmanda cathartica was identified in the garden of Sri Devaraj Urs Medical College, Kolar. During flowering, the plant material was authenticated by Horticulture College, Kolar. Actively growing younger twigs were collected in morning hours at the time of pruning. Milky white latex was collected from these twigs and stored at 4 °C.

Synthesis of green NPs
For the synthesis of metal NPs, 3% aqueous latex solution was made with distilled water and stored at -20 °C till further use [20]. To 20 mL of 1 mM silver nitrate solution, equal volumes of 3% latex solution were mixed thoroughly and incubated at 37 °C for 24 h at 135 rpm in the shaker cum incubator. After incubation, development of reddish brown color was observed and therefore indicated the formation of green silver NPs. These particles were stored in 4 °C till further analysis.

Characterization of NPs
Ultraviolet-visible (UV-visible) spectrophotometer allows identification, characterization and analysis of metallic NPs. UV-visible absorption spectra were obtained from Nanodrop 8000, (UV-visible spectrometer, Thermo Scientific) in 200-700 nm wavelength range. Scanning and transmission electron microscopy (SEM and TEM) methods are common for surface and morphological characterization at nanometer to micrometer scale. SEM and TEM were carried out at Department of Microbiology and Cell Biology (MCBL), Indian Institute of Science, Bangalore. Sample preparation for SEM was carried out according to Banerjee et al. [18,21] with a little modification. Briefly, about 2 mL of the green NP solution was centrifuged at 4000 rpm for 20 min. The pellet was dried and used for SEM. The pellet was scrapped carefully with a forceps and transferred onto a clean glass slide; the images were obtained in a scanning electron microscope, Quanta 200 (FEI). TEM analysis was carried out with carbon coated copper 300 mesh grids. The sample was prepared by placing 10 μL of green silver NP solution on metal-coated copper grids and dried under lamp. The images were obtained from Tecnai G2 Spirit BioTWIN at a voltage of 120 kV. The X-ray diffraction (XRD) pattern measurements of drop-coated films Ag NPs on aluminum foil substrate were recorded in a wide range of Bragg angle 2θ at a scanning rate of 2/min, and carried out on a spectroscope by using Seifert Rayflex 300TT X-ray diffractometer with CuK (λ = 1.542 Å) radiation that was operated at a voltage of 40 kV and a current of 30 mA with Cu k-α radiation (1.5405 Å). Particle size and zeta potential measurement experiments were carried out by using a Nanopartica (HORIBA Instrument). Fourier transform infrared spectroscopy (FTIR) is useful to reveal the organic functional groups that are attached to the surface of NPs. FTIR of the particles was carried out in Spectroscopy Analytical Test Facility, Society for Innovation and Development (SID), Indian Institute of Science, Bangalore with Nicolet 6700 FTIR. The scan range was 4000/cm to 400/cm by potassium bromide (KBr) pellet method.

Collection of peripheral blood and isolation of lymphocytes
The PBMCs were isolated from the left over blood collected in heparin vacutainers from the Central Diagnostic Laboratory of Sri Devaraj Urs Medical College, after getting ethics clearance (SDUAHER/ KRL/Res. Project/128/2017-'18) from the institute. To about 1 mL of the blood was added equal volume of cold PBS (pH 7.0), which was mixed properly and carefully layered onto 2 mL Ficol in a falcon tube without getting mixed up. The contents were spun down at 3000 rpm for 30-45 min at room temperature. Without disturbing the buffy coat layer, plasma was removed, and then the buffy coat was aspirated. The buffy coat was washed with PBS. The growth medium was prepared by mixing RPMI medium, FBS and anitbiotic/antimycotic. The details of the samples and controls are given in Table 1. About 1 mL of RPMI medium was dispensed into falcon tubes, and into it were added 30 µL of PHA and 100 µL of PBMCs. The number of cells was counted in 10 µL and incubated with an atmosphere of 95% air and 5% CO 2 at 37 °C for 4 h [19,22].

Treatment of lymphocytes with green NPs
After 4 h of incubation, the lymphocytes were inoculated with 20, 40, 60, 80 and 100 µg/mL of the green NPs from 1 mg/mL stock. Lymphocytes in culture media without particles were considered as controls. All the treatments were performed in triplicate, and the mean (± standard error) was calculated in each case. The tubes were kept in a CO 2 incubator at 37 °C for 72 h and monitored regularly.

Trypan blue dye exclusion method
Trypan blue dye exclusion is a cell viability assay based on the ability of the live cells to exclude the vital dye, trypan blue [20,23]. Equal volumes of cell suspension and trypan blue (0.4% trypan blue in balanced salt solution) were mixed and left for 10 min at room temperature. For viability, about 10 µL trypan blue treated cell solution was counted onto the haemocytometer and observed under microscope. Percentage viability was calculated by applying the following formula:

DNA fragmentation assay
About 10 4 cells were taken further for DNA fragmentation assay. DNA fragmentation assay was done with Tris-EDTA and SDS (TES) lysis buffer [24]. Solutions were prepared as mentioned in the protocol viz. 1 M Tris, 20 mM EDTA and 1% SDS. Aliquoted 200 µL of lymphocytes were centrifuged at 2000 rpm for 5 min, and then the supernatant was discarded. To the pellet, 20 µL of TES buffer was added to lyse the cells by mixing properly. To this, 10 µL of RNase was added and was incubated for 1 h at 37 °C. After incubation, 10 µL of proteinase K was added and incubated at 50 °C for 1.5 h. The entire volume was loaded in 1% agarose gel using 6 × blue DNA loading dye and ran the gel at 80-100 V. After the run, the gel image was taken by using UV-trans illuminator and the image was documented.

Statistical analysis
All the experiments were conducted in triplicates and standard errors were calculated wherever needed.

Results and Discussion
In the present study, silver NPs were synthesized by using aqueous extract of Allamanda cathartica latex. When 3% latex was mixed with 1 mM silver nitrate solution, within 20 min, the solution turned pale yellow. This was in agreement with Allamanda leaf extract [7,8,11,25], and upon overnight incubation there developed clear reddish brown, indicating the formation of silver NPs due to the conversion of Ag + ions to elemental Ag by extracellular latex (Fig. 1) on account of the surface plasmon resonance (SPR) phenomenon. During the process of reduction, the silver nitrate solution was reduced to silver ions (Ag + ), i.e. silver NPs (Ag NPs) with the help of phytoconstituents present in the extracellular latex extract of Allmanda cathartica. The color change from pale coloration of the solution to dark color was read in UVvisible spectrophotometer for further confirmation.

UV-visible spectrophotometry (UV-Vis)
The UV-visible spectra (Fig. 2) of the reaction solution showed an absorbance peak at 455 nm, which is characteristic SPR peak of Ag NPs, and hence confirmed their synthesis.  This strong absorption peak in the visible range was due to surface plasmon vibrations excited. It is known that the size and shape of NPs reflect the absorbance peak. In fact, the size has a linear correlation with the peak intensity, while the number of NPs does not have such linear correlation [26]. SPR peak could correspond to the spherical shape of metal NPs. This SPR peak was due to the different metabolites and proteins present in the aqueous extract. UV-visible spectroscopy of Ag NPs showed maximum absorption at critical wavelength, corresponding to the absorption maxima of Ag NPs, i.e., at 455 nm. This was in agreement with Harekrishna et al. [20] and other reported results [10,11]. Further FTIR analysis was carried out to confirm which functional groups had participated in the reduction of green silver NPs.

Fourier transforms infrared spectroscopy (FTIR) analysis
FTIR detected inorganic and organic functional groups of the particles. The FTIR spectrum of latex green NPs is presented in Fig. 3.
The peak at 3150.98 indicates O-H; the peak at 2922.65 is C-H; the peak at 2849.40 is C-H; the peak at 2351.91 is C== C; the peak at 1730.13 indicates either C-C or C-O or C-N; and the peak at 1402.33 indicates C-N or C-O or C-C. The FTIR spectrum of silver NPs from the above figure reveals that the hydroxyl group with the stretch of (O-H, 3150.98; C-H, 2922.65, 2849.40) and the carbonyl group with the stretch of alkenes groups (C == C, 2351.91; C==O, 1730.13/cm; C-N 1402.33) were involved in the reduction of Ag + to Ag. A similar type of results was also presented in the research by Litvin and Minaev [27,28]. Therefore, it may be concluded that the water soluble essentials secondary metabolites are responsible for capping and efficient stabilization [8,22].

M o r p h o l o g y o f g r e e n s i l v e r N P s w i t h transmission electron microscopy (TEM)
TEM provides the images of a sample by scanning it with a focused beam of electrons. When there is an interaction between electrons of the metal and electrons of the sample, various signals can be detected and they contain the information about the sample's surface topography and composition. TEM provides the morphology, i.e., the shape and size details of the particles. From the instrument, thirteen particles were captured, and by using ImageJ software, the area, angle, length and diameter of the particles were measured ( Table 2, Fig. 4 and 5).
The average diameter of the particles was 34.69 nm. The shape of the particles was spherical, rectangular, round and angular. TEM images showed that the particle size ranged from 19-52 nm. The particles are spherical, rectangular, a few with striations, uniformly distributed without any aggregation [9].

Scanning electron microscopy (SEM) analysis
Morphology of synthesized NPs was characterized by SEM analysis. Fig. 6 shows the SEM images obtained upon equal volumes of 3% latex and 1 mM silver nitrate solution. SEM revealed that the NPs were mostly spherical, polydisperse, and not in direct contact with each other even within the aggregate. This indicated that the synthesized NPs were stabilized   by a capping agent (Fig. 5). The similar result has been observed with silver NPs synthesized with plant extracts [7,9] and fungal extract [29].

XRD analysis
The crystalline nature of the green synthesized Ag NPs by latex of Allamanda was also confirmed by XRD pattern analysis. The XRD pattern in Fig. 6 indicated that the particles had a face-centered cubic structure. There were three distinct reflections in the

Particle size determination
Particle size of the biosynthesized green Ag NPs was determined by laser diffraction intensity which revealed that the particles obtained were polydisperse in nature with particle size ranging from 22.2 to 35 nm (Fig. 7). The Z-average diameter of the particles was found to be 41.1 nm. The results were similar to the results obtained in TEM analysis.

Zeta potential measurement
The zeta potential of the synthesized Ag NPs was found to be -27.6 mV (Fig. 8) in water as dispersant. On the other hand, the electrostatic repulsive forces between the NPs when they were negatively charged possibly protected them from forming an association. This prevented the particles from agglomeration in the medium, leading to long term stability.
The high value of zeta potential confirmed the repulsion among the particles which thereby increased the stability of Ag NPs and their formulation [8]. The Ag NPs in the present study were negatively charged with a zeta potential of -27.6 mV, which proved evident that the particles were dispersed in the medium.

Cytotoxicity of particles on PBMCs
The PBMCs were treated with 20, 40, 60, 80 and 100 µg/mL of green synthesized NPs from 1 mg/mL stock. After 72 h, trypan blue dye exclusion method was used to reveal the percentage cell viability of both control and treated cells. The results showed dose dependent response. As the concentration of green NPs increased, the percentage of dead cells increased and the percentage of live cells decreased. In the control wherein only PBMCs were present, the percentage viable cells and percentage non-viable were noted as 67 and 33%. In other controls, only minimum and maximum concentrations were taken into consideration. With the control of PBMCs with DMSO, 100 µL showed higher dead cell percentage (71%) than 20 µL (33%). With AgNO 3 metal solution at 1 mM concentration, both 20 and 100 µL showed almost the same percentage of viable and non-viable cells, i.e., 71 and 75%. Similar to the metal control, PBMCs with 3% latex showed similar percentage viability and non-viability for 20 and 100 µL as of 39% and 36% respectively ( Table 3, Fig. 9).
These results demonstrated the Ag NPs mediated concentration-dependent toxicity on PBMCs. This provides a basis for understanding the toxicity of latex  NPs and cellular uptake [30]. Several studies explained that the Ag NPs disrupted normal cellular functions, influencing the membrane integrity and inducing programmed cell death [31].

DNA fragmentation assay
We examined the influence of Ag NPs on DNA fragmentation. The induction of DNA fragmentation is a predicted marker for DNA damage of PBMCs. In the present study, NPs showed apoptotic fragmentation of lymphocyte DNA (Fig. 10). This might be due to interaction between silver atoms and the functional groups of proteins and DNA. DNA fragmentation assay indicates the extraction of DNA from lysed cells which is followed by Agarose gel electrophoresis. We examined the effect of silver NPs on DNA fragmentation. The gel electrophoresis revealed that the intensity of DNA was diminished because of the cleavage of DNA so fragments of DNA were noted. The NPs produce oxidative stress which causes DNA damage. Excessive production of ROS,is known to induce apoptosis [32].

Conclusions
Allmanda cathartica latex extract was found to be suitable for quick synthesis of green Ag NPs by green synthesis within 20 min to overnight. Spectral analysis of green Ag NPs by UV-Vis, FTIR, SEM, TEM, particle size and zeta potential support the formation and stability of the bio-synthesized green Ag NPs. The average size of green silver NPs was found to be 35 nm. Hence, the current study also reveals that green Ag NPs have very good genotoxicity and cytotoxicity activity in PBMC. As leukemia leads to development of high numbers of white blood cells, to overcome this problem, we made an attempt to see the efficacy of latex green silver NPs on PBMCs and DNA fragmentation, which leads to the development of future therapeutic drugs.