Introduction

In recent years, nanoscience has emerged as an important multidisciplinary section that deals with research and advancement in material sciences. Nanotechnology deals with the production and utilization of nanomaterial from 1 to 100 nm size range (Akintelu et al. 2020). In present day, nanotechnology is an important tool for manufacturing various kinds of biologically active pharmaceuticals and materials in the nano-range using metallic or alloy-based nanoparticles that have novel optical, chemical, and physical traits (Ijaz et al. 2021; Kumar et al. 2018).

Metallic nanoparticles can be produced by several approaches comprising physical, chemical and biological systems, but the biological or ‘green chemistry’ oriented synthesis technique is the most fascinating synthesis process. The biological synthesis methods have unique features and advantages as compared to other conventional synthesis methods which involve various parameters, such as high pressure, inert gases, high temperature, laser radiation and toxic by-products that cause harmful effects to environment and human health (Garg et al. 2020; Chung et al. 2016).

Silver (Ag) is the basic element and used as an effective antimicrobial agent from early medical practice by humans in contrast to other metals and their large surface area to volume ratio that furnishes greater contact with target microorganisms. Silver nanoparticles (AgNPs) are the metallic nanoparticles with unique and distinctive properties, including high conductivity, high stability and flexibility that makes them the most desired one’s to be used in divergent fields, such as optics, telecommunications, electronics, textiles, pharmaceuticals, biosensors, waste water treatment, agriculture and food processing and other industries (Garg et al. 2020; Velidandi et al. 2020). Apart from these, AgNPs are extensively used as antifungals, antivirals, anti-inflammatory, antioxidant, anti-angiogenesis, anti-platelet and anticancer agents in the field of medicine and biomedical sciences (Tadele et al. 2021; Thirumal, 2021).

Biogenic synthesis of AgNPs can be accomplished by utilizing various life forms such as bacteria (Javaid et al. 2018; Singh et al. 2015), fungi (Khan et al. 2018; Zhao et al. 2017), actinomycetes (Kumari et al. 2020a, b; Manivasagan et al. 2014), algae (Al-Nadhari et al. 2021; Sharma et al. 2016), and plants (Tarannum and Gautam 2019; Rajeshkumar and Bharath 2017), which contain enough metabolites to effectively reduce and stabilize AgNPs. However, plants contain numerous bioactive secondary metabolites like alkaloids, flavonoids, terpenoids, carbohydrates, proteins, glycosides and saponins that naturally reduce AgNPs during the synthesis (Ahmed et al. 2016). The plant-mediated metallic nanoparticles synthesis is more advantageous as the characters of nanoparticles are majorly regulated by the physical conditions, adsorption process of stabilizing agent, and the rate at which metal ions interact with reducing agents (Ahmad et al. 2019; Restrepo and Villa 2021).

The need for a broad-spectrum antibiotic is increasing at an immense pace as many of the human pathogenic bacteria are acquiring multi-drug resistance. The age old knowledge of silver or silver-coated antimicrobials can come in handy for the preparation of such clinical antibiotic to fight severe diseases (Ahmad et al. 2019). As silver is toxic to bacteria, fungi and yeast, the use of AgNPs as an antimicrobial agent may help control the resistant pathogenic microbes. Cancer is considered as a deadly disease taking countless lives every year with the prostate cancer being the third most dangerous, contributing to 1,414,259 (7.3%) of incidence and 375,304 (3.8%) of mortality worldwide in 2020 (Sung et al. 2021). There is an immediate need to cure cancer in a lesser painful and adverse manner; AgNPs are the promising alternatives to replace chemotherapy to achieve that need.

Artocarpus lakoocha belongs to Moraceae family and the fruits have been proven to be used for antibacterial, antifungal, antiviral, antitubercular, antiplatelet, antiarthritic and other medicinal purposes. Therefore, the current research was focused on the biological synthesis, characterization and potential antibacterial and anticancer activity of Artocarpus lakoocha fruit extract-mediated AgNPs.

Materials and methods

Collection of materials

Artocarpus lakoocha Roxb. (A. lakoocha) fruit was collected from local market near Dharwad, Karnataka, India and validated by referring literature and herbarium collection at Plants and Animals museum, Karnatak University Dharwad (Fig. 1A). Silver nitrate was acquired from Sigma-Aldrich laboratories and microbial pathogens were obtained from National Collection for Industrial Microorganisms (NCIM), Pune, India.

Fig. 1
figure 1

Visual observations; A A. lakoocha fruit, B dried fruit powder, C colour change from light yellow to dark brown after AgNO3 addition

Full size image

Preparation of fruit extract

The collected fruits were washed thoroughly to remove adhered unwanted materials and cut into smaller pieces. The dried pieces were ground into a powder (Fig. 1B) and 10 g of dried A. lakoocha fruit powder loaded into the thimble placed inside the soxhlet extractor; methanol was added to the round bottom flask. Once the process was finished methanol was evaporated using rotary evaporator leaving extracted plant material in the round bottom flask and that was stored in separate vial.

Synthesis of AgNPs

About 1.0 mM AgNO3 solution was prepared by solvating 0.0169 g of AgNO3 in 100 ml of distilled water. 25 ml of A. lakoocha fruit methanol extract was mixed with 75 ml of AgNO3 solution in the ratio of 1:3, and the pH was adjusted to 9.0. Thus prepared solution was incubated for 24 h in dark and then subjected to centrifugation for 10 min at 10,000 rpm. The residue was air dried and the AgNPs were collected and stored in Eppendorff tubes to carry out further analysis.

Characterization of AgNPs

The spectrophotometric study of biosynthesized AgNPs was analysed in the wavelength range of 200–700 nm by using double beam UV–visible spectrophotometer (UV-9600A, Metash Instruments Co. Ltd., Shanghai, China). The functional groups/biomolecules accountable for the bio reduction and capping of AgNPs was studied from FTIR analysis (Nicolet 6700, Thermo Fisher Scientific, Waltham, Massachusetts, USA) by scanning the KBr mixed sample pellet from 400 to 4000 cm−1. A thin film of dried AgNPs powder was prepared on a clean glass slide and scanned with atomic force microscope (Nanosurf Flex-AFM, Liestal, Switzerland) to ascertain the surface topology and distribution of the AgNPs. The morphological details and the elemental compositions of the AgNPs were investigated by subjecting the sample to SEM with EDS analysis (JEOL JSM-IT 500LA, USA). The atomic arrangements at interfaces and the crystalline structure of synthesized AgNPs were determined by XRD analysis (Rigaku SmartLab SE, Japan). The determination of shape, size and distribution nature of the synthesized AgNPs was done by TEM analysis (FEI, TECNAI G2, F30, China).

Antibacterial activity of AgNPs

The antibacterial potential of the synthesized AgNPs was verified against pathogenic bacteria, such as Streptococcus pneumoniae (MTCC1935), Staphylococcus aureus (MTCC6908), Klebsiella pneumoniae (MTCC9238), Bacillus subtilis (MTCC6633), Shigella flexneri (MTCC1457), and Escherichia coli (MTCC40) using agar well diffusion method by making 6 mm diameter wells with the help of cork borer on nutrient agar (NA) media plates. The stock AgNPs solution of 1 mg/ml concentration was prepared and 25, 50, 75 and 100 μl (1 μg/μl each) of solutions were filled into corresponding wells; for positive and negative controls, streptomycin and sterile distilled water were used, respectively. The plates were incubated overnight at 37 °C and the clear zone formed around each well was measured as diameter in mm.

Determination of MTT cytotoxic activity of AgNPs

To determine the anticancer potential of AgNPs, human prostate adenocarcinoma (PC-3) cell lines were procured from NCCS, Pune, India and sub-cultured on DMEM-High Glucose (#AL111, Himedia) medium. During analysis, medium with cells but without the AgNPs/anticancer drug was taken as negative control and medium with cells and 12.5 μg/ml of cisplatin (#PHR1624, Sigma) was taken as positive control; AgNPs solution with concentrations of 6.25–100 μg/ml was filled into respective wells. The plates were incubated in a humidified 5% CO2 atmosphere (Healforce, China) for 24 h at 37 °C to allow cells to grow. After incubation, MTT reagent (# 4060 Himedia) was added by removing the used-up media; again after 3 h of incubation, 100 μl of DMSO (#PHR1309, Sigma) was added and MTT reagent was removed. Finally, the absorbance at 570 nm was read on an ELISA reader by taking 630 nm as reference wavelength and the IC50 value was calculated.

Evaluation of apoptosis using flow cytometry analysis

The apoptosis/necrosis study of the synthesized AgNPs was done by employing FITC Annexin V (Annexin V)/ Propidium Iodide (PI) expression assay. Annexin V (Cat No: 51-65874X, BD Biosciences) was used to discover the percentage of cells undergoing apoptosis, whereas PI (Cat No. 51-66211E, BD Biosciences) was used to differentiate viable from nonviable cells. During this study, 6-well plate (Biolite-ThermoNunc) was used to culture PC-3 cells and kept for overnight incubation at 37 °C in a humidified CO2 incubator. The cells were treated with 30.62 µg/ml (IC50) concentration of AgNPs. The flow cytometry analysis was done by following the standard protocol suggested by manufacturers of FITC Annexin-V apoptosis detection kit (BD FACS calibur, Biosciences, USA). Finally, the preparation was analysed using flow cytometer immediately after the addition of PI (5 μl) and 1× binding buffer (400 μl) to each tube. The images were assessed by using BD Cell Quest Pro Ver.6.0 software.

Statistical analysis

All the analysis were performed in triplicates and the data are represented as mean ± standard deviation. The statistical analysis was performed by using Graph Pad Prism 4.0 software (San Diego, CA, USA).

Results

Synthesis of AgNPs

The initial indication on formation of AgNPs was evidenced by colour change in the reaction mixture (Fig. 1C). The AgNPs synthesized by using A. lakoocha fruit methanol extract was slightly yellow in colour; when the extract was added with AgNO3 the observed colour change was from light yellow to dark brown. The colour change was due to the bio-reduction of ionic silver (Ag+) to elemental silver (Ag0) by the combined action of biomolecules present in the A. lakoocha fruit extract.

Characterizations of AgNPs

The bio-reduction of Ag+ ion was recorded with the use of UV–visible spectroscopy by recording the absorption spectral wavelength between 200 and 700 nm. UV–visible spectral analysis showed that maximum absorption was at 415 nm (Fig. 2A), that reasserted the synthesis of AgNPs in the solution. The single characteristic absorption peak was obtained due to the surface plasmon resonance of excited particles. The FTIR analysis exhibited several bands/peaks at around 3412, 2925, 2853, 2426, 1732, 1585, 1384, 1305, 1245, 1078, 903, 840, and 619 cm−1 wave numbers (Fig. 2B). The broad absorption band observed at 3412 cm−1 was correlated to O–H stretching alcohol and the absorption bands at 2925 and 2853 cm−1 corresponded to the symmetrical and asymmetrical stretching of alkane molecules respectively. The band at 1732 cm−1 is for stretching of aldehyde molecules and band at 1585 cm−1 attributed to N–H bending amine. The bands at 1384 cm−1 and 1245 cm−1 showed the S=O stretching of sulphate and C–N stretching amine. The bands at 1078, 903 and 619 cm−1 corresponded to C–O stretching primary alcohol, C=C bending alkane and C–Br stretching halo compounds, respectively. The peaks at 2426 and 1305 cm−1 were due to the absorbed atmospheric CO2.

Fig. 2
figure 2

Characterization of A. lakoocha fruit extract AgNPs; A UV–vis. analysis, and B FTIR analysis

Full size image

Morphological characterization

AFM analysis gave an insight to the surface morphology, distribution and distance between AgNPs by displaying two and three dimensional topographical views (Fig. 3A–C). AFM data revealed that the synthesized AgNPs were polydispersed with less or no agglomeration, spherical and diameter ranging from 10 to 30 nm. SEM image displayed relatively spherical-shaped AgNPs formed with polydispersed nature (Fig. 3D). The EDS spectrum exhibited peaks for various elements, such as C, O, Na, and Al along with the typical peak for elemental silver (Agº) at 3 keV (Fig. 3E). The peaks for C, O and Na were emerged due to the surface bound biomolecules, whereas peak for Al was due to sample grid holder.

Fig. 3
figure 3

Morphological characterization of A. lakoocha fruit extract AgNPs; A 2-D AFM view, B particle size distribution, C 3-D AFM view, D SEM micrograph, and E EDS spectrum

Full size image

Structural characterization

The XRD patterns of A. lakoocha AgNPs showed the synthesis of crystalline structured nanoparticles and the XRD peaks were obtained at 38.13°, 44.11°, 63.82°, 77.48°, and 81.49°. The obtained diffraction intensities can be assigned to the (111), (200), (220), (311), and (222) Bragg’s diffraction planes of the metallic silver having face- centered cubic (FCC) crystalline structure (Fig. 4A).

Fig. 4
figure 4

Structural characterization of A. lakoocha fruit extract AgNPs; A XRD pattern, B TEM micrograph, and C TEM image showing smallest particle

Full size image

The image obtained from HR-TEM analysis illustrated the morphological details of the synthesized AgNPs along with accurate size range of the particles. The micrograph displayed the spherical, polydispersed AgNPs ranging from 6.59 to 25 nm in size (Fig. 4B), and the smallest size of the particle was found out to be 6.59 nm (Fig. 4C).

Antibacterial activity of AgNPs

The potential of synthesized AgNPs to inhibit bacterial growth was analysed against human pathogenic bacteria; among the tested pathogens, S. pneumoniae was the most sensitive with measured inhibition zones of 14 ± 0.6, 16 ± 0.5, 17 ± 0.5, and 20 ± 0.4 mm at 25, 50, 75, and 100 μl of AgNPs treatment. Similarly, S. aureus was observed to be the least sensitive with inhibition zones of 13 ± 0.5, 14 ± 0.4, 14 ± 0.5, and 15 ± 0.6 mm at 25, 50, 75, and 100 μl of AgNPs solutions.The AgNPs displayed antibacterial activity by increased zones of inhibition with increased AgNPs volume. All the other pathogenic bacteria like K. pneumoniae, B. subtilis, E. coli and S. flexneri exhibited moderate zones of inhibition at different concentrations of AgNPs (Fig. 5A–G).

Fig. 5
figure 5

Antibacterial activity of A. lakoocha fruit extract AgNPs against pathogens; A S. aureus, B K. pneumoniae, C B. subtilis, D S. pneumoniae, E E. coli, F S. flexneri, and G Graph showing zones of inhibition formed against pathogenic bacteria

Full size image

Determination of MTT cytotoxic activity of AgNPs

The bio-fabricated AgNPs exhibited increased cytotoxic activity with increase in concentration to prevent the growth of cancer cells by inducing apoptosis; that is marked by the difference in cell shape, size, and other morphological details (Fig. 6A–G). The AgNPs effectively inhibited PC-3 cells by displaying decreased % cell viability of 96.34 ± 0.5%, 84.06 ± 0.6%, 57.90 ± 0.6%, 32.43 ± 0.5%, and 11.07 ± 0.4% at 6.25, 12.5, 25, 50, and 100 μg/ml of AgNPs concentrations indicating the dose-dependent activity (Fig. 6H). The IC50 value of 30.62 μg/ml against PC-3 cell line indicated the higher potential of A. lakoocha AgNPs to inhibit the proliferation of tumour cells.

Fig. 6
figure 6

MTT cytotoxic activity of A. lakoocha AgNPs against PC-3 cell line; A untreated, B standard, C 6.25 µg/ml, D 12.5 µg/ml, E 25 µg/ml, F 50 µg/ml, G 100 µg/ml, and H graph showing % cell viability of PC-3 cells at different concentrations of AgNPs

Full size image

Evaluation of apoptosis using flow cytometry analysis

In Annexin V/PI expression study, the cells in early apoptosis stained positive for Annexin V and negative for PI, while the cells positively stained for both Annexin V and PI were in late apoptosis or already dead. The viable cells or cells with no measurable apoptosis were stained negatively for both Annexin V and PI after 24 h of treatment with AgNPs. The flow cytometer analysis unveiled 48.11 ± 0.7% of early apoptosis (lower right), 24.92 ± 0.5% of late apoptosis (upper right), 25.78 ± 0.6% of viable cells (lower left) and 1.19 ± 0.4% of necrotic cells (upper left) as depicted in the quadrangular plot for both control (Fig. 7A) and treated cells(Fig. 7B). The obtained results specified that AgNPs can prevent proliferation of cancer cells by increasing apoptosis and the possible mechanism of action of AgNPs on cancer cells leading to cell death is shown in Fig. 7C.

Fig. 7
figure 7

Flow cytometry analysis of A. lakoocha AgNPs against PC-3 cell line; A untreated PC-3 cells, B treated PC-3 cells, and C possible mechanism of action of AgNPs against PC-3 cell line

Full size image

Discussion

The change in colour after adding AgNO3 to extract indicates about initiation of AgNPs synthesis; the dark brown colour was attributed to the intense absorption of light in the specific wavelength range leading to surface plasmon resonance effect from the combined oscillation of induced free electrons (Hemmati et al. 2018). The previous studies regarding UV–vis. analysis of green AgNPs also reported the similar results, where absorption spectra of AgNPs present in the aqueous solution displayed absorbance peak around 400–440 nm and with an increase in the reaction time a swift increase in the synthesis of AgNPs was observed (Hemmati et al. 2018; Salayova et al. 2021). The formation of stabilized AgNPs was because of the presence of biochemical functional groups and the role of plant extract that contain biomolecules namely phenolics, terpenoids, flavonoids, polysaccharides, alkaloids, enzymes, amino acids, proteins and alcoholic compounds, which were accountable for the synthesis of stable AgNPs (Salayova et al. 2021; Arumugam et al. 2017). The AFM analysis for A. lakoocha AgNPs depicted the spherical shape, polydispersion of AgNPs; and the obtained results were well supported by the earlier studies on biosynthesized AgNPs investigated using AFM analysis, where the biosynthesized AgNPs were spherical, polydispersed with less evidence of agglomeration (Bhat et al. 2021). The EDX spectrum of A. lakoocha AgNPs exhibited various peaks corresponding to various elements; however, a strong absorption peak was obtained at 3 keV depicting silver. The earlier findings suggested that metallic silver exhibits typical absorption peak at 3 keV because of surface plasmon effect. According to them, AgNPs synthesized using plant extracts were stable in solution several months after synthesis due to the encircled biomolecules (Bhat et al. 2021; Umoren et al. 2014).

The XRD pattern of A. lakoocha AgNPs displayed five diffraction planes specific for metallic silver at five different 2θ angles and was compared with the International Center for Diffraction Data (ICDD) of silver to confirm the FCC structure; the result was also supported by findings of the earlier studies, where biosynthesized AgNPs exhibited same pattern of Bragg’s diffraction peaks characteristic for metallic silver at similar range of 2θ angles (Acharya et al. 2020; Nayaka et al. 2020a). In TEM analysis, the AgNPs showed spherical shape, small size and less agglomeration. Similarly, earlier studies using TEM analysis of AgNPs suggested similar results regarding shape, size and distribution of AgNPs; the results suggested the synthesis of spherical AgNPs of 10–50 nm in size with demonstrating some amount of agglomeration and mostly dispersed nanoparticles (Bhat et al. 2021; Nayak et al. 2020). The green AgNPs from A. lakoocha fruit methanol extract exhibited increased antimicrobial activity with increase in concentration. The previous reports about the antimicrobial activity of green AgNPs suggested that, cell membranes of microorganisms are negatively charged because of the presence of amino acids, phosphates, and carboxyl groups. The slightly positively charged AgNPs were attracted towards cell membrane and attach to bacterial cell wall. The AgNPs migrate through membranes and cell walls, alter the permeability of plasma membrane and affect cellular homeostasis of the microorganism to induce the chain of events that cause cell death (Nayaka et al. 2020a; b).

The synthesized AgNPs demonstrated dose-dependent anticancer activity in vitro against PC-3 cell line indicating the potential of AgNPs to inhibit the tumour growth by altering the structure and function of cancer cells. Similar observations displayed dissembled gaps between neighbouring cells and deformation of cells were reported in the previous studies involving MTT assay; where, the AgNPs prepared by Hyptis suaveolens callus, Asafoetida, and Pinus roxburghii extracts showed notable anticancer activity againstPC-3, MDA-MB-231, HepG2 and A549 cell lines (Botcha and Prattipati 2020; Satsangi, 2020; Kumari et al. 2020a, b). According to previous findings, the AgNPs attach to an end consisting of thiol groups of enzymes and bring about the unfolding, aggregations and deformation of proteins inside the cancer cells and lead to induction of apoptosis. The flow cytometry-assisted apoptosis studies were reported indicating the % of early apoptosis, late apoptosis, necrosis and viable cells, when cancer cell lines were treated with bio-fabricated AgNPs. These results suggested that AgNPs can induce apoptosis and prevent cellular multiplication by generating reactive oxygen species (ROS) leading to cellular DNA damage. The increased level of ROS induces oxidative stress causing early apoptosis; meanwhile, the ROS generation increases due to the reduced membrane potential of mitochondria that acts as a secondary messenger simultaneously in many signaling pathways. The cascade of events play predominant role in apoptosis by controlling the activity of specific enzymes concerned in the apoptotic pathways (Vasanth et al. 2014; Almalki and Khalifa 2020; Amini et al. 2021).

Conclusion

As awareness increasing towards green nanotechnology, use of green route for metal nanoparticles synthesis is leading to a desire to establish eco-friendly technique; AgNPs synthesis from plant extract is beneficiary over other sources as the former is energy efficient, cost effective, and less affective to human health and environment. Biosynthesized AgNPs from A. lakoocha exhibited high antibacterial activity against human pathogenic bacteria indicating its possible use as an alternative to antibiotics in coming years after successful clinical trials. The AgNPs displayed dose-dependent anticancer activity on PC-3 cells suggesting the possible use as an anticancer agent to cure prostate cancer. Therefore, the findings of the present study conclude that, the green AgNPs can replace the clinical or biomedical agents to cure microbial and cancer-related diseases in near future.