Multifaceted phytogenic silver nanoparticles by an insectivorous plant Drosera spatulata Labill var. bakoensis and its potential therapeutic applications

The current investigation highlights the green synthesis of silver nanoparticles (AgNPs) by the insectivorous plant Drosera spatulata Labill var. bakoensis, which is the first of its kind. The biosynthesized nanoparticles revealed a UV visible surface plasmon resonance (SPR) band at 427 nm. The natural phytoconstituents which reduce the monovalent silver were identified by FTIR. The particle size of the Ds-AgNPs was detected by the Nanoparticle size analyzer confirms that the average size of nanoparticles was around 23 ± 2 nm. Ds-AgNPs exhibit high stability because of its high negative zeta potential (− 34.1 mV). AFM studies also revealed that the Ds-AgNPs were spherical in shape and average size ranges from 10 to 20 ± 5 nm. TEM analysis also revealed that the average size of Ds-AgNPs was also around 21 ± 4 nm and the shape is roughly spherical and well dispersed. The crystal nature of Ds-AgNPs was detected as a face-centered cube by the XRD analysis. Furthermore, studies on antibacterial and antifungal activities manifested outstanding antimicrobial activities of Ds-AgNPs compared with standard antibiotic Amoxyclav. In addition, demonstration of superior free radical scavenging efficacy coupled with potential in vitro cytotoxic significance on Human colon cancer cell lines (HT-29) suggests that the Ds-AgNPs attain excellent multifunctional therapeutic applications.

sis A.Fleischm.&Chi C.Lee plants (Fig. 1A) were collected from the nitrogen deficient lands located at Thottambedu, Srikalahasti, Chittor district, Andhra Pradesh. (12°37′-14°8′ north latitudes and 78°3′-79°55′ east longitudes). The identified plant was confirmed and authenticated [Herbarium No.NN2120/PURSESVU/2021] by Dr. N.Nagaraju, Associate Professor and Head Retd, Department of Botany, Sri Venkateswara Arts and Science College, Tiruapti, Andhra Pradesh and a voucher specimen has been deposited in the department. The plant height was 2 cm, the root depth was 0.50 cm and it was highly associated with soil particles while the plant flowered part diameter measured was 1.8 cm. Wild D. spatulata var. bakoensis plant materials were collected from the place which was the open space and belong to the private land, which the main author has obtained oral permission from them. All the methods used in plant collection and authentication was appropriate with standard guidelines. There are no unethical practices involved in any of the methods in plant collection and preparations. The plant materials were thoroughly washed with distilled water until the roots were completely dissociated from the soil particles. Then these plants were dried in the shade at room temperature for 6 to 9 days and the dried plant material is kept in the hot air oven at 40 °C temperature for overnight to remove any moisture left in the sample. The dried plant samples were ground into fine powder which was brownish in color and was used to carry out further studies. The collection of plant material was made with relevant institutional, national, and international guidelines and legislation. The Fig. 1A photographs were taken by the author and are not copied from any other sources.
Green synthesis of silver nanoparticles. The D. spatulata var. bakoensis plants extract was prepared by taking 5 g of fine plant powder with 250 mL of Milli Q water in a sterile 500 mL conical flask and steadily mixed the mixture solution and kept in water bath at 60 °C for 30 min, soon after than 30 min the prepared extract was filtered through sterile muslin cloth followed by Whatman no.1 filter paper. The filtrate solution was used to carry out the green synthesis of silver nanoparticles. 5 mL of plant extract was diluted to 20 mL with sterile Milli Q water and then added 50 mL of 0.002 M AgNO 3 solution . The reaction mixture was left at room temperature and observe for color change of the solution, after few minutes the color changed to pale brown and subsequently to dark brown color within 10 min (Fig. 1Ba,b). The color change of the reaction solution is due to the reduction of AgNO 3 to silver ions. In the current study, the AgNPs have been biosynthesized by using whole plant extract of D. spatulata var. bakoensis without any toxic chemicals. Hence this method is an environmentally safe "Green method"; hence the biosynthesized nanoparticles can be referred as Ds-AgNPs.  www.nature.com/scientificreports/ Purification of biosynthesized silver nanoparticles or green Ds-AgNPs. The purification Ds-AgNPs was carried out with modified procedure of Sucharitha et al. 40 . The biosynthesized Ds-AgNPs solution containing silver nanoparticles were centrifuged at 15,000 rpm for 30 min to obtain the pure Ds-AgNPs pellet. The Ds-AgNPs pellets were re-dispersed in 15 mL of sterile Milli Q water to get rid of unbound plant extract residues and biological molecules. The process of centrifugation and re-dispersion of Ds-AgNPs in sterile Milli Q water was repeated thrice to obtain pure green Ds-AgNPs. The purified green Ds-AgNPs pellets were used for consecutive studies like FTIR, EDX, XRD and other advanced spectroscopic methods like TEM and AFM.
Spectral characterization of Ds-AgNPs. The biosynthesized Ds-AgNPs by the total plant extract of D.
spatulata var. bakoensis was analyzed using Nanodrop 8000 (UV-visible spectrophotometer, Thermo Scientific). The optical absorbance of the Ds-AgNPs was recorded at 220-750 nm wavelength range from time-to-time sampling 1 µl of sample and the reading were carried out at room temperature with 1 nm resolution 54 . The Fourier transformed infrared (FTIR) spectrum analysis was carried out for biosynthesized Ds-AgNPs using Alpha T model, FTIR spectrophotometer, Bruker Company, to reveal the potential bioactive components of plant extract of D. spatulata var. bakoensis responsible for the bio-reduction and stabilization of Ds-AgNPs. The particle size and zeta potential of the biosynthesized nanoparticles were determined by using Dynamic light scattering technique on Nanopartica analyzer SZ-100, Horiba scientific, Japan. The size, exterior surface morphology and topology of the green synthesized Ds-AgNPs were analyzed using Atomic Force Microscope (AFM-Solver Next, NT-MDT, Russia), by coating a thin film of Ds-AgNPs on sterile glass piece and air-dried completely prior to analysis. Transmission electron microscopy (TEM) analysis was carried out to identify the exact size and morphology of the synthesized Ds-AgNPs. The sample for TEM analysis was prepared by placing a drop of the purified Ds-AgNPs solution on a carbon-coated copper grids and allowed to dry completely and TEM analysis was carried out using FEI Tecnai F12 (Philips Optics Ltd, Holland) operated at 100 kV 38 TEM was also used to study the selected area electron diffraction (SAED) pattern of the Ds-AgNPs and to get best SAED pattern, the image mode was maintained by Z control (sample height) while the objective lens and standard current were kept. The diffraction spots and the size of the Ds-AgNPs were determined using SIS imaging software (Munster, Germany). X-Ray Diffractometry (XRD) analysis was done to verify the crystalline nature of the Ds-AgNPs using Cu 2+ Kα radiation source on an Ultima IV X-ray powder diffractometer (Rigaku Ltd, Tokyo, Japan). Energy dispersive X-ray (EDAX) was also performed by Oxford Inca Penta FeTX3 EDS instrument attached to Carl Zeiss EVO MA 15 Scanning Electron Microscope (200 kV) machine with a line resolution 2.32 (in Å). The analysis was done by coating a drop of purified Ds-AgNPs on an aluminum foil.
In vitro antioxidant activity. Free radical scavenging activity of Ds-AgNPs by DPPH method. Free radical scavenging activity of the Ds-AgNPs was determined by using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay according to the method described by Mittal et al. 27 . The DPPH stock solution was prepared by dissolving 4 mg of DPPH in100 mL of methanol and stored at 20 °C. DPPH solution (2 mL) was added to 1 mL of methanol solution containing test samples of D. spatulata var. bakoensis plant extract and biosynthesized Ds-AgNPs at different concentrations of 25 μg/mL, 50 μg/mL, 75 μg/mL and 100 μg/mL. The radical scavenging activity (RSA) was calculated by determining the absorbance at 517 nm, by using standard ascorbic acid, the antioxidant activity was expressed as IC 50 . The IC 50 is the half maximum inhibitory concentration of any substance inhibiting a specific biological process or function. www.nature.com/scientificreports/ individual microorganisms were prepared by spreading 200 μl of culture on the nutrient agar plate with the help of sterile glass spreader. Discs were prepared by using Whatman No.1 filter paper. The discs were placed on agar plates and sample plant extract of D. spatulata var. bakoensis 30 μl and biosynthesized Ds-AgNPs 15 μl and 30 μl (μl = mcg) were added on the disc with the help of micropipette. Amoxyclav (Himedia SD063, 30 mcg) disc was used as reference drug. The plates were incubated at 37 °C overnight in an bacteriological incubator. The zones of inhibition (ZOI) of Ds-AgNPs along with standard drug were measured and tabulated.
Antifungal activity. The antifungal activity of Ds-AgNPs was evaluated against two fungal species Aspergillus niger and Penicillium sp. at concentration of 25 mcg by disc diffusion method along with the reference drug Nystatin, (SD025, HiMedia). The ZOI of Ds-AgNPs, ZOI of standard drug Nystatin, (SD025, HiMedia) and the ZOI of plant extract of D. spatulata var. bakoensis and 0.002 M silver nitrate solution were analyzed, and the results were tabulated.
Cell culture. MTT in vitro assay for cytotoxicity of Ds-AgNPs. The cytotoxic activity of biosynthesized Ds-AgNPs was measured by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay 29 . Human colon cancer cell lines (HT-29) were obtained from the National Centre for Cellular Sciences, Pune, India. Cells were cultured in Eagle's Minimum Essential Medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM NaHCO 3 , 100 μg/mL streptomycin and 100 units/mL penicillin. The cell lines were maintained at 37 °C in presence of 5% CO 2 atmosphere in an incubator. The cytotoxic assay was carried out by our previous protocol explained in recent research articles 35

Results and discussion
Spectral characterization of Ds-AgNPs. UV-visible spectral analysis. UV-Vis spectroscopy is the key method recommended to find out the possible characteristics of biofabricated silver nanoparticles (AgNPs). In the current investigation, color less plant extract of D. spatulata var. bakoensis color was changed to pale reddish brown and later to murky brown in color within couple of minutes after addition of 0.002 M silver nitrate solution shown in Fig. 1Ba,b. The above reaction clearly proves that the plant extract consists of several bioactive components which have a very good potential in the reduction of 0.002 M silver nitrate into silver nanoparticles i.e. Ds-AgNPs. The SPR spectrum of biosynthesized Ds-AgNPs was detected at 427 nm (Fig. 1C, Ds-Plant Extract, Ds-AgNPs). The metallic silver nanoparticles have SPR region in-between 390 and 470 nm. Earlier reports reveal that the AgNPs having the SPR spectrum in the region in-between 390-420 nm and 410-450 nm revealed to have small size nanoparticles around 25-50 nm and spherical in shape 30,31 . The plant bioactive compounds such as flavanoids, phenols, alkaloids and sugars, etc.are responsible for capping and stabilizing of metal nanoparticle by using biological extracts 32 .
FTIR analysis of biosynthesized Ds-AgNPs. FTIR analysis of D. spatulata var. bakoensis plant extract and Ds-AgNPs were analyzed separately to investigate the functional groups responsible for bio-reduction and stabilization of Ds-AgNPs and the results are shown in Fig. 2a www.nature.com/scientificreports/ Particle size and zeta potential of biosynthesized Ds-AgNPs. The particle size and zeta potential of the biosynthesized Ds-AgNPs was measured using dynamic light scattering (DLS) method to determine the size distribution and electrical charge present on the surface of Ds-AgNPs. Our results revealed that the size distribution of biosynthesized Ds-AgNPs was between 15 and 40 nm with an average size of 21.9 nm (Fig. 3a) indicating that biosynthesized Ds-AgNPs are poly dispersed in nature. The zeta potential of Ds-AgNPs was detected to be − 32.6 mV (Fig. 3b), due to its high negative potential the nanoparticles are well dispersed in biosynthesized colloidal solution. The stability of the Ds-AgNPs was due to their similar charges on the surface which will oppose the agglomeration. When the zeta potential is lower the attraction between nanoparticles exceeds repulsion and promotes aggregation of the nanoparticles. The zeta potential of silver nanoparticles using other plants also exhibit similar type of results 37,38 . Therefore, the Ds-AgNPs are highly stable and well dispersed in the biosynthesized colloidal solution.
Transmission electron microscopic analysis of biosynthesized Ds-AgNPs. TEM analysis provides the images of nanoparticles, and these images are helpful to find out the size, shape, surface morphology, texture, and distribution of nanoparticles. The results revealed that the Ds-AgNPs were roughly sphere-shaped with variations in www.nature.com/scientificreports/ size distribution, The bio-nanoparticles Ds-AgNPs are in the range from 5 to 40 nm at different magnifications ( Fig. 4a-d). The Ds-AgNPs TEM images revealed that the nanoparticles are poly dispersed in nature without any agglomeration. The variation in size and shape of biosynthesized nanoparticles may be due to the presence of bioactive molecules of the plant extract present on the surface of the Ds-AgNPs. The Ds-AgNPs crystal analysis at 2 nm resolution revealed the crystal lattice fringes with the d spacing value of 0.223 nm (Fig. 4e). Later on, the crystal analysis done by SAED pattern (Fig. 4f) showed evidently Debye-Scherer rings of different planes of face centric cubic structures. With the above results it is concluded that the biosynthesized Ds-AgNPs are crystalline in nature, the results were similar to the earlier reports of biosynthesized AgNPs by leaf extracts of Andrographis serpyllifolia and also fruit extract of Terminalia belerica 39,40 .
XRD analysis of biosynthesized Ds-AgNPs. X-ray diffraction analysis of Ds-AgNPs was done to find out the crystalline characteristics of nanoparticles (Fig. 5a). The spectral analysis of the Ds-AgNPs revealed diffraction peaks at 38.7°, 44.9°, 65.3° and 78.4° and were respectively indexed to planes of face centered cubic (FCC) Energy dispersive X-ray spectroscopy (EDX) analysis of biosynthesized Ds-AgNPs. The EDX analysis of biosynthesized Ds-AgNPs revealed that Ds-AgNPs exhibited very strong signal of silver (Fig. 5b), oxygen and weak signals Si and chlorine peaks, indicating the complete reduction of silver ions to elemental silver. Likewise, the previous reports on silver nanoparticles revealed similar results in EDX analysis. The Ds-AgNPs showed high emission energy at 3 keV for silver and insubstantial signals for other elements.

Atomic force microscopy analysis (AFM) analysis of biosynthesized Ds-AgNPs.
The AFM analysis of biosynthesized Ds-AgNPs was done to detect the surface morphology and topology of nanoparticles. The results indicated that the Ds-AgNPs are spherical in shape and the size of the Ds-AgNPs were in the range of 5 nm to 30 nm and the average grain size detected as 23 ± 5 nm (Fig. 6a,b). Further, we have also carried out Z-coloration analysis with 3D image of Ds-AgNPs to find out the distribution of different size of biosynthesized Ds-AgNPs. The results clearly indicate that the Ds-AgNPs are in the range of 5 nm to 30 nm (Fig. 6c), It is concluded that the size of Ds-AgNPs almost similar to that of Particle size analysis and TEM analysis.    (Fig. 8a,b). So, it is clearly understood that the Ds-AgNPs comprise higher inhibitory activity then the standard antibiotic Amoxyclav. Though there are several reports on antimicrobial activity of silver nanoparticles which were bio-fabricated or green synthesized by different parts of plant materials, revealed that in most of the cases the plant extracts show negligible amount of antimicrobial activity or no inhibition of bacteria, while the biosynthesized AgNPs revealed enhanced and exceptional antimicrobial activity. In the present study also, the Ds-PE showed minimum inhibition zones of 07 mm, 08 mm, and 06 mm respectively, but the biosynthesized Ds-AgNPs revealed superior antibacterial activity when compared to Ds-PE alone. When the bacterial cells are treated with AgNPs, which are in nano-size particles will come in contact with bacterial cell wall and cause cell damage by penetrating into the cell wall due to electrostatic forces and endocytosis, subsequently cause bacterial cell death by production of reactive oxygen species which cause inflammation, lead to DNA damage and other cell organelles, it's also depends upon the concentration of silver nanoparticles used for the assay, higher the concentration of AgNPs superior zone of inhibition was observed by various researchers. Similar type of results was observed AgNPs biosynthesized with turmeric extracts 41 . Other recent study by root extract of Salvadora persica mediated silver nanparticles, also reveals that ZOI will also depend upon the size of the nanoparticles and concentration of nanoparticles. Smaller the size of nanoparticles more easily the nanoparticles will percolate through the cell membrane and induce toxicity and cause cell death in both gram positive Staphylococcus epidermidis and gram negative Escherichia coli bacteria 42   www.nature.com/scientificreports/ AgNPs against Escherichia coli and Staphylococcus aureus also reveals similar results in which protein synthesis is arrested due AgNPs 43,44 . In another study silver nanoparticles synthesized using Cynodon dactylon leaves also revealed that, the AgNPs will reacts with proteins of bacterial wall and penetrate to cells and cause DNA Antifungal activity of Ds-AgNPs. The biosynthesized Ds-AgNPs have proved to be exceptionally lethal against two fungal species Aspergillus niger and Penicillium sp. at the concentration of 25mcg (Fig. 8c). The outcome of the inhibitory activity of biosynthesized Ds-AgNPs were tabulated in (Table 3) and shown in (Fig. 8d) (Table 3), from the above results, it is concluded that the biosynthesized Ds-AgNPs have excellent antifungal activity than the standard antifungal drug Nystatin. Therefore, the biosynthesized Ds-AgNPs can be useful as effective and excellent antifungal agent and can be widely used in pharmaceutical industries for the development of antifungal ointments and other formulations 47 . Similar type of results were reported recently by seed extract mediated AgNPs by Plantago major, in which the silver nanoparticles revealed good antifungal activity against Penicillium sp. and also good antibacterial activity against E. coli also 48 .

In vitro antioxidant activity of Ds-AgNPs by DPPH, H 2 O 2 and nitric oxide (NO) methods.
Anticancer activity of biosynthesized Ds-AgNPs. The ability of NPs to make a way into cells primarily depends on the physiochemical properties including shape, size and surface net charge. In the current study, the size of biosynthesized Ds-AgNPs is in-between 12 and 40 nm in size with average size of 21.9 nm. It is well known fact that the nanoparticles having the size below 100 nm are said to be of biomedical importance. Similarly, the nanoparticles having size < 50 nm are said be very efficient in anticancer activity because of their minute size they can easily taken up and penetrate deeply into the cancer cells by endocytosis subsequently and cause cell death or apoptosis and eradicate tumorogenic cells. Whereas the nanoparticle having size > 50 nm cannot penetrate easily in the cancer cell and cannot completely damage the cancer cells or tumorogenic cells. So it is concluded that nanoparticles of minute size have three important properties by which they can easily diffuse, deep infiltration and improved accumulation can results in complete eradication of cancer cells or tumorogenic cells 49 .
In the present study, anticancer activity of the Ds-AgNPs was evaluated against HT29 human colon cancer cells by MTT assay. The results revealed that the Ds-AgNPs induced cytotoxicity in HT29 cancer cells by concentration dependent approach (Fig. 9a). Higher the concentration of Ds-AgNPs decreases the cell viability of cancer cells. Ds-AgNPs showed maximum inhibition of 92% against HT29 colon cancer cells. IC 50 values of the Ds-AgNPs were found to be 52.81 µg/mL against HT29 colon cancer cells. The (Fig. 9b) reveals the cytotoxic effects of Ds-AgNPs at concentration of 50 µg/mL which almost equivalent IC 50 value at different time interval up to 72 h to observe the morphological changes in the cancer cell due cytotoxic effect of the biosynthesized Ds-AgNPs. It is clearly evident that the HT 29 cancer cells completely dead after 72 h no viable cells were observed, which proves the efficacy of biosynthesized Ds-AgNPs as an anticancer agent this may due to the minute size of Ds-AgNPs. Recently, Alqahtani et al. also reported efficacy of biosynthesized AgNPs by lichens on HCT-116 (Human colorectal cancer cell) and MDA-MB-21 (Breast cancer cell) and FaDU (Pharynx cancer cell) by in vitro cytotoxic MTT assay also revealed similar results to our present study which is size and dose dependent 50 . Kabir et al. also conducted cytotoxic studies in different cancer cell lines which were similar to the above results 51 , an another study by photosynthesized AgNPs using Ficus religiosa also proved have substantial cytotoxicity against COLO205 cells 52 , other study using silver nanoparticles by M. koenigii have also proven to an effective applicable  53 . Finally it could be concluded that the results of cytotoxic activity of biosynthesized Ds-AgNPs are in line with previous reports and any difference in cytotoxic assay results could vary due size, shape and concentration and different cancer cell lines.  www.nature.com/scientificreports/ www.nature.com/scientificreports/

Summary and conclusion
In this report it was demonstrated the biofabrication of nanosized Ds-AgNPs using insectivorous plant Drosera spatulata Labill var. bakoensis extract, being the first of its kind. The biosynthesized Ds-AgNPs are spherical, well-dispersed with average range size of 10 to 20 nm and exhibited outstanding antimicrobial activities against clinical bacteria and fungi isolates. Furthermore, Ds-AgNPs also demonstrated superior free radical scavenging activities and strong cytotoxic potential against HT-29 colon cancer cell lines, indicating the possibility of employing and developing Ds-AgNPs as multifaceted therapeutic agents for various clinical conditions (Supplementary Information).