Cryptolepis buchanani, is a medically important plant belonging to the Apocynaceae family. C. buchanani is commonly habitant in hot, deciduous forests and distributed throughout India . The plant is used traditionally as medicine in loss of appetite, fever, skin diseases, curing bone fracture, diarrhea, and cough. It is considered as anti-inflammatory, antibacterial, blood purifier, and prescribed to children for rickets [2-5]. The plant C. buchaniis full of various phytochemicals like nicotinoyl glucoside , buchanin, sarvero-genin, isosarverogenin glycosides, cryptosin, cryptolepain.
Previous studies explored some interesting properties of C. buchani plants. Ethanolic extract of stem has anti-inflammatory potential , root extract has immunopotentiating properties , plant extract has analgesic, anti-inflammatory, and chondroprotective activities , antioxidant and hepatoprotective activity , insecticidal activity , anti-dermatophyte activity .
Enrichment of phytochemicals in C. buchanani plant extract seeking attention for its alternative application. Recent study showed that gold nanoparticles from C. buchanani antimicrobial and catalytic Properties . Plant materials are widely used for green synthesis of nanoparticles [17-22] as organic compounds act as capping and reducing agents. Green synthesis of nanoparticles is inexpensive, eco-friendly, and preferable than the traditional physical or chemical process . Nanoparticles have a large surface to volume ratio, resulting from their very small sizes, making them more active than the bulk materials. Due to their enhanced physicochemical properties and biological activities in comparison with the bulk materials, nanoparticles are effectively used in a wide range of areas, like medicine, engineering, catalysis, and environmental remediation [24,25]. In this study silver nanoparticles were prepared from C. buchanani leaves extract, characterized, and evaluated its biological and photocatalytic activity.
MATERIALS AND METHODS
The Plant materials (leaves of Cryptolepis buchanani) were collected from Amlachati, Pashchim Medinipur, West Bengal, India (22˚22΄36˝ N, 87˚02’36” E) during the season of summer season. The plant was identified by the experts of the “Ex-situ conservation site of Medicinal plant species” (Amlachati, West Bengal, India). The voucher specimen (herbarium code:PBC/15-16/Phd-109) was archived at the Department of Botany, Panskura Banamali college.
Synthesis of silver nanoparticles
The nanoparticles from plant leaves were synthesized by the green synthesis method described earlier  with modifications. The plant materials were cleaned thoroughly with running water for 15 minutes, followed by distilled water for 5 minutes, and allowed to dry in air. 10 g of the dry chopped leaves were homogenized with 100 mL of deionized water and boiled at 80 °C in a water bathfor 1 h to prepare leaf extract. The extract was filtered through 0.6 μm filter papers. One mM AgNO3 and aqueous leaf extracts was mixed in 1:9 v/v ratio and incubated at 90 ⁰C for 1 hour. Changing of color from light to deep brown during the reaction confirmed synthesis of the silver nanoparticles (AgNP) . After reaction, the synthesized AgNPs were separated from colloidal suspension by centrifugation, and washed as described earlier. [27,28]. Finally, the pellet was dried to obtain the AgNPs.
Unlike other methods, this method neither required any hazardous chemicals like hydrazine, sodium borohydride, etc., nor to maintain any physical and chemical parameters like temperature, pressure, or pH. In this approach, the phytochemicals and other organic compounds present in plant extract were reduced the Ag+ ions, and act as a stabilizing agent. In contrast, highly toxic chemicals used in chemically synthesis processes may be absorbed on the surface of nanoparticles, which might be toxic to health and the environment. These advantages make the single-step green synthesis process is inexpensive, less toxic, less harmful, and eco-friendly process.
Characterization of silver nanoparticles
UV-Vis spectrum was recorded from 300 nm to 800 nm with a resolution 1 nm [27, 29] by using UV-Vis spectrophotometer (Eppendorf BioSpectrometer). The shape and particle size and shape of the synthesized AgNPs was studied by Transmission electron microscopy (TEM) . In this study, JEM-1200EX electron microscope (JEOL, Tokyo, Japan) was used and operated at an accelerating voltage of 120 kV. Particle sizes were analyzed by Image J v 1.53a software. The size distribution and potential stability in colloidal suspension of the synthesized AgNPs were studied by Dynamic light scattering (DLS) and Zeta potential respectively [30, 31]. Fourier transform infrared spectroscopy  (FTIR) spectrum was recorded by the Perkin-Elmer spectrometer FTIR Spectrum Two within the range of 4000 to 400 cm-1 with a resolution of 1 cm-1. X-ray diffraction (XRD) analysis was conducted by X’pert-pro X-ray diffractometer at a voltage of 40 kV. Current of 30 mA in 2θ angle pattern. Cu K, radiation is used to record the spectrum in the range of 20° to 100° [31-32].
HeLa, MCF7, and HEK-293 cell lines were obtained from the National Centre for Cell Science (Pune, India), and maintained as per instructions. The cytotoxicity of the synthesized AgNPs was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay described before .
HeLa cells were seeded plated in a six well animal cell culture plate and incubated overnight. Next day, cells were treated with different concentrations of the AgNPs after 4 h of serum starvation, and incubated for 24 h. After the incubation, the cells were washed after the incubation with phosphate buffered saline (PBS), and stained with acridine orange (AO)/ propidium iodide (PI) (1:1, 20 µM) . After 30 min of incubation at dark, the cells were observed under microscope, photographs were taken, and analyzed by ImageJ v 1.53a software.
Hemolysis activity of the silver nanoparticle was evaluated as follows . Two percent of human erythrocyte (RBC) in PBS, pH 7.2 were treated with different concentrations of AgNPs, and incubated at 37 ℃ for 24 h. TritonX 100 (5%) used as positive control, and PBS used as solvent control. After incubation, the reaction mixtures were centrifuged to separate unlysed cells from supernatant, and measured absorbance at 490 nm against PBS as blank.
The % of RBC lysis = (A- A0)/(A1-A0) ✕ 100
Where A is the absorbance of the test samples, A0 is the absorbance of the solvent control, A1 is the absorbance of positive control.
Photocatalytic activity of the synthesized nanoparticles was evaluated using methylene blue (MB) as described previously . Briefly, 10 µg mL-1 solutions of the MB were prepared. The AgNPs (200 µg mL-1) was added to the solution, and continued the reaction in presence of light with constant stirring. Periodically, the solutions were scanned using UV-Vis spectrophotometer (Eppendorf BioSpectrometer). The concentration of dye is directly proportional to the absorbance in the UV-Vis spectrophotometer . The percentage of dye degradation, and reaction kinetics were calculated as described earlier .
All the experiments of the present study were performed in triplet/ or more as mentioned, and the data of the experiments were expressed as mean ± standard deviation (SD). Variance <5% (i.e. P<0.05) is considered as a positive result.
RESULTS AND DISCUSSION
C. buchanani leaves are enriched with phytochemicals. During the preparation of nanoparticles, these compounds act as capping and reducing agents . Free electrons present in metal nanoparticles yielding a surface plasmon resonance (SPR) absorption band . Silver nanoparticles exhibit a sharp band around 420 - 440 nm . Fig. 1(A) shows a band at 432 nm, which confirmed the presence of silver nanoparticles in solution.
The TEM result confirms the synthesized silver nanoparticles are round shaped, and around 17.05±5.27 nm in size (Fig. 2(A)). More than 90% particles are distributed between 10-25 nm size. The result is similar to the DLS result (Fig. 4(A)). The nanoparticles are small in size which make it chemically active [38-40].
The functional groups that are present in plant extract acted as reducing agents for the reduction of Ag+ion. In this FTIR spectrum, major peaks were observed at 3417, 1635, 1393 cm-1[Fig. 3(A)]. The broad peak shown at 3417 cm-1 was denoted the O-H bond stretch or H bonding. The peak at 1635 cm-1was assigned for the N-H bending of amine, and the peak at 1393 cm-1indicated the presence of the phenol groups. Other minor peaks below 1300 cm-1 might be due to the various organic groups present in plant extract .
X-ray powder diffraction (XRD) analysis confirms the crystalline nature of the synthesized AgNPs was confirmed by. The distinct diffraction peaks at 2θ values of 38.02, 44.54, 64.46 and 77.52 could be indexed to the (111), (200), (220) and (311) reflection planes of face centered cubic structure of silver (Fig. 3(B)). The small peaks might be caused by impurities present in biological extract. Early reports also demonstrated the same type of peaks during the synthesis of silver nanoparticles [42-43]. The 10 sharp peaks shown at 2θ = 27.85, 32.3, 46.3, 54.9, 57.47, 67.56, and 77.52 can be indexed to 111, 200, 220, 311, 222, 400 and 420 planes of the cubic AgCl-NPs (ICDD file no 00-001-1013). The peaks at 38.2, 44.54 and 64.46 are corresponds to the planes of 111, 200, 220 which represent small amount of cubic Ag (ICDD file number 00-087-0718) (Fig 2B). This data also indicates that the synthesis of AgNP is a result of the reduction of Ag+ by the presence of the organic compounds in the plant extract.
The short- and long-term stability of emulsions relates to the value of the zeta potential. Emulsions with high negative/positive zeta potential are electrically stable, while emulsions with low zeta potentials tends to coagulate or flocculate . Synthesized AgNPs have a high zeta value of 65 ± 5 mV, and are highly stable in emulsion (Fig 4(B)). In summary the synthesized nanoparticles are round shaped, having an average size of 17.05±5.27 nm, crystalline in nature with a positive surface charge.
Biological activity of the synthesized nanoparticles
Cells from HeLa, MCF-7, and HEK-293 cell lines were treated with different concentrations (0-25 μg mL-1) of the synthesized nanoparticles in-vitro. Results showed that the nanoparticles were specifically more toxic to the HeLa cells. At highest concentration (25 μg mL-1), more than 70 % of the MCF-7 population survived, indicating the nanoparticles have very less toxicity against MCF-7 (Fig. 5 (A)), and not considered for further study. In case of HeLa cells, the nanoparticles killed 26.6, 32.8, 52.0, 82.5, and 91.0 % of the population at 1, 2.5, 5, 10, and 25 μg mL-1 concentration respectively (Fig. 5 (A)). The LD50 value in our study found 3.98 μg mL-1 for HeLa cells. In contrast, the nanoparticles killed 0, 17.5, 35.7, 44.7, 77.9 % of HEK-293 populations, which is a non-cancer cell line, at 1, 2.5, 5, 10, and 25 μg mL-1 concentration of the nanoparticles respectively (Fig. 5 (A)), with a 9.45 μg mL-1 of LD50 value. The LD50 value is almost three times higher than the LD50 value found in HeLa, making it unique for its potential application. The nanoparticles lysed less than 2.5 % of human red blood cells at 24 h at 10 μg mL-1 (Fig 5. (B)), at the same conditions only ~18% HeLa cells survived.
As the nanoparticles specifically showed better toxicity to the HeLa cells, the nuclear morphology was further studied by propidium iodide (PI), and AO double staining method. The HeLa cells were treated with 1, 5, and 25 μg mL-1 of the AgNPs for 24 h, and stained with acridine orange (AO) and propidium iodide (PI) nuclear staining dyes. AO is green fluorescent dye, and permeable to both live and dead cells membrane. PI, which generates red fluorescence, is permeable to dead, and dying apoptotic and necrotic cells membrane. The untreated cells have intact morphology and highly organized nuclei fluorescing green color (Fig. 6) as PI not permeable to live cell membrane. Cells with increasing concentration of the AgNPs showed morphological the changes in the cell nuclei morphology change which relate to apoptosis fluorescing orange to red. In lower concentrations of the nanoparticles, fewer cells fluoresce orange indicating early apoptosis. At highest concentration almost all nuclei fluoresce red which is the indication of late apoptosis (Fig. 6) [45,46]. Decreasing of cell number is observed in case of treated cells as compared to control, which is proportional to the concentration of the AgNPs, also validates MTT assay results. The above results showed that the synthesized AgNPs have promising characteristics and might be studied further for its potential application in the field of cancer biology.
In the presence of light, we study the photo catalytic activity of the AgNP using methylene blue. The concentration of the dye in this solution is equal to the absorbance of the solution at 660 nm. The result showed increasing the exposure time of the solution of dye and AgNP in presence of light, the pick is decreasing due to the degradation of dye (Fig. 7(A)). The AgNP degraded 49% dye in 1 h exposure, and 60% dye in 3.5 h exposure. After 8.5 h of incubation, more than 90% dye was degraded (Fig. 7(B)). The presence of the nanoparticles along with light, accelerated degradation rate of the methylene blue, in comparison when only light is present (Fig. 7 (D)). (Fig X (E)) represents the pseudo 1st order reaction model, which is not a good fit due to low correlation coefficient value (R2 = 0.9029). A liner plot was observed when t/qt was plotted against time. Higher value of correlation coefficient (R2 = 0.9759), which is closed to 1, indicating the reaction followed pseudo 2nd order kinetic (Fig. 7(E)) .
In conclusion, this is the first study that attempted to synthesize silver nanoparticles from C. buchanani plant material, which is enriched with different phytochemicals. The small size of the synthesized nanoparticles makes it chemically active. The nanoparticles showed two distinct properties, it killed specifically HeLa cells in vitro, probably by inducing apoptosis. It is also capable of degrading more than 90 % of methylene blue in presence of light at 8.5 h. Both characteristics make the nanoparticles promising for future study.
Author is grateful to DBT-BOOST (Govt. of West Bengal) and DST-FIST (Govt. of India) for instrumentation support.
CONFLICT OF INTEREST
The authors declare that no competing interest exists.
SP- Experimental sections, AP- Hemolysis, AO/PI staining, KK- Conceptualization, Cytoto-xicity study, IC- Manuscript preparation, BP- Conceptualization, NB- Conceptualization, super-vise, preparation of final manuscript.