Bioengineering of Piper longum L. extract mediated silver nanoparticles and their potential biomedical applications

Materials Science & Engineering C 104 (2019) 109984

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Bioengineering of Piper longum L. extract mediated silver nanoparticles and their potential biomedical applications Renuka Yadava, Himanshu Sainia, Dinesh Kumara, Shweta Pasib, Veena Agrawala, a b

T

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Department of Botany, University of Delhi, Delhi 110007, India National Institute of Malaria Research, Dwarka, New Delhi 110077, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Leaf extract AgNPs Piper longum Green synthesis Antioxidant Anticancerous Larvicidal

The present investigation highlights the strong antioxidant, anticancer and larvicidal potential of green synthesized silver nanoparticles (AgNPs) using aqueous leaf extract of Piper longum L. for their diverse therapeutic applications. The optimum conditions for the synthesis of AgNPs were recorded as 1 mM AgNO3, 60 ± 2 °C at pH 6 for 120 min. Synthesized AgNPs proved to be highly stable and monodispersed as characterized through various techniques. UV–Vis spectrum of biosynthesized AgNPs showed a maximum absorption peak at 420 nm. Field emission-Scanning electron microscopy (FE-SEM) and High resolution-Transmission electron microscopy (HR-TEM) micrographs showed the spherical shape of AgNPs with mean diameter size of 28.8 nm. Existence of crystallographic AgNPs was proved by X-ray diffraction (XRD) pattern analysis. Presence of phenolics, terpenoids and flavonoids compounds which act as bioreducing agents were confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. Furthermore, the AgNPs and leaf extracts prepared individually in different solvents such as methanol, ethyl acetate, chloroform, hexane and aqueous were assessed for their bio-efficacies. AgNPs showed the enhanced antioxidant (IC50 67.56 μg) and radical-scavenging activities (IC50 196.8 μg) as compared to the crude leaf extracts. Anticancer activity revealed the strong and dose-dependent cytotoxic effect of AgNPs against the HeLa cells showing maximum IC50 value being 5.27 μg/mL after 24 h and was also found to be non-toxic to normal cells (HEK). The AgNPs induced the fragmentation of DNA in the cells, indicating the occurrence of apoptosis and necrosis. Subsequently, an efficient larvae mortality was also recorded against Anopheles stephensi having LC50 and LC90 values being 8.969 and 16.102 ppm, followed by Aedes aegypti (LC50;14.791 and LC90;28.526 ppm) and Culex quinquefasciatus (LC50;18.662 and LC90;40.903 ppm) after 72 h of exposure. Besides, they showed no toxicity against Mesocyclops thermocyclopoides (non-target organism). This is the first report showing strong anti-tumorous and larvicidal activity of AgNPs synthesized using P. longum leaf extract against cervical cancer cell line and mosquito vectors causing dengue, malaria and filariasis. Based on our findings, we suggest that AgNPs derived using P. longum leaf extract possessed excellent anti-cancerous and mosquito larvicidal potential and therefore, can be bioprospected further for the management of these hazardous health diseases. This study has given a new insight for the novel drug designing after conducting experiments on the in vivo models.

1. Introduction Nanobiotechnology is a unifying field of science and engineering which deals with the synthesis, strategy, and manipulation of particle's structure ranging from approximately 1 to 100 nm in size. The term “nano” is derived from the Greek word nanos, which means “dwarf”. Due to their unique physiochemical and optoelectronic properties, nanoparticles (NPs) are attracting the attention in various fields such as catalysis, health care, sensors, drug delivery, electronic devices, medical diagnostic imaging, antimicrobial activity [1–3] and now extending

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their applications to cancer therapy [4]. The versatile performance of NPs critically depends on their size, shape, distribution, surface chemistry and large surface area to volume ratio [5]. Synthesis of nanoparticles with desired shape and size can be achieved through various physical and chemical methods [6,7]. Nowadays, synthesis of NPs using biological agents (fungus, bacteria and plant extracts) are preferred as an alternative to traditional methods due to their easy modification, rapidness, high drug loading capacity, stability, non-pathogenic, costeffective and eco-friendly nature [8,9]. In the last few years, cancer and vector-borne diseases (malaria,

Corresponding author at: Medicinal Plant Biotechnology Lab, Department of Botany, University of Delhi, Delhi 110007, India. E-mail address: [email protected] (V. Agrawal).

https://doi.org/10.1016/j.msec.2019.109984 Received 6 November 2018; Received in revised form 17 June 2019; Accepted 17 July 2019 Available online 18 July 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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dengue and filariasis) are considered as the most challenging diseases across the world [10,11]. There is a continuous struggle around the world to explore novel aspects to decrease the morbidity and mortality caused by these diseases. Currently, there are several anti-malarial and anti-cancer synthetic drugs are available in the market for combating these diseases. In the case of mosquito vectors, various synthetic chemicals and repellents such as DDT, organochlorine, malathion and synthetic insecticides are used [8]. Such conventional insecticides cause severe threat to the ecosystem and non-target organisms, including humans due to their toxic and hazardous nature [12]. Similarly, for the treatment of cancer, the available management options include chemotherapy (synthetic drugs such as methotrexate and trimetrexate), radiotherapy or surgery [13]. These treatments are expensive, cause severe side effects such as hair loss, fatigue and also damage the healthy cells along with cancer cells in the body. Realizing the current scenario, there is an extreme exigency in the search for a reliable and alternative approach for the management of these diseases. Over the years, plants have a reputable history to be a good source of chemotherapeutic agents. Several studies have been reported on the anti-malarial and anti-cancer activities of numerous plant species such as Callicarpa maingayi, Piper nigrum, Tribulus terrestris, Centella asiatica and Catharanthus roseus [14]. Plant-based drugs and insecticides are preferred because of their biodegradable nature, least side-effects, non-toxic to non-targeted organisms and highly effective due to synergistic and multi-targeted action [15]. Despite these, it is hard to develop resistance towards plant bioactive compounds due to their complex structures [8]. Piper longum L. (Indian long pepper or Pippali), a member of the family Piperaceae, is considered one of the most important species which has been used traditionally for the treatment of various ailments such as gonorrhoea, menstrual pain, tuberculosis, respiratory tract infections and arthritic conditions [16,17]. Various biological activities such as anti-inflammatory, anti-platelet aggregation, anti-leishmanicidal, hepatoprotective and CNS depressant have been reported for P. longum [18–20]. Being a rich source of various bioactive compounds, the plant extract has already shown promising anti-oxidant, anticancer, and anti-inflammatory potential. Considering its immense medicinal uses, the current investigation has been designed with the following objectives (i) synthesis of silver nanoparticles (AgNPs) using aqueous leaf extract of P. longum (ii) characterization of AgNPs through various techniques such as UV–Vis spectroscopy, field emission scanning electron microscope, energy dispersive X-ray spectroscopy, transmission electron microscope, X-ray diffraction, dynamic light scattering and Fourier transform infrared spectroscopy and (iii) evaluation of bioefficacy of biosynthesized AgNPs and leaf extract prepared in different solvents against the cervical cancer cell line (HeLa) and the 3rd instar larvae of Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus and non-target organism (Mesocyclops thermocyclopoides) and cells (HEK 293).

methanol and distilled water individually. The solvent-tissue extract was kept on an incubator shaker for continuous shaking at room temperature for 48 h. These extracts were then filtered individually through Whatman filter paper No. 1. For preparing the aqueous extract, 20 g of the aforesaid powder was boiled with 200 mL of distilled water for 1 h and kept on incubator shaker for continuous shaking for 48 h at room temperature and filtered using Whatman filter paper No. 1. Subsequently, all the filtrates were air-dried and stored at 4 °C for further experiments. 2.2. Fabrication of silver nanoparticles (AgNPs) For the green synthesis of AgNPs, 5 mL of fresh leaf extract (prepared above) was added to 45 mL of 1 mM aqueous silver nitrate solution at pH 7.5. The mixture was then heated at 40 ± 2 °C with continuous stirring for 20 min. After 10 min of heating, the gradual change in the color of the reaction mixture was observed. The conversion of solution color showed the reduction of silver ions to silver nanoparticles. Subsequently, the reaction mixture was centrifuged at 12,000 rpm for 20 min. The supernatant was discarded and the pellet was collected. The pellet containing nanoparticles was washed with double distilled water and air-dried. The dried AgNPs powder was used for the characterization of AgNPs and their bio-efficacy experiments. 2.2.1. Parameters affecting the synthesis of AgNPs In order to determine the optimum conditions for the biosynthesis of AgNPs, various parameters such as concentration of AgNO3 solution, temperature, incubation period and pH of the solution were analyzed. To determine the optimum concentration of AgNO3 for the biosynthesis of silver nanoparticles, different concentrations (0, 0.5, 1, 2, 3, 4 and 5 mM) of AgNO3 solution were used. Similarly, to study the effect of temperatures, synthesis of AgNPs was carried out at different temperature 40, 60, 80 and 100 °C. Subsequently, the effect of incubation period was also determined by measuring the absorption spectra of the solution at a different time interval of 0, 5, 30, 60, 90, 120 and 240 min. To investigate the effect of pH on the biosynthesis of AgNPs, the reaction was carried out by varying the pH (3, 6, 9 and 12) of the solutions. The pH of the solutions was adjusted before incubation by adding 0.1 N HNO3 or 0.1 N NaOH. 2.3. Characterization of AgNPs 2.3.1. Ultraviolet-Visible spectroscopy (UV–Vis) analysis Primarily, the change in color of the reaction mixture from colorless to brownish yellow was observed by naked eye which showed the preliminary confirmation of bio-reduction of silver nitrate and then further confirmed by UV–Vis spectrophotometer (Shimadzu 250 1 PC, version 2.33) and their spectral analysis was done at the range of 360 to 800 nm wavelengths.

2. Materials and methods

2.3.2. Field emission scanning electron microscope (FE-SEM) and energy dispersive X-ray spectroscopy (EDAX) measurement The morphological features and elemental composition of synthesized silver nanoparticles were monitored under the FE-SEM (TESCAN MIRA-3) coupled with EDAX. Few drops of nanoparticles solution were loaded on the sputter-coated copper stub and air-dried completely. After that samples were characterized under FE-SEM operated at 20 kV voltages.

2.1. Collection of plant materials and preparation of leaf extracts The plants of Piper longum L. were procured from Herbal Garden and Herbarium, Research Institute in ISM, Joginder Nagar (31.98°N, 76.78°E), Mandi, Himachal Pradesh, India and were established in the Greenhouse of Department of Botany (28.68°N, 77.21°E), University of Delhi, Delhi, India, during the month of August (Fig. 1A). The fresh green leaves were collected and washed thoroughly thrice under running tap water to remove the adherent impurities. The leaves were then shade dried at room temperature (28 °C) for 48 h. The dried leaf samples were coarsely ground into rough powder using mortar and pestle and separated into five parts of 20 g each for making extracts with different solvents. Subsequently, 20 g of leaf powder was extracted with 200 mL of different solvents such as chloroform, ethyl acetate, hexane,

2.3.3. High resolution-transmission electron microscope (HR-TEM) and selected area electron diffraction pattern (SAED) analysis The size, shape and crystallinity nature of the nanoparticles were measured under HR-TEM (Technai G2 T30, U Twin). A drop of synthesized nanoparticles sample was loaded on a carbon-coated copper TEM grid. The extra amount of sample was removed using blotting paper and the grid was allowed to evaporate for complete dryness at 2

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Fig. 1. (A) Piper longum L. (B) Chromatic variation of the P. longum aqueous leaf extract observed after addition of 0.5, 1, 2 and 4 mM AgNO3 solution heated at 60 ± 2 °C, pH 6.5 for 120 min. (C) UV–Vis spectrum of silver nanoparticles synthesized using P. longum aqueous leaf extract by varying concentrations of AgNO3 solution (0.5, 1, 2, 3, 4 and 5 mM) at 50-60 °C for 20 min. (D) Absorption spectrum of silver nanoparticles prepared at different reaction temperatures (40, 60, 80 and 100 °C) with 1 mM aqueous solution of AgNO3 for 20 min. (E) UV–Vis absorption spectrum of synthesized nanoparticles prepared using plant aqueous leaf extract with 1 mM AgNO3 solution obtained at different time intervals (5, 30, 60, 90, 120 and 240 min) (F) UV–Vis spectra of silver nanoparticles recorded at different pH values (pH 3, 6, 9 and 12) of the solution.

room temperature. Micrographs of AgNPs were recorded on an acceleration voltage of 50–300 kV.

monitored by XRD analysis (Bruker D8 Discover) which was operated under the following conditions: step size, 0.02/θ; 2θ range, 10-80ᴼ; voltage, 40 kV; current, 40 mA with Cu kα radiation of 0.1541 nm wavelength.

2.3.4. X-ray diffraction (XRD) analysis In order to confirm the crystalline nature of AgNPs, the powder was 3

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650 nm using UV–Vis spectrophotometer. The linear calibration curve of gallic acid was obtained (for concentrations ranging 31.25 μg/mL to 500 μg/mL) having R2 = 0.999. The total phenolic content was expressed as μg of gallic acid equivalents (GAE)/mg of dry extract (μg GAE/mg).

2.3.5. Dynamic light scattering (DLS) analysis To measure the particle size distribution and zeta potential of prepared silver nanoparticles, DLS analysis was performed using Zetasizer Nano ZS (Malvern Instruments Ltd., USA) at 25 °C. 2.3.6. Fourier-transform infrared spectroscopy (FTIR) analysis To identify the presence of functional groups majorly involved in reduction and capping of AgNPs, a drop of synthesized silver nanoparticles solution and few drops of 1 mg/mL of plant leaf extract were put on the KBr plates separately and subjected to FT-IR analysis (Perkin Elmer, Spectrum RXI, Detector: LitAO3, resolution: 400 cm−1).

2.4.4. Total flavonoid content determination The total flavonoid content determination (TFC) of each of the leaf extract prepared individually in different solvents (methanolic, aqueous, ethyl acetate, chloroform and hexane) in comparison to AgNPs was estimated using the aluminium chloride colorimetric method. Quercetin was used as a standard. In a reaction mixture, 250 μL of each test sample was added to the test tube containing 75 μL NaNO2 (5% w/ v) solution. The mixture was then shaken well and incubated for 6 min at room temperature. After incubation, 150 μL AlCl3 (10% w/v) solution was added followed by 500 μL NaOH (1 N). The final volume of the reaction mixture was made up to 2.5 mL by adding distilled water. The absorbance of the reaction mixture was recorded using UV–Vis spectrophotometer at 510 nm and the results were expressed as μg of quercetin equivalents (QE)/mg of dry extract (μg QE/mg). A linear calibration curve of quercetin was obtained (concentrations ranging from 31.25 μg/mL to 1000 μg/mL) having R2 = 0.94.

2.4. Antioxidant assay 2.4.1. DPPH radical scavenging assay For the determination of antioxidant activity, DPPH (2,2-diphenyl1-picrylhydrazyl) free radical scavenging assay was performed according to the modified protocol of MacDonald-Wicks et al. [21]. The reaction mixture was prepared by adding 100 μL of different concentrations of the plant solvents extract and synthesized AgNPs with 300 μL of methanol and subsequently, 400 μL of 0.2 mM concentration of DPPH solution was added. The mixture was then vortexed for 10 s and further incubated for 30 min at room temperature. Absorbance was then recorded at 517 nm using the spectrophotometer. Ascorbic acid (Vitamin C) was taken as the standard antioxidant and lineared between 10 and 50 μg/mL (R2 = 0.95). The experiment was done in triplicates. The antioxidant potential of the leaf extracts prepared in different solvents and AgNPs was calculated as the percentage inhibition of DPPH discoloration using the equation below:

2.5. Evaluation of the anti-cancer potential of AgNPs 2.5.1. Cancer cell lines and cell culture HeLa (Cervical cancer) and normal cell line, HEK 293 (Human Embryonic Kidney) were procured and authenticated from National Centre for Cell Sciences (NCCS), Pune, India. The cells were cultured in their respective media (Sigma, USA) augmented with 10% heat-inactivated fetal calf serum (Gibco, USA) and 1% antibiotic solution (HiMedia, India). The cells were maintained in a humidified CO2 incubator (Shell Lab, USA) consisting of 5% CO2 at 37 °C. For experiments, cells less than five passages were used.

Percent Inhibition = 100 × (1 − A sample/Ablank), where, Ablank is absorbance of the solution when nothing was added, Asample is absorbance of the solution when extract or AgNPs was added. 2.4.2. Phosphomolybdenum assay In order to determine the total antioxidant capacity (TAC), phosphomolybdenum assay was performed according to the modified protocol of Eddine et al. [22]. For sample preparation, 1 mg of different solvents leaf extracts/AgNPs was dissolved in 10 μL DMSO (dimethyl sulphoxide) and volume was made up to 1 mL using methanol. Ascorbic acid was taken as standard or positive control. A stock solution (1 mg/ mL) of ascorbic acid was prepared using methanol. In the reaction mixture, 100 μL of the test samples were added in the test tube containing 1 mL of freshly prepared Phosphomolybdate reagent solution (28 mM sodium dihydrogen orthophosphate, 0.6 mM sulphuric acid and 4 mM ammonium molybdate tetrahydrate) and shaked vigorously. The test tubes were then capped with aluminium foil and incubated in a water bath at 95 °C for 90 min. After incubation, the test tubes were allowed to cool at room temperature for 15 min. The absorbance was measured at 695 nm using UV–Vis spectrophotometer. Reaction mixture containing methanol was taken as negative control. The results were expressed as μg/mL of ascorbic acid equivalents (AAE) using the calibration curve of ascorbic acid. The linear range of the calibration curve was 7.18–125 μg/mL (R2 = 0.932).

2.5.2. Cytotoxicity assay The cytotoxic effect of biofunctionalized AgNPs on HeLa cancer cells was assessed by MTT (4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium Bromide) assay. The monolayer of cells was trypsinized to the single cell suspension and viable cells were counted using a haemocytometer. For the experiment, the cells were seeded onto 96-well plates, maintaining the plating density as 5000 cells/well. The cells were then incubated for 24 h at 5% CO2 at 37 °C for cell adhesion to the bottom of the wells. The cells were then treated with different concentrations of AgNPs and leaf extract prepared in different solvents (5, 10, 20, 40, 60 and 80 μg/mL) and positive control [methotrexate (10–60 μg/mL)], keeping alongside vehicle control (10 μL DMSO in 990 μL in the respective incomplete media of the cell lines) and control (without drug) in triplicates for 24, 48 and 72 h separately. After drug treatment, 20 μL of MTT (Sigma, USA) reagent (5 mg/mL) was added to each well, followed by incubation for 4 h. Subsequently, the supernatant was removed and 150 μL of DMSO was added to solubilize the formazan crystals. The plates were then recorded at 540 nm (reference: 630 nm) using Elisa multiwell plate reader (Epoch 2.0, USA). 2.5.3. Morphological analysis In each 40 mm petri-dish, 0.1 × 106 cells were seeded. The cells were then exposed to different concentrations of AgNPs and aqueous leaf extract (5–80 μg/mL) along with controls for 24, 48 and 72 h. The cytotoxic effect of AgNPs and leaf extract on the morphology of HeLa cells were observed and photographed using an inverted microscope (Nikon, Japan).

2.4.3. Total phenolic content determination To determine total phenolic content (TPC), Folin-Ciocalteu assay was followed with a slight modification in the protocol of Slinkard and Singleton, [23]. Aliquots of 100 μL of test samples were taken and volumes were made up to 3 mL using distilled water. 0.5 mL of FolinCiocalteu reagent was further added to the reaction mixture in the dark conditions, mixed thoroughly and allowed to stand for 3 min. 2 mL of 20% (w/v) Na2CO3 (anhydrous) solution was added and mixed thoroughly. The reaction mixture was then incubated for 60 min in dark. For the blank, an equal volume of methanol was taken instead of the test sample in the reaction mixture. The absorbance was recorded at

2.5.4. Trypan blue exclusion assay HeLa cells (0.1 × 106) were seeded in each of the 40 mm petriplates and incubated for 24 h. Cells were then treated with the given 4

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concentrations of AgNPs and plant extract (10, 20, 40, 60 and 80 μg/ mL) for 24, 48 and 72 h. The treated and group of controls (positive control, vehicle control and control) were further trypsinized and washed. The harvested cells were then stained with trypan blue (Sigma Aldrich, USA) dye in 1:1 ratio. The live and dead cells were then visualized and counted under a light microscope (Carl Zeiss, Germany) at 10 x.

was evaluated using the protocol of [26]. In a 500 mL of plastic bowl, twenty-five numbers of M. thermocyclopoides were exposed to 250 mL of test solution of AgNPs for 72 h at 28 °C RT. Each set of experiments were performed in three replicates with a set of dechlorinated water as the control. Mortality was recorded after 72 h of treatments.

2.5.5. DNA fragmentation DNA fragmentation assay was assessed according to the modified protocol of Saadat et al. [24]. Cells were seeded at a density of 0.1 × 106 cells in each 40 mm of petri-dishes for 24 h. Subsequently, these cells were then treated with different concentrations of AgNPs (5–80 μg/mL) and leaf extract (10–100 μg/mL) for 24 h. The monolayer of cells from treated and control petri-dishes were harvested for DNA isolation. Isolated DNA was resolved on 1.2% (w/v) agarose gel (Sigma, USA), at 90 V for 45 min and visualized and photographed under gel documentation system (Alpha Innotech Corporation, San Leandro, USA).

The larval and cancer cells mortality rate was recorded after 24, 48 and 72 h of AgNPs and different solvents leaf extract treatment. Their corresponding lethal doses (LC50 and LC90 values), Chi-square values, upper and lower confidence limits (UCL-LCL), regression equations and confidence limits were calculated using Probit Analysis [27]. Data were analyzed using the SPSS software (IBM 21). The p ≤ 0.05 were considered to determine the statistical significance. Corrected percentage mortality was calculated using Abbott's formula. Calculation of IC50 values and plotting of sigmoidal curve for cancer activity was done using Graph Pad Prism 7 software.

2.7. Statistical analysis

3. Results and discussion 2.6. Evaluation of larvicidal potential 3.1. Biosynthesis and UV–Vis spectrometric analysis of silver nanoparticles 2.6.1. Mosquito rearing Cyclic colonies of Aedes aegypti Linnaeus, Anopheles stephensi Liston and Culex quinquefasciatus Say were maintained in an insectory of National Institute of Malaria Research (NIMR), Dwarka, Delhi, India, at 28 ± 2 °C temperatures and 70–80% relative humidity with a photoperiod of 14 h light and 10 h dark. The adult mosquitoes were kept in an iron frame of 30 cm × 30 cm × 30 cm which was tied with organdy cloth cages. Soaked raisins and 10% glucose solution were offered to adult mosquitoes as a source of energy. The females were fed on rabbit blood for ovarian development. The gravid females oviposited their eggs on the water surface in small plastic containers lined with filter paper. The eggs were kept undisturbed for hatching for 48 h. The hatched larvae were reared in enamel trays and fed with fish food and dog biscuits (4:6). The water in the trays was changed daily as these species of mosquito prefer fresh water. The larvae then developed to pupae and lasted at the pupal stage for about 48 h. Before the emergence of an adult, the pupae were then transferred into the organdy cloth cages.

Silver nanoparticles were successfully synthesized when the 5 mL of leaf extract of P. longum was added to 45 mL of 1 mM of silver nitrate solution and heated at 40 °C for 20 min. A change in the color of solution from yellow to bright yellow and then to brown designated the formation of silver nanoparticles due to the reduction of silver ions (Ag+) to zero-valent silver nanoparticles (Ag0). The change in color occurred due to the excitation of surface plasmon resonance (SPR) bands of silver nanoparticles and thus directly correlated to the synthesis of AgNPs. The oscillation of free electrons (e−1) of nanoparticles in resonance to lightwave resulting in the rise of SPR absorption bands [28]. Further validation of synthesis was done using UV–Vis spectrophotometer in the range of 300–800 nm. A strong and distinct SPR peak of silver nanoparticles was recorded at a wavelength of 420 nm, which is a characteristic feature of well-dispersed AgNPs. Our observation is similar to the work of Kumar et al. [29], while working on Holarrhena antidysenterica (L.) Wall. synthesized AgNPs reported the optimum absorption peak at 420 nm. Thus, it can be concluded that P. longum leaf extract has the potential to reduce the silver ions into AgNPs. The shape, size, and composition of nanoparticles are proportionally linked to the ratio of salt, plant extract, pH and temperature which automatically facilitate their diversity in applications [30]. Therefore, to optimize the synthesis of AgNPs with smaller size and high monodispersity, synthesis was performed under different experimental conditions with various parameters.

2.6.2. Larval bioassay Larvae of A. aegypti, A. stephensi and C. quinquefasciatus were reared at insectory of National Institute of Malaria Research (NIMR), Delhi, India for the regular availability for bioassays. Larval susceptibility bioassays were performed according to the standard protocols of the World Health Organization [25]. Stock solutions were prepared in ethanol for each extract (chloroform, ethyl acetate, hexane, methanol and aqueous) and AgNPs by dissolving 1 g of each test samples in 50 mL of ethanol. The stocks were further diluted with de-chlorinated water to prepare a range of test concentrations in ppm. Batches of 25 early third instar larvae of A. aegypti, A. stephensi and C. quinquefasciatus were exposed to five various concentrations ranging from 700 to 1500 ppm in 500 mL beakers containing 1 mL test concentration and 249 mL of water. Each set of experiments was carried out in triplicates, along with the controls (de-chlorinated water, solvent concentration and 1 mM silver nitrate) in each series at 27 ± 2 °C temperature and 85 ± 5% relative humidity.

3.2. Physical parameters affecting the synthesis of nanoparticles Various physical factors were found to be responsible for the reduction of Ag+ to Ag0 using aqueous leaf extract of P. longum. In the present investigation, different parameters affecting the synthesis of AgNPs such as time, concentration, temperature and pH of the reaction mixture were studied and optimized. 3.2.1. Effect of varying concentration and temperature During the present study, an increase in the SPR absorption peaks of silver nanoparticles was observed with increase in the concentrations of AgNO3 (0.5–5 mM) solution. The color intensity of the mixture also increased proportionally to the concentration of the AgNO3 solution which is presented in Fig. 1B. The UV–Vis spectrum of aforesaid concentrations showed absorbance peaks in the range of 410–480 nm. It is therefore suggested that the synthesis of nanoparticles increases continuously with the increasing concentrations of silver nitrate due to the enhanced availability of the substrate. A variation in the range of metal

2.6.3. Bioassay for non-targeted organisms, Mesocyclops thermocyclopoides Harda Toxic effect of green synthesized silver nanoparticles was also evaluated against the non-targeted organism, Mesocyclops thermocyclopoides. These species were collected from ponds, ditches and pools of Burari village (North Delhi) and further acclimatized to the laboratory of NIMR, Delhi. Toxic effect of silver nanoparticles concentrations (LC50 and LC90 values of A. aegypti, A. stephensi and C. quinquefasciatus) 5

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pH (9 and 12) due to the enhanced rate of reduction reaction. Similar to our results, Phull et al. [38] while working on the crude extract of Bergenia ciliata reported that the intensity of absorption peaks of synthesized nanoparticles was increased with increasing pH (4–12) of the reaction mixture. Similarly, Ahmed et al. [1] also reported that slightly neutral pH was optimum for preparation of silver nanoparticles using Azadirachta indica aqueous leaf extract. Further, it was observed that when the pH of the colloidal solution was increased, the size of the nanoparticles was also increased. Our observations suggested that pH 6 was optimum for the biosynthesis of silver nanoparticles using P. longum aqueous leaf extract whereas acidic and alkaline conditions lead to the inhibited and enhanced reduction of silver nitrate, respectively. Thus, it can be concluded that the intensity of the absorption peaks and color of the mixture were pH dependent.

salt and biological sample concentrations contributed to control the size of nanoparticles [31]. Similar to our results, Venkatesan et al. [32] while working on marine algae Ecklonia cava also stated that higher concentration of metal salt enhanced the synthesis of nanoparticles. Our results showed that, as the concentration of AgNO3 was increased, a red shift (towards higher wavelength) of UV–Vis spectrum peaks was also observed showing the larger size of nanoparticles (Fig. 1C). According to our observations, it was concluded that AgNO3 concentration is directly proportional to the size and peak intensity of AgNPs. In order to optimize the temperature for the best synthesis of AgNPs, the reaction was carried out using P. longum leaf extract at different temperatures (40–100 °C). It was observed that the colloidal solutions which were incubated at 40, 60 and 80 °C revealed the blue shift of the SPR peaks towards lower wavelengths from 420 to 408 nm (Fig. 1D), indicating the decrease in the size of nanoparticles whereas the mixture incubated at 100 °C showed the distorted peak revealing an uneven synthesis of nanoparticles. Similar to our finding, Verma and Mehta, [33] while working on AgNPs synthesized using neem leaves also observed the shift of absorption peaks towards lower wavelengths from 433 to 397 nm at different temperatures (10–50 °C). The kinetic energy of the molecules increases with an increase in the temperature thus leads to the maximum reduction of silver ions which further reduces the possibility of growth of the size of AgNPs [34]. Thus, it is concluded that 40 °C is the optimum temperature for the synthesis of smaller sized uniform AgNPs using P. longum leaf extract and higher temperature enhanced the synthesis of AgNPs.

3.3. Characterization of silver nanoparticles The size and surface morphology of the synthesized nanoparticles were determined by FE-SEM analysis. Visual observations of the micrograph obtained by FE-SEM illustrates that biosynthesized nanoparticles were monodispersed and completely spherical in shape as depicted by the SPR absorption bands in UV–Vis spectrum (Fig. 2A). The images obtained by FE-SEM were then analyzed using Image J software which revealed that the average diameter of the nanoparticles was in the range of 25–32 nm as shown in Fig. 2B. Agglomeration of nanoparticles was also observed due to the uneven mixing of the mixture before plating on a stub. According to the previous studies, micrographs of bio-synthesized silver nanoparticles obtained by FE-SEM analysis showed spherical morphology of the silver nanoparticles with an average diameter range of 5–60 nm [39–41]. HR-TEM analysis of the green synthesized silver nanoparticles confirmed their spherical nature with the average diameter of 28 nm (Fig. 2C). Besides this, no agglomeration was seen as they were distant from each other by an interparticle distance. Further, the observed selected area electron diffraction (SAED) pattern of silver nanoparticles showed the polycrystalline nature of the biosynthesized silver nanoparticles (Fig. 2D). Thus, it can be concluded that silver nanoparticles synthesized using P. longum aqueous leaf extract were mono-dispersed, crystalline and predominantly spherical in nature. A strong absorption peak was recorded at 3 keV in EDX micrograph (Fig. 2E) due to the SPR of silver, which confirmed the existence of elemental silver in the sample and formation of silver nanoparticles. The elemental profile of silver nanoparticles showed the maximum proportion of silver only. The analysis of the XRD pattern of synthesized silver nanoparticles confirmed their crystalline nature (Fig. 2F). The four distinct Bragg's diffraction peaks of 38.02°, 44.16°, 64.12°, and 76.56° were observed at 2θ corresponding to (111), (200), (220), and (311) sets of crystal reflection planes which are assigned to four faces of face-centered cubic (fcc) structure of silver. Our results are consistent with previous reports [34,42,43]. Besides this, due to some unidentified impurities in the powder of AgNPs, the XRD spectra showed few smaller peaks. The size of the crystallite was calculated using full width at half maximum of the dominant peak (111) by Scherrer formula. The DLS analysis was performed in order to ensure the mean size and zeta potential of the silver nanoparticles derived using P. longum aqueous leaf extract. Various sizes of the synthesized nanoparticles ranging from 10 to 73 nm were observed as shown in Fig. 3A. The results showed that the average size of nanoparticles was 28 nm with narrow size distribution due to the less difference between the largest and smallest size of the nanoparticles. In addition to this, the surface zeta potential value was observed as −24.5 mV at a standard deviation of 4.26 mV (Fig. 3B). The zeta potential more than −20 mV showed that the synthesized nanoparticles have sufficient electrostatic repulsion between each other to remain stable in the solution [26]. The negative potential value of the silver nanoparticles was might be acquired due to the capping action of biomolecules present in aqueous leaf extract of P. longum. Thus, it can be concluded that biosynthesized

3.2.2. Effect of varying incubation time and pH For the complete reduction of AgNPs, incubation period also plays a crucial role. In order to confirm the optimum range of time period required for the best synthesis of AgNPs, 45 mL of 1 mM silver nitrate solution was incubated with 5 mL of plant extract for various time intervals (5–240 min). A gradual change in the color of the colloidal mixture was started to visualize after 8 min of incubation period and reaction continued till 240 min, showing the complete synthesis of AgNPs. We also observed that the intensity of the SPR absorbance peaks was increasing with an increase in the incubation time up to 240 min as shown in Fig. 1E. After that, no increase in the intensity of the absorbance peak and color was observed indicating the complete reduction of the substrate by aqueous leaf extract. It was observed that 120 min incubation time was proved to be the best for the synthesis of AgNPs using leaf extract of P. longum. A similar observation was also recorded by Ibrahim, [35] while working on banana peel extract synthesized nanoparticles. A shift in absorbance bands towards higher wavelength was also observed when the incubation time increased from 5 to 240 min showing the increase in the size of nanoparticles. A similar shifting in peak position was also reported by Veisi et al. [36] while working on Thymus kotschyanus mediated AgNPs revealed that 72 h (optimum time) were required for the complete reduction of the silver nitrate. Thus, it can be deduced that the incubation time required for complete reduction of the substrate depends on the reduction potential of the plant species used. pH of the reaction mixture is another parameter which plays a vital role in the manipulation of surface morphology, shape, size, texture and aggregation of nanoparticles by altering the charge of phytoconstituents present in the aqueous extract. This alteration might affect their capping and stabilizing potential [33]. A change in absorption peak intensity and wavelength was recorded on varying pH of the reaction mixture (3 to 12). A red shift in absorption maximum was observed from 323 to 468 nm, as the pH of the colloidal solution increased from 3 to 12 (Fig. 1F). At pH 3, a flat spectrum of SPR and no change in the color of the reaction mixture was observed because, at low pH, nucleation and agglomeration take place which lead to the production of large sized nanoparticles [37]. Hence, synthesis of nanoparticles was suppressed at acidic conditions. While an intense absorption peak and a characteristic color was recorded at neutral conditions (pH 6) whereas the absorption peaks get distorted at alkaline 6

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Fig. 2. Characterization of silver nanoparticles synthesized using P. longum aqueous leaf extract with 1 mM solution of AgNO3 at optimum conditions. (A) Micrograph showing the morphological features of synthesized AgNPs obtained through Field emission scanning electron microscope, the scale bar corresponds to 200 nm. (B) Histogram obtained by Image J analysis depicting the range of mean particle size of AgNPs synthesized using aqueous leaf extract. This data is made on the image analysis of > 600 particles. (C) High resolution transmission electron microscopy image of AgNPs derived from P. longum aqueous leaf extract confirmed their spherical shape, the scale bar corresponds to 50 nm. (D) Selected area electron diffraction (SAED) pattern of synthesized AgNPs showing the crystalline nature of nanoparticles. (E) Micrograph obtained by EDAX of silver nanoparticles showing the composition of elements of synthesized AgNPs. (F) XRD pattern of AgNPs synthesized using leaf extract of P. longum.

nanoparticles are represented in Fig. 3C. It was observed that the FTIR spectra of plant aqueous extract and their silver nanoparticles were similar with minimal shifts. Table 1 summarizes the absorbance peaks and shifts observed between FTIR spectra of plant aqueous extract and their synthesized nanoparticles. FTIR spectra of synthesized silver nanoparticles scanned in the range of 600–4000 cm−1, showed prominent

silver nanoparticles were strongly anionic due to which they are highly important in various biological applications. Furthermore, to ensure the possible functional groups present in aqueous leaf extract of P. longum and their derived silver nanoparticles, which plays a vital role in reducing, stabilizing and capping the silver nanoparticles, FTIR analysis was performed. The FTIR spectrum of plant extract and silver 1100 400

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Table 1 Table showing the shift in the FTIR peaks after synthesis of nanoparticles using the P. longum leaf extract. Wavelength (cm−1) extract

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3316.43 2166.28 2146.28 1636.20 1011.03 602.89

3315.73 2173.98 2127.37 1636.39 1019.28 575.78

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Alcohol, phenol/aromatic secondary amines Alkyne Isothiocyanate Proteins Vinyl ether Aliphatic bromo- and chloro-compounds

synthesized AgNPs (200.54 μg GAE/mg of dry extract) was found to be highest followed by methanolic, aqueous, ethyl acetate, chloroform and hexane extract showing 195.14, 186.39, 93.85, 90.91 and 70.73 μg GAE/mg of dry extract, respectively. The total phenolic content of AgNPs and leaf extract prepared in different solvents are presented in Fig. 4C. The total flavonoid content (TFC) of AgNPs was found to be 1026.65 μg QE/mg of dry extract as compared to leaf extracts prepared individually in methanol, aqueous, ethyl acetate, chloroform and hexane having TFC as 1014.87, 1006.46, 857.79, 756.16 and 638.88 μg QE/mg of dry extract, respectively as shown in Fig. 4D. Similar to our findings, Phull et al. [38] showed the enhanced TPC and TFC values of AgNPs synthesized using Bergenia ciliata compared to the crude extracts. From the above results, a strong correlation was observed between TFC, TPC, TAC and DPPH values and thus, concluded to the fact that the presence of high amount of phenolics and flavonoid content in synthesized AgNPs are responsible for their pronounced antioxidant activity.

peaks at 3315.73, 2173.98, 2127.37, 2022.44, 1636.39, 1019.27, 575.78, 502.75 cm−1 corresponding to NeH stretch of aromatic secondary amines or OeH stretch of alcohols or phenols, CeH stretch of alkynes, N=C=S stretch of isothiocyanate, C]O stretch in proteins, CeO, NeH stretch of vinyl ether, CeCl or CeBr stretch of aliphatic bromo- and chloro-compounds and CeI stretch of aliphatic iodo compounds, respectively which is in good correlation with other reports [41]. The investigation revealed that hydroxyl, carboxyl and amide groups on the surface of plant extract may be responsible for the synthesis of silver nanoparticles [35] and the presence of proteins on the surface of nanoparticles increases their stability [45]. Besides this, flavonoids and terpenoids which are present in P. longum leaf extract also proved to act as capping and stabilizing agents [45]. 3.4. Antioxidant potential In the current investigation, DPPH and phosphomolybdenum assays were conducted to analyze the antioxidant potential of the AgNPs in comparison to crude extracts prepared in different solvents individually. In DPPH assay, the nitrogen atom of DPPH which contains the odd electron gets reduced by accepting the hydrogen ion and free electron from the anti-oxidant compounds by converting the purple colored DPPH to yellow colored hydrazine molecule. A dose-dependent DPPH free radical scavenging activity of the test samples expressed in percentage were recorded as shown in Fig. 4A. The results showed an increase in the scavenging activity with an increase in the concentration of the samples. Among the various treatments, it was observed that the AgNPs exhibited highest antioxidant activity (78.64%) followed by methanolic extract, aqueous, ethyl acetate, chloroform and hexane extract showing 73.6, 72.32, 65.05, 50.36 and 43.33% scavenging activity at 1 mg/mL dose of test samples, respectively. Our results thus confirmed the radical scavenging activity of AgNPs synthesized using aqueous leaf extract of P. longum. The strong radical-scavenging activity of AgNPs may be due to the presence of substantial amount of phenols and flavonoids in the aqueous extract which play role as capping and stabilizing agent. Similar to our results, Abel-Aziz et al. [46] also showed the higher antioxidant activity of AgNPs-containing leaf extract as compared to the leaf extract alone. Further, AgNPs and plant extracts prepared in different solvents were assessed for the total antioxidant capacity (TAC) using phosphomolybdate assay. In this assay, molybdenum (VI) gets reduced to molybdenum (V) in the presence of antioxidants (reducing agents). The results showed the enhanced antioxidant capacity of AgNPs (225.7 μg/mL) in comparison to plant crude extracts prepared in methanol followed by aqueous, chloroform, ethyl acetate and hexane having 210.1, 201.45, 173.26, 140.52 and 103.61 μg/mL of AAE at 1 mg/mL concentration of samples, respectively. Fig. 4B depicts the dose-dependent increase in the total antioxidant activity of the tested samples. Similar to our results, Nakkala and Sadras [47] showed higher antioxidant activity of AgNPs synthesized using P. longum fruit extract when compared to the standard rutin. Recent reports highlighted that the enhanced antioxidant activity of AgNPs may be due to the attachment of phenol and flavonoid compounds widely existing in the plant extract with metal [48]. In this regard, the total phenolic and flavonoid content of AgNPs and plant extracts prepared in different solvents were determined. The TPC of

3.5. Cytotoxicity assays against cancer cell line In the recent study, the cytotoxic effect of AgNPs and crude leaf extract of P. longum prepared separately in different solvents were tested against cervical cancer (HeLa) cell line using MTT, trypan blue exclusion and DNA fragmentation assays and compared with the standard methotrexate that commercially available in the market as an anticancer drug. In MTT assay, the aqueous solution of MTT (yellow colored) gets reduced to formazan crystals (purple colored) by the enzyme mitochondrial dehydrogenase which is present in metabolically active cells. The intensity of color was then determined using the spectrophotometer. A linear relation was observed between the number of viable cells and absorbance obtained. A dose and time- dependent decrease in HeLa cells was observed by sigmoidal curve (Graph Prism 7, CA) when treated with different concentrations (10, 20, 40, 60, 80 and 100 μg/mL) of plant crude extract and their derived AgNPs for 24, 48 and 72 h (Fig. 5C). The IC50 value as obtained through the absorbance versus dose plot for plant crude extract was 12.61 ± 0.026 (R2 = 0.96), 3.25 ± 0.097 (R2 = 0.95), and 2.36 ± 0.042 μg/mL (R2 = 0.96) for 24, 48 and 72 h, respectively. The maximum cytotoxic effect was observed by AgNPs with IC50 value being 5.27 μg/mL as compared to extracts individually prepared in hexane, chloroform, ethyl acetate, methanol and aqueous where the IC50 values were 221.4, 35.6, 17.8, 12.9 and 8.8 μg/mL, respectively after 24 h of treatment (Fig. 5D–E). To best of our knowledge, this is our first study to report the excellent cytotoxicity of P. longum aqueous leaf extract mediated AgNPs against cervical cancer (HeLa) cell line at minimum doses. Since, AgNPs caused strong inhibition on the growth of HeLa cells, its effect on the morphology of the cells was also observed. As shown in Fig. 5(A–B), the hallmark of apoptosis such as decreased cell growth, shape distortion, blebbing and cell shrinkage was observed in HeLa cells treated with 5 μg/mL dose of AgNPs, as compared to the control. The cytotoxic effect was due to the production of ROS (reactive oxygen species) in tumor cells which eventually caused dysfunctioning of mitochondria, activation of caspases, damage of the cellular DNA and proteins which further leads to the cell mortality. Piperlongumine is one of the major and active phytoconstituent of the plant P. longum which contributes to 9

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Fig. 5. (A, B) Effect of AgNPs on morphology of HeLa cells after 24 h, Order: Control and 5 μg/mL of AgNPs at 20× and 10×, the scale bar corresponds to 100 and 50 μm, respectively. (C) Dose and Time dependent effect of P. longum leaf extract (10–100 μg/mL) on viability of HeLa cells after 24, 48 and 72 h of treatment. (D) Sigmoidal curve of P. longum methanol, aqueous leaf extract and their derived AgNPs dose (5–100 μg/mL) response for HeLa after 24 h. (E) Sigmoidal curve showing the comparative dose (5–100 μg/mL) response of P. longum leaf extracts prepared in different solvents (chloroform, hexane, ethyl acetate and methanol) for HeLa after 24 h. (F) Sigmoidal curve depicting the dose (10–100 μg/mL) response of standard compound (Piperlongumine) for HeLa cells after 24 h. (G) Sigmoidal curve of P. longum leaf extract, their derived AgNPs and piperlongumine dose (10–100 μg/mL) response for HEK 293 (normal cell line) after 24 h of treatment.

against all the three vectors. The maximum larvicidal activity was recorded with the synthesized AgNPs against A. stephensi showing LC50 and LC90 values being 8.969 and 16.102 ppm followed by A. aegypti (LC50 as 14.791 and LC90 as 28.526 ppm) and C. quinquefasciatus (LC50 as 18.662 and LC90 as 40.903 ppm) after 72 h of treatment (Fig. 7A–C; Tables 2–4). As compared to the aqueous extract and leaf extracts prepared in different solvents, these were the minimum doses showing maximum vector mortality by synthesized AgNPs after 72 h of treatment. However, 0% mortality was observed with controls (1 mM silver nitrate, respective solvents and distilled water) against all the three respective mosquito vectors. Similar to our results, Kumar et al. [34] also observed high mortality rates in larvae of A. aegypti (LC50; 5.53 ppm and LC90; 12.01 ppm) and C. quinquefasciatus (LC50; 9.3 ppm and LC90; 19.24 ppm) exposed to AgNPs synthesized using Holarrhena antidysenterica bark extract. Morejon et al. [58] also reported the maximum toxicity of Ambrosia arborescens extract mediated AgNPs (LC50; 0.28 ppm and LC90; 0.43 ppm) as compared to the aqueous extract (LC50; 1844.61 ppm and LC90; 6043.95 ppm). This is our first report showing the efficient larvicidal activity of P. longum aqueous leaf extract synthesized AgNPs and crude leaf extracts prepared in different solvents. In consonance to our results, Madhu et al. [59] reported petroleum ether extract of fruits of P. longum to be the most effective against C. quinquefasciatus having LC50 and LC90 being 1.03 and 2.04 ppm, respectively. The probable mechanism by which AgNPs inhibit the growth of mosquito larvae could be due to the small size of AgNPs which helps them to penetrate through insect cuticle and bind to the phosphorus and sulphur of DNA and proteins, respectively, causing the denaturation of proteins, organelles protein and alteration of their DNA. Subsequently, the membrane permeability decreased which further caused disturbance in proton motive force and leading to the larvae mortality [60–62]. However, Singh et al. [2] hypothesized that AgNPs act as soft acid due to the presence of silver and cell acts as natural base because of availability of phosphorus and sulphur in their cytoplasm. The reaction between the acid and base leads to disruption of midgut and consequently leads to larval death. In contrary to this, bioassays conducted with the leaf extracts of P. longum prepared in different solvents showed the moderate toxic effect against the 3rd instar larvae of A. aegypti, A. stephensi and C. quinquefasciatus. However, the maximum mortality was achieved with the methanolic leaf extract of P. longum followed by ethyl acetate and hexane showing LC50 as 687.175, 1065.035, 1209.158 ppm and LC90 values as 878.98, 1427.349, 1881.729 ppm against A. aegypti. Similarly, the maximum larvicidal mortality against A. stephensi and C. quinquefasciatus was also observed with methanolic leaf extract showing LC50 and LC90 values being 883.532, 1238.161 ppm and 758.894, 990.975 ppm, respectively. Similar to our results, Govindarajan et al. [63] while working with extracts of Cassia fistula prepared in different solvents reported methanolic extract as a potent extract exhibiting good larvicidal potential against A. stephensi and C. quinquefasciatus showing LC50 values as 17.97 and 20.57 mg/L. Thus, of the different type of solvents used, methanolic extract proved to be an ideal solvent used for the extraction of larvicidal compounds from the leaf extract of P. longum. Kamaraj et al. [64] also reported a good larvicidal activity of methanolic extracts of Cassia auriculata L., Leucas aspera and Rhinacanthus nasutus (L.) Kurz against A. subpictus Grassi and C. tritaeniorhynchus Giles, as compared to the other solvents. It was observed that leaf extracts prepared in ethyl acetate, chloroform and hexane showed less toxicity against 3rd instar larvae of A. aegypti (LC50;

its immense therapeutic potential [16]. The previous literature reported the strong anti-cancerous activity of piperlongumine on various cancer cell lines [49–53]. Contrary to the above reports, we observed the less cytotoxicity of the piperlongumine (IC50 = 174.6 μg/mL) as compared to AgNPs against HeLa cell line (Fig. 5F). Thus, confirmed the enhanced cytotoxic activity of AgNPs which might be due to the synergistic effect of the various bioactive compounds present in the plant. However, AgNPs (IC50 = 1844 μg/mL) and leaf crude extract showed very minimal toxic effects on normal epithelial cells (HEK 293) as compared to piperlongumine where IC50 was 475.5 μg/mL (Fig. 5G). Similar to our results, Sharma and Deswal [54] reported the potent anticancer activity of Hippophae rhamnoides berry extract mediated AgNPs (IC50 18.48 μg) when compared to leaf extract showing IC50 value being 73.53 μg. In yet another study, Kanipandian et al. [44] showed good cytotoxicity of AgNPs synthesized using Cleistanthus collinus against lung cancer cell line (A549) where IC50 was found to be 30 μg/mL. Arunachalam et al. [55], also showed an efficient anti-cancerous property of the plant extract of Gymnema sylvestre and their synthesized nanoparticles against HT29 human colon adenocarcinoma cells showing 95% and 30% of cell growth inhibition at 85 μg/mL concentration of AgNPs and crude extract, respectively. Similar to this, Ahmadian et al. [56] revealed that the hepatocellular carcinoma cell proliferation was inhibited by the silver nanoparticles (IC50–75 μg/mL) via induction of apoptosis. To further validate the above results of MTT assay, trypan blue exclusion assay was further performed. In correlation to the above results, similar result was observed showing a sharp decline in live cell count with respect to the increasing concentrations and time duration of drug treatments. As compared to control, 50% reduction in cell count was observed at 10 μg/mL and 20 μg/mL dose of AgNPs and aqueous crude leaf extract, respectively as shown in Fig. 6(A–B). However, no significant difference in viable cell number was recorded between control and vehicle control. Degradation of nucleic acid is considered as a hallmark of apoptosis in the cell. DNA fragmentation assay was further performed and confirmed the apoptotic and necrotic mode of cell death in AgNPs and leaf crude extract-treated HeLa cells. AgNPs induced apoptosis in HeLa cells was observed at minimum dose concentration (5 μg/mL) as shown in Fig. 6(C–D). An intact DNA was observed in control and vehicle control whereas cells treated with increasing concentrations of AgNPs exhibited fragmented ladder-like smear. In consonance to our result, Kapoor et al. [15] also reported the concentration-dependent fragmentation of DNA induced by plant-based drugs. Similar to this, Pandurangan et al. [57] also showed nanoparticles induced necrosis and apoptosis in tumor cells. Thus, fragmentation in cancer cells proved that AgNPs prevent the proliferation of cancer cells by inducing apoptosis. In the present investigation, it is therefore validated that biologically synthesized nanoparticles using P. longum aqueous leaf extract exhibited excellent cytotoxic potential against human cervical cancer cell line as compared to crude leaf extracts and standard.

3.6. Larvicidal activity of AgNPs and leaf extracts In the present investigation, the bio-efficacy of leaf extract of P. longum prepared in different solvents viz. methanol, ethyl acetate, chloroform, hexane, aqueous and leaf aqueous extract mediated AgNPs were evaluated against the 3rd instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The bioassays were carried out with above-mentioned samples showed differential larvicidal activities 12

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1092.05

990.98

1018.95

1000

758.89

500

18.66 40.9

0 Aqueous

Chloroform

Ethyl acetate

Hexane

Methanol

AgNPs

Fig. 7. Graphical representation of lethal doses (LC50 and LC90) causing mortality against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae using crude leaf extract prepared in different solvents and AgNPs synthesized using aqueous leaf extract of P. longum. (A) A. aegypti, (B) A. stephensi and (C) C. quinquefasciatus. 14

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Table 2 Larvicidal activity of P. longum leaf extracts and their synthesized silver nanoparticles against Aedes aegypti showing log probit and regression analysis. Larvae Aedes aegypti

Extracts Methanol

Ethyl acetate

Chloroform

Hexane

Aqueous extract

AgNPs

Time

Regression equations

χ2 (d.f.)a

LC50b (LCLc and UCLd) ppm

LC90

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

y = −17.403 + 5.516x y = −20.783 + 7.098x y = −34.008 + 11.987x y = −21.701+ 6.777x y = −23.685 + 7.711x y = −30.605 + 10.109x y = −16.971 + 5.277x y = −22.924 + 7.308x y = −22.201 + 7.182x y = −22.740 + 6.990x y = −19.127 + 6.077x y = −20.566 + 6.672x y = −36.488 + 11.308x y = −13.461 + 4.097x y = −12.605 + 4.023x y = −4.538 + 2.724x y = −3.911 + 2.952x y = −5.257 + 4.493x

0.632 1.891 0.272 0.321 0.832 0.392 1.505 0.818 0.166 1.081 0.374 1.660 1.024 0.959 0.388 7.539 6.364 4.652

1428.355 (1260.079–1867.713) 847.463 (732.132–933.488) 687.175 (557.131–746.396) 1592.525 (1399.462–2293.931) 1179.136 (1081.790–1301.617) 1065.035 (987.364–1144.317) 1645.557 (1397.419–2810.208) 1371.317 (1247.023–1615.019) 1234.185 (1127.257–1389.274) 1729.72 (1513.986–4352.510) 1404.338 (1199.995–1616.285) 1209.158 (1098.447–1368.127) 1685.64 (1504.594–4034.029) 1929.798 (1502.085–7636.516) 1359.265 (1165.603–1975.791) 46.352 (37.416–57.422) 21.125 (0.760–43.481) 14.791 (11.604–17.667)

2438.201 (1865.392–5300.828) 1284.186 (1144.809–1594.170) 878.980 (805.943–1084.870) 2461.264 (1891.376–6104.552) 1729.607 (1501.134–2268.006) 1427.349 (1302.954–1663.977) 2877.586 (2030.865–11,287.289) 2057.900 (1705.691–3264.424) 1861.720 (1588.524–2618.814) 2734.522 (1966.571–19,393.680) 2281.079 (1805.254–4269.662) 1881.729 (1589.342–2714.249) 2188.081 (1760.546–15,493.034) 3964.454 (2331.960–105,090.465) 2830.460 (1956.793–11,233.507) 136.950 (94.416–161.389) 57.399 (32.723–75.345) 28.526 (23.475–39.137)

(3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)

e

(LCL and UCL) ppm

Control, Zero percent mortality (1 mM silver nitrate, respective solvents and distilled water-not shown in Table 2); aDegree of freedom; blethal concentration that kills 50% of the exposed larvae; c95% lower confidence limit (LCL); d95% upper confidence limit (UCL); elethal concentration that kills 90% of the exposed larvae; χ2 = chi square, (α = 0.05); d.f. = dilution factor. Bold letter (LC50 and LC90)-maximum larvicidal activity at minimum concentration. Table 3 Larvicidal activity of P. longum leaf extracts and their synthesized silver nanoparticles against Anopheles stephensi showing log probit and regression analysis. Larvae Anopheles stephensi

Extracts Methanol

Ethyl acetate

Chloroform

Hexane

Aqueous extract

AgNPs

Time

Regression equations

χ2 (d.f.)a

LC50b (LCLc and UCLd) ppm

LC90

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

y = −14.689 + 4.622x y = −23.012 + 7.679x y = −25.747 + 8.739x y = −19.774 + 6.125x y = −24.276 + 7.916x y = −24.842 + 8.235x y = −21.701 + 6.777x y = −25.208 + 8.186x y = −31.770 + 10.507x y = −17.389 + 5.287x y = −21.408 + 6.817x y = −30.569 + 10.075x y = −17.655 + 5.205x y = −24.387 + 7.628x y = −17.141 + 5.509x y = −5.695 + 4.070x y = −4.081 + 3.631x y = −4.804 + 5.043x

1.245 (3) 0.625 (3) 1.853 (3) 0.358 (3) 0.311 (3) 0.312 (3) 0.321 (3) 0.884 (3) 0.504 (3) 0.404 (3) 1.016 (3) 3.704 (3) 3.097 (3) 0.375 (3) 1.457 (3) 11.197 (3) 3.524 (3) 0.155 (3)

1508.417 (1291.177–2291.833) 992.293 (898.213–1081.517) 883.532 (795.899–958.183) 1691.613 (1445.751–2923.845) 1166.124 (1070.78–1281.197) 1039.003 (950.156–1128.363) 1592.525 (1399.462–2293.931) 1201.159 (1105.822–1321.871) 1056.027 (979.923–1132.036) 1945.842 (1549.779–7564.374) 1382.324 (1248.912–1661.106) 1082.051 (1004.066–1163.042) 2465.331 (2203.343–2757.44) 1575.516 (1402.133–2157.301) 1293.227 (1153.383–1564.942) 25.064 (20.371–29.655) 13.308 (9.359–16.529) 8.969 (4.969–11.217)

2856.527 (2012.516–9643.929) 1457.354 (1298.544–1798.443) 1238.161 (1123.017–1466.075) 2738.497 (1988.717–10,098.283) 1693.576 (1486.801–2183.874) 1487.388 (1331.859–1812.299) 2461.264 (1891.376–6104.552) 1722.607 (1512.306–2227.423) 1398.325 (1281.957–1617.064) 3399.055 (2172.33–65,604.45) 2130.961 (1739.445–3593.268) 1450.371 (1260.378–1575.422) 4345.898 (3576.597–4735.675) 2319.154 (1838.413–5031.426) 2209.994 (1747.264–4062.674) 51.749 (43.834–55.011) 30 (24.023–43.63) 16.102 (12.957–26.987)

e

(LCL and UCL) ppm

Control, Zero percent mortality (1 mM silver nitrate, respective solvents and distilled water-not shown in Table 3); aDegree of freedom; blethal concentration that kills 50% of the exposed larvae; c95% lower confidence limit (LCL); d95% upper confidence limit (UCL); elethal concentration that kills 90% of the exposed larvae; χ2 = chi square, (α = 0.05); d.f. = dilution factor. Bold letter (LC50 and LC90)-maximum larvicidal activity at minimum concentration.

longum aqueous leaf extract and their potential as strong antioxidant, anticancer and larvicidal agent. Shape and size of nanoparticles can be manipulated by altering the conditions such as the concentration of AgNO3, temperature, incubation time and pH of the reaction mixture. AgNPs synthesized using this technique were mostly spherical in shape with an average diameter size of 28 nm. The nature of synthesized AgNPs was found to be crystalline and stable due to negative zeta potential. In vitro antioxidant activity of AgNPs showed their powerful scavenging and antioxidant potential. From the cytotoxicity assay on HeLa cell line, AgNPs proved their strong anti-cancerous property with 50% inhibition at 5.27 μg/mL, which is minimal as compared to different solvents extract. In order to see the occurrence of apoptosis in tumor cells, DNA degradation was analyzed by agarose gel electrophoresis. A significant larvicidal activity of AgNPs was also found against the early 3rd instar larvae of three mosquito vectors (A. aegypti, A. stephensi and C. quinquefasciatus) with LC50 values in the range of 8.969 to 18.662 ppm after 72 h of treatment. Despite the latter effects, silver nanoparticles showed no toxicity towards normal cell line (HEK 293) and non-targeted organism (M. thermocyclopoides). To the best of

1065.035, 1234.185, 1209.158 ppm and LC90; 1427.349, 1861.720; 1881.729 ppm), A. stephensi (LC50; 1039.003,1056.027, 1082.051 ppm and LC90; 1487.388, 1398.325, 1450.371 ppm) and C. quinquefasciatus (LC50; 1018.949, 1092.053, 1135.101 ppm and LC90; 1418.331, 1570.465, 1548.459 ppm), respectively. The availability of larvicidal agents in the plant and their synergistic effect are mainly responsible for the potent larvicidal activity of the AgNPs and the different solvents leaf extract. The current investigation proved that P. longum leaf extract mediated AgNPs showed excellent larvicidal activity against the third instar larvae of Anopheles stephensi followed by A. aegypti and C. quinquefasciatus at minimum doses as compared to the plant extracts prepared in different solvents. Besides this, AgNPs synthesized using aqueous leaf extract of P. longum showed no toxic effects against nontargeted organism (M. thermocyclopoides). 4. Conclusions The present investigation revealed a simple, eco-friendly, non-hazardous and biological system for the synthesis of AgNPs using Piper 15

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Table 4 Larvicidal activity of P. longum leaf extracts and their synthesized silver nanoparticles against Culex quinquefasciatus showing log probit and regression analysis. Larvae Culex quinquefasciatus

Extracts Methanol

Ethyl acetate

Chloroform

Hexane

Aqueous extract

AgNPs

Time

Regression equations

χ2 (d.f.)a

LC50b (LCLc and UCLd) ppm

LC90

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

y = −19.868 + 6.2953x y = −27.794 + 9.350x y = −31.852 + 11.059x y = −22.699 + 7.068x y = −25.122 + 8.240x y = −26.845 + 8.925x y = −20.640 + 6.450x y = −23.483 + 7.612x y = −24.671 + 8.120x y = −11.838 + 3.450x y = −28.699 + 9.178x y = −29.001 + 9.494x y = −17.649 + 5.213x y = −20.229 + 6.302x y = −18.928 + 6.113x y = −4.896 + 2.956x y = −4.747 + 3.472x y = −4.779 + 3.760x

1.517 1.064 0.203 0.285 0.093 2.546 0.711 0.531 1.373 0.986 1.384 3.602 1.185 0.798 0.900 2.279 5.538 2.397

1433.369 (1279.276–1799.241) 938.987 (858.188–1012.930) 758.894 (670.702–821.632) 1628.562 (1426.607–2431.568) 1119.08 (1027.743–1218.989) 1018.949 (935.155–1100.458) 1586.516 (1389.121–2301.536) 1216.171 (1115.059–1353.673) 1092.053 (1001.66–1189.911) 2702.485 (1543.565–3695.279) 1339.297 (1238.973–1502.82) 1135.101 (1052.216–1227.139) 2429.299 (1555.958–3883.902) 1620.548 (1407.759–2479.723) 1249.191 (1126.61–1450.501) 45.3 (36.042–71.565) 23.297 (8.971–39.603) 18.662 (14.626–22.397)

2290.094 (1816.636–4259.766) 1287.212 (1172.855–1505.950) 990.975 (908.951–1164.799) 2471.281 (1896.405–6495.453) 1600.494 (1422.644–1994.8) 1418.331 (1281.889–1688.304) 2505.295 (1906.539–6420.399) 1792.664 (1550.226–2417.773) 1570.465 (1396.197–1949.05) 6356.397 (4045.091–9437.216) 1847.737 (1608.969–2522.525) 1548.459 (1395.956–1865.911) 4278.839 (3140.009–6463.12) 2588.366 (1936.856–7422.11) 2024.845 (1661.884–3243.081) 122.918 (75.848–481.413) 54.502 (34.25–1408.347) 40.903 (32.884–59.486)

(3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)

e

(LCL and UCL) ppm

Control, Zero percent mortality (1 mM silver nitrate, respective solvents and distilled water-not shown in Table 4); aDegree of freedom; blethal concentration that kills 50% of the exposed larvae; c95% lower confidence limit (LCL); d95% upper confidence limit (UCL); elethal concentration that kills 90% of the exposed larvae; χ2 = chi square, (α = 0.05); d.f. = dilution factor. Bold letter (LC50 and LC90)-maximum larvicidal activity at minimum concentration.

our knowledge, this is the first report revealing the excellent and diverse biomedical applications of the synthesized AgNPs prepared using P. longum extract. Needless to say, this study will lead to the advancement of a new technology to resist these deadly diseases.

[12]

[13]

Declaration of Competing Interest

[14]

We declare that we have no conflict of interest.

[15]

Acknowledgement [16]

Authors are grateful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST) Government of India for the Funding of major Research Project (Grant No. EMR/2016/ 001673) to Veena Agrawal, and to University of Delhi for providing DST PURSE (Promotion of University Research and Scientific Excellence) Grant. Renuka Yadav is grateful to Delhi UniversityUniversity Grants Commission (UGC) for awarding UGC Non-National Eligibility Test Fellowship.

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