Bio‐inspired synthesis of silver nanoparticles from leaf extracts of Cleistanthus collinus (Roxb.): its potential antibacterial and anticancer activities

Abstract

In this investigation, the biological synthesis method was adopted to synthesise silver nanoparticles (AgNPs) by using the leaf extracts of Cleistanthus collinus (C. collinus). This plant has traditionally been used to remove the harmful pest from the agriculture field. Leaf extract of C. collinus was used as bioreductant on the precursor solvent of AgNO₃. The synthesised AgNPs were characterised by spectroscopic method such as UV–vis spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, dynamic light scattering and microscopic method by field‐emission scanning electron microscopy analysis. The AgNPs were studied for both antibacterial and antifungal activities and found to exhibit potential antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa. The anticancer activity of AgNPs was screened against A‐431 osteosarcoma cell line by [3‐(4, 5‐dimetheylthiazol‐2)‐2, 5 diphenyl tetrazolium bromide] assay and the IC₅₀ value was found to be 91.05 ± 1.53 μg/ml. This trend of eco‐friendly stable synthesis of AgNPs could prove a better substitute for the chemical methods and offer greater opportunity to use these nanosilvers in agricultural and biomedical sectors.

1 Introduction

Nanobiotechnology is the integrative field of both nanotechnology and biological sciences, which is highly concerned about the utilisation of biological systems for synthesising biofunctional organic and inorganic nanoparticles (NPs) materialised into nanomaterials. This nanobiotechnology approach to the biology has created a platform to review the cell as a biological molecular tool for preparation of nanomaterial [1, 2, 3]. Biological systems especially plants often feature natural capability in the fabrication of NPs and nanomaterials on its own. The novel characteristics of plant derived molecules can be utilised by the researchers to overcome the distinct impediments associated with synthesis of NPs by chemical and physical methods. This use of plants for the synthesis of NPs is regarded to be more beneficial because of its eco‐friendly and economical attributes. Besides this, the applications of plants for large‐scale synthesis of metal NPs are highly appreciated and beneficial as it produces on less input of energy, low pressure and low temperatures [3, 4]. This biosynthesis technique is the more promotive over physical and classical chemical methods due to inadequacy of toxic by‐products and ensuring the decrease in degradation of the products. Plants can provide an excellent platform for reducing, capping and stabilising properties of synthesised NPs, which confers as an ideal candidate for metal NP synthesis [5]. The relatively higher stability of NPs synthesised by plant sources as compared to other biological sources is the better option in favour of synthesis of silver NPs (AgNPs). Biosynthesis of AgNPs using ethanolic leaf extracts from Callistemon lanceolatus, Decaspermum parviflorum, Eucalyptus citriodora, Melaleuca cajuputi, Rhodomyrtus tomentosa, Syzygiupam campanulatum, and Xanthostemon chrysanthu has been successfully reported having potential antibacterial properties [6]. Moreover, the Tamarix gallica leaf extract was used for synthesis of AgNPs and evaluated for its potential antibacterial activity [7].

AgNPs have immense potential for dynamic utilisation in diverse fields such as textiles, water purification, electronics, cosmetics and medicines because of their exceptional physical, chemical, and biological properties [5, 8, 9]. AgNPs are well notable to possess antibacterial properties which may provide a sustainable explanation to the increasing trend of antibiotic intransigence displayed by pathogenic bacteria [10, 11]. Nanobiotechnology is the only area of application where AgNPs can prove to be valuable to the biomedical sector.

Cleistanthus collinus (C. collinus) (Roxb.) Benth ex Hook. f. is an extremely toxic poisonous plant. The leaf contains toxic principles viz. arylnaphthalene lignan lactones – diphyllin and its glycoside derivatives Cleistanthin A and B [12] and was reported for the anticancer, cytotoxicity and larvicidal properties [13, 14]. It is also used as washing agent for clearing septic wounds, cure fungal and bacterial diseases [15]. The leaves are commonly used for poisoning humans (suicide or homicide) and animals (cattle and fish poison), more specifically as an abortifacient, especially in rural South India [16]. Extracts of leaves, roots, and fruits have been used in acute gastrointestinal disorders [17].

In this study, an attempt has been made to demonstrate the synthesis of AgNPs using C. collinus leaf extract. The structural, morphological, and elemental studies of the synthesised AgNPs were carried out by UV–vis spectroscopy, dynamic light scattering (DLS), Fourier transform infrared (FTIR) and field‐emission scanning electron microscopy (FE‐SEM) analysis, respectively. In vitro antimicrobial activity of synthesised AgNP was carried out against a group of pathogenic microorganism including both Gram‐negative, Gram‐positive and Candida sp., additionally, anticancer activity also been investigated against A431 osteosarcoma cell lines. To our perception, this is a novel approach in synthesis of AgNPs using poisonous plant like C. collinus and evaluation of biomedical efficacy. Hence, the output of this study will be more useful for synthesising the AgNPs and their application in the biomedical sector.

2 Materials and methods

2.1 Chemicals used

The chemicals used during the experiments such as 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT), Mueller Hinton medium and silver nitrate (AgNO₃) were purchased from Hi‐media. Bisbenzimide H 33342, 1% penicillin–streptomycin solution and Dulbecco's Modified Eagle Medium (D‐MEM) supplemented with 10% foetal bovine serum (FBS) were purchased from Sigma‐Aldrich.

2.2 Preparation of leaf extract

Leaves of C. collinus Roxb. (Family – Phyllanthaceae) were collected from the dense forest of Mayurbhanj, Odisha, India. The plant (Voucher specimen No‐NOU 1115) was identified and deposited in the P.G. Department of Botany, North Orissa University. The shed dried fresh leaves were powdered separately using a mechanical grinder and then were passed through sieve (mesh size 40 µm) in order to maintain the uniform powder size. Five grams of leaf powder was added to 50 ml of deionised water and sonicated for 5 min (5000 rpm). The sonicated mixture was filtered through Whatman® grade no. 1 filter paper and stored at 4°C for future use.

2.3 Biosynthesis of AgNPs

A reaction mixture was prepared by adding 1 ml of aqueous leaf extract with 9 ml of 1 mM AgNO₃ solution in a test tube for the biosynthesis of AgNPs. Simultaneously, control was taken by addition of 9 ml sterile deionised water with 1 ml leaf extract [5]. The reaction mixture was kept at different temperatures, pH and incubation time till the turning up the dark reddish brown colour for optimisation of the process. The colour change confirmed the synthesis of NPs and solutions with NPs were centrifuged at 10,000 rpm for 45 min (C24‐BL centrifuge, REMI, India) thrice with successive washing with Mili Q water to expel any trace of un‐exploited leaf phyto‐compounds. The precipitate pellet was lyophilised and kept in 4°C for further characterisation and application studies.

2.4 Characterisation of AgNPs

2.4.1 UV–vis spectra analysis

The UV–vis spectrophotometer (Lambda 35®, Perkin Elmer, Waltham, MS, USA) was employed to study the optimal temperature, pH and time appropriate for the synthesis of AgNPs. The reaction mixture (plant extract and AgNO₃) was monitored regularly by scanning in the wavelength spectrum from 400 to 600 nm.

2.4.2 DLS spectroscopy

Zeta sizer (ZS 90, Malvern Instruments Ltd, Malvern, UK) was employed to study the hydrodynamic size, polydispersity index (PDI) and surface charge of the synthesised AgNPs.

2.4.3 Field‐emission scanning electron microscopy (FE‐SEM)

The surface morphology of the biosynthesised AgNPs was investigated by FE‐SEM (Jeol 6480LV JSM USA). The NPs were properly fixed and coated with platinum using platinum sputter module in a higher vacuum evaporator. The NPs were performed at 20 kV to observe in different magnifications.

2.4.4 Attenuated total reflection Fourier transform infrared (ATR‐FTIR) spectroscopy

The probable part of discrete phytochemicals present in the leaf extract, on the surface alternation of the biosynthesised AgNPs were investigated by ATR‐FTIR spectroscopy (Bruker ALPHA Spectrophotometer, Ettlinger, Germany) with a resolution of 4 cm⁻¹. The samples were scanned in the spectrum ranges from 4000 to 500 cm⁻¹.

2.5 Analysis of phytochemicals

Phytochemical analyses were carried out qualitatively by following the methods described in Govindachari et al., (1969) and Maji et al., (2010) [14, 15].

2.6 Antimicrobial activity

2.6.1 Microbial strains

Four different strains of bacteria viz. Bacillus subtilis (B. subtilis) (MTCC 736), Staphylococcus aureus (S. aureus) (MTCC 737), Pseudomonas aeruginosa (P. aeruginosa) (MTCC 424), Escherichia coli (E. coli) (MTCC 443); three fungal strains viz. Candida albicans (MTCC 227), C. kruseii (MTCC 9215), C. viswanathii (MTCC 1929) and T. mentagrophytes (MTCC 8476) were used as test organisms. The bacterial strains were sub‐cultured on Mueller–Hinton Agar (MHA) while fungus on Potato Dextrose Agar (PDA) and stored in a cold condition (4°C).

2.6.2 Agar well diffusion (AWD) method

The AWD method was used to study antimicrobial activity following standard protocols [16]. The MHA plates were seeded with overnight culture (100 µl) of each test bacterial strains, while PDA was seeded with 200 µl of overnight grown fungal strains. After seeding, wells were punched with a sterile cork borer (8 mm) to make well. Forty microlitres of synthesised AgNPs were filled to each well. The 1 mM AgNO₃ solution without plant extracts serve as a control while standard antibiotics – gentamycin (for bacterial strain) and clotrimazole (for fungal strain) were used as a reference control. After 24 h of incubation at 37°C, the inhibition zones (mm) were measured. Each experiment was done in triplicates.

2.7 Anticancer activity by MTT assay (A‐431 cell line)

2.7.1 Cell culture

The osteosarcoma cell line (A‐431) was procured from National Centre for Cell Science, Pune (India).Cells were seeded in a flask with D‐MEM (Minimal Essential media) medium supplemented with FBS (10%), Foetal Calf Serum (FCS) (2–10%), penicillin–streptomycin solution (1%) and incubated at 37°C in a 5% CO₂. After the incubation period of 24 h, the cells were trypsinised for 3–5 min to form a monolayer and centrifuged (1200 rpm, 6 min). The viability of cells was counted by nucleocounter and again seeded in 96 well microtriter tissue culture plates with 3000 cells/well. The plate was kept for incubation (24 h, 5% CO₂, 37°C) [17].

2.7.2 AgNPs treatment

As our main objective to observe the anticancer activity of biosynthesised AgNPs, the NPs with varying concentrations viz. 0, 50, 100, 150, 200 and 250 μg/ml were treated in triplicates in the wells having grown cells (1 × 10⁴ cells/well) and incubated for 48 h with same experimental conditions optimised for this type of cell line.

2.7.3 Cell viability test by MTT assay

The cell viability was measured MTT calorimetric method. MTT solution of concentration (0.8 mg/ml) was prepared without FBS. The 200 μl of MTT solution was added to each well and further incubated for 5 h. After the incubation, the MTT solution was dispensed with 200 μl of dimethyl sulfoxide (DMSO, molecular grade) in each well followed by incubation of 10–15 min for complete solubilisation of crystals. The amount of formazen was measured at 595 nm in multi‐well spectrophotometer (Biorad, USA).

2.8 Statistical analysis

All experiment sets were carried out in triplicate and the results were given as mean ± standard deviation. The experimental results were also interpreted by using SPSS with acceptance of statistical significance at a level of p  < 0.05 to calculate IC₅₀ values.

3 Results and discussion

3.1 UV–vis spectra analysis

The UV–vis spectroscopy is the most efficient method to monitor the conversion of Ag⁺ from AgNO_3. The change in the colour from light brown to dark brown due to the formation of AgNPs was observed with absorbance maxima in the range of 420–460 nm (Fig. 1). For efficient synthesis of AgNPs separate parameters were taken into account such as temperature, pH and incubation time along with their optimisation. Fig. 1 signifies the gradual momentum of AgNPs synthesis with an increase in the temperature when the reaction mixture was incubated at 25, 40, 60 and 80°C. The significant AgNPs synthesis was optimised and found to be 80°C. Another essential parameter like an incubation period for AgNPs synthesis was monitored after endorsement of optimised temperature (80°C). During the incubation time the absorbance was measured continuously at different time intervals (15, 30, 45, 60, 75 and 90 min), till the manifestation of a broad absorbance peak for the NPs. Within the 90 min of incubation, the appearance of absorption peak at 426 nm, suggested the process to be speedy and is due to the surface plasmon resonance property of AgNPs (Fig. 2). At the same time no colour change was detected in the control without AgNO₃ solution. Hence, the results are vibrant in the rapid synthesis of AgNPs. We also demonstrated how pH plays the crucial role in balancing the reaction conditions during the process of synthesis of AgNPs. The reaction mixture was incubated at an optimised temperature at 80°C and time at 90 min at different pH conditions (pH 2–10) and the rate of AgNPs were monitored. Swift synthesis of NPs was monitored at alkaline pH 8 by UV–vis spectroscopy (Fig. 3); but just in higher alkaline condition the rate of synthesis is decreased. Similar kind of results regarding the effect of alkaline pH in NP synthesis was earlier described and similar findings were also reported by other researchers [18, 19]. The optimised reaction condition for AgNP synthesis was set to be of temperature 80°C, pH 8 and incubation time 90 min.

Fig. 1.

UV–vis spectra of the synthesised AgNPs at different temperature from the leaf extracts of C. collinus

Fig. 2.

UV–vis spectra of the synthesised AgNPs with respect to different time duration at 80°C from the leaf extracts of C. collinus

Fig. 3.

UV–vis spectra of the synthesised AgNPs with respect to different pH at 80°C from the leaf extracts of C. collinus

3.2 DLS studies

The hydrodynamic size, surface zeta potential and PDI of AgNPs were determined by DLS analysis. The hydrodynamic size of the particle was measured by employing the Stokes–Einstein equation, where the translational diffusion co‐efficient of particle is calculated [20]. Fig. 4 a represents the average size of the AgNPs synthesised from leaf of C. collinus (100.8 ± 0.431 nm). To correspond the actual impact of incubation temperature in NP size during the synthesis, the AgNPs formed in different temperature were taken for determination of average hydrodynamic size and surface zeta potential (Figs. 5 a and b). In the graph, it is clearly revealed the continuous increase of surface zeta potential with a decrease in NP size with respect to hike in temperature. This result illustrates the impact of temperature in controlling the NP size. The width of the particle size distribution is determined as PDI, where the PDI value ‘0’ means the monodispersity and ‘1’ means for the polydisporsity nature of particles [21]. Briefly, the PDI is measured from a cumulate analysis of intensity autocorrelation function determined by DLS where the size of a single particle is presumed and a single exponential fit is enforced to the autocorrelation function. The PDI value ‘0’ represents the monodisperse distribution whereas value ‘1’ represents the polydisperse distribution. Fig. 4 b illustrates the surface zeta potential (−19.8 ± 4.03 mV) of the biosynthesised AgNPs. It is one of the crucial parameters for depicting the stability of NPs for different biological interactions in the colloidal environment [22].

Fig. 4.

Dynamic light scattering spectroscopic analysis

(a) Size distribution of synthesised AgNPs, (b) Zeta potential of synthesised AgNPs

Fig. 5.

Effect of different temperatures on synthesis of AgNPs

(a) Mean particle size, (b) Mean surface zeta potential of the synthesised NPs

3.3 FE‐SEM

The surface morphology of AgNPs was determined by FE‐SEM (Fig. 6). The average sizes of the particles were found and the irregularity on the spherical surface of biosynthesised AgNPs was clearly illuminated in the FE‐SEM images.

Fig. 6.

FE‐SEM image of synthesised AgNPs

3.4 ATR‐FTIR analysis

The FTIR spectra of both plant extract and the synthesised AgNPs were recorded in order to identify the functional group of the biomolecules involve in the synthesis and stabilisation of the NPs. Fig. 7 clearly indicates the FTIR peaks at 3740, 2345, 1708, 1529, 1073, and 779 cm⁻¹ for synthesised AgNPs and at 3744, 2202, 1676, and 1199 cm⁻¹ for plant extract. A strong intensive band was detected at around 3740 cm⁻¹ of AgNPs and 3744 cm⁻¹ of plant extract resembling to the O–H stretching, which indicates the existence of polyphenols. The bands at 2325 and 2202 cm⁻¹ which corresponds to C = N stretching (nitriles) for both synthesised AgNPs and plant extract, respectively. A medium band was observed about 1708, 1676 and 1529 cm⁻¹ in synthesised NPs representing the C=O stretching (α, β‐unsaturated aldehydes, ketones) and N–O asymmetrical stretching (nitro compounds), respectively. A single band at 1073 and 1199 cm⁻¹ pointed out the C–N stretching (amine groups) for both cases. The presence of N–H wags at around 779 cm⁻¹ confirms the positive attachment of primary and secondary amines in the AgNPs.

Fig. 7.

FTIR result analysis of AgNPs synthesised by C. collinus, X‐axis represents spectra (wave number cm⁻¹) and Y‐axis represents % of transmission

FTIR bands of both plant extract and AgNPs represent the characteristic functional groups of alcohols, aldehydes, flavonoids, phenols terpenoids, primary and secondary amines as phytochemicals in the leaves of C. collinus that play active participation in the synthesis of AgNPs as strong bio‐reducing agent. Although, the actual mechanism in the synthesis of AgNPs is not well understood, but it has been assumed to be the involvement of free amino groups from the plant extract which may reduce the silver ions and may act as a capping agent for stability [22, 23]. Our present FTIR results also revealed the involvement of free amino acids and the carboxyl groups of proteins act as surfactant on the surface of the NPs results in their stabilisation during the synthesis [24]. Diverse natural reductase enzymes such as NADH‐dependant and nitrate‐dependant reductases exist in the biological system channelise the electron transfer system to produce AgNPs.

3.5 Analysis of phytochemicals and antimicrobial activities

Preliminary phytochemical screening from aqueous extracts of leaves revealed the presence of major phyto‐constituents such as tannins and phenolic compounds, flavonoids, saponins, glycosides and triterpenoids (Table 1). This is in conformity with earlier observations made by Pratheepa [12] and Suman et al. [24]. Their results revealed that the presence of phyto‐constituents in organic solvents is more than the aqueous extracts. Maji et al. [15] studied the antimicrobial activity of C. collinus leaf extracts and reported that different organic and aqueous extracts have antimicrobial activity with a zone of inhibition >11 mm against E. coli (MDR), S. aureus (MDR), Klebsiella pneumoniae, B. cereus, Vibrio cholerae and Candida albicans. However, our study showed that aqueous extract of C. collinus leaf is able to inhibit S. aureus while unable to inhibit E. coli, B. subtilis, P. aeruginosa, Candida sp. and T. mentagrophytes. However, the AgNPs synthesised by leaf extract of C. collinus have strong inhibitory activity against B. subtilis, P. aeruginosa and S. aureus (Fig. 8 and Table 2). Moreover, the antimicrobial activity in terms of percentage (%) of inhibition was also determined with respect to size of AgNPs (Fig. 9). It was shown that the % of inhibition is more with respect to smaller the size of AgNPs. This result indicated that, the small size NPs have high efficiency of antagonistic activity against pathogenic bacteria. This is due to active transport and invading of AgNPs into the cell membrane of bacteria and directly affects the metabolism of the cell. Such antimicrobial activity of biosynthesised AgNPs proved the utility of particles as strong antimicrobial agent against several bacterial pathogens. Similar observations were originated by Suman et al. [24] against bacterial pathogens like E. coli, S. typhi, K. pneumoniae and P. aeruginosa. Though the antimicrobial activity of this plant has been reported by several researchers, but the mechanism of the action of this plant is not yet fully known. Moreover, Pratheepa [12] reported a drug candidate ‘dioctyl phthalate’ by GC‐MS from leaves of C. collinus and reported that the compound is a highly potential therapeutic agent for the people who are suffering from cancer, infected by deadly pathogens. In addition, few more compounds such as ellagic acid, diphyllin (IIIa), cleistanthin and collinusin, have been isolated from C. collinus [14].

Table 1.

Preliminary phytochemical screening from C. collinus leaf extracts

Name of the phyto‐constituents	Observation
alkaloids	—
tannins and phenolic compounds	+++
flavonoids	+++
saponins	++
glycosides	+
steroids and sterols	—
triterpenoids	+++

Fig. 8.

Antibacterial activity of AgNPs synthesised by C. collinus against

(a) P. aeruginosa, (b) S. aureus

Table 2.

Antimicrobial activity (zone of inhibition in mm)

Extract/NPs	B. subtilis	S. aureus	E. coli	P. aeruginosa
aqueous extract	—	10 ± 0.05	—	—
AgNPs	12 ± 1.05	17 ± 1.00	—	24 ± 1.20

Fig. 9.

Antimicrobial activity (% of inhibition) with respect to size of the NPs

3.6 Anticancer activity by MTT assay

AgNPs have promising medicinal effect in breast cancer, skin cancer and wound healing. To test the potentiality of AgNPs as antiproliferative agent, the anticancer study was executed against the A431 osteosarcoma cell line. The cell viability of A431 cell line with treated AgNPs was evaluated by analysing the spectroscopic absorbance data of MTT assay. The (%) inhibitions of the cells treated with discrete concentrations of AgNPs are represented in Fig. 10. The IC₅₀ values were determined to be 84.55 ± 1.52, 91.05 ± 1.53 and 80.271 ± 0.99 μg/ml for the leaf extract, AgNPs and AgNO₃, respectively. The anticancer or cytotoxic activity against A‐431 of biosynthesised AgNPs exhibited nearly similar IC₅₀ values with the results published earlier [25, 26, 27, 28, 29, 30]. The results of this study clearly revealed the significant cytotoxic effect of AgNPs against the osteosarcoma cell lines due to the combinatorial effect of nanosize and bioactive molecules adhered on the surface of the biosynthesised NPs. Deciphering the cellular and molecular mechanism of AgNP towards anticancer activity is highly indispensable for utilising the NPs in cancer treatments.

Fig. 10.

Cytotoxic effect of AgNPs from the leaf extracts of C. collinus against A431 osteosarcoma cell lines

4 Conclusion

The experimental achievement of bioreduction of AgNPs by C. collinus leaf extract without using any chemical entities has a greater advantage of nanosynthesis process being cost effective and its environmental concerns. The respective NPs were found polydispersed in nature. The reducing properties of leaf extract were associated to phyto‐compounds such as phenolic, flavonoids and triterpenoid as evident by the phytochemical analysis before and after synthesis.

The synthesised AgNPs exhibit a strong antibacterial activity against both Gram‐positive B. subtilis and Gram‐negative P. aeruginosa. Besides this, AgNPs also showed potential anticancer activity against osteosarcoma cell line which encourages the application in cancer treatment.