Green synthesis, characterisation and biological studies of AgNPs prepared using Shivlingi (Bryonia laciniosa) seed extract

Abstract

Green synthesis of silver nanoparticles (AgNPs) using Shivlingi (Bryonia laciniosa) seed extract was carried out. Characterisation of synthesised nanoparticles was accomplished through the optical absorption and photoluminescence spectrum, X‐ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The XRD analysis further confirmed the size of nanoparticles ∼15 nm. TEM images revealed homogeneous spherical ∼10 nm Bryonia extract capped AgNPs. The biological studies indicated that both Bryonia seed extract and the nanoparticles lack anti‐microbial activity; however, the nanoparticles had better cytotoxicity and total antioxidant activity. The Lethal concentration (LC)₅₀ value of water extract and the nanoparticles were found to be 1091 and 592 μg/ml, respectively. The lower LC₅₀ of nanoparticles indicates that it is more cytotoxic than the crude extract. The results indicate that the Bryonia seed is safe to be used as a medicine and the formation of their nanoparticle has further enriched the chemical reactivity, energy absorption and biological mobility.

1 Introduction

Shivlingi known as Bryonia laciniosa Linn. (Cucurbitaceae) is categorised as Vrishyarasayana (for maintaining fertility and sexual performance) in Ayurveda. The seeds of B. laciniosa are important constituent of Ayurvedic formulation ‘Strirativallabhpugpak’ described in the ancient text to improve sexual behaviour and are traditionally being used as an aphrodisiac and reproductive tonic [1] and also possesses anti‐inflammatory, anti‐diabetic, anti‐microbial, analgesic and anti‐pyretic activities [2, 3, 4, 5].

Green synthesis of nanoparticles is in vogue these days and the micro‐organisms and plants are replacing expensive, energy consuming and potentially toxic chemical/physical methods for the production of nanoparticles. Plant synthesised green silver nanoparticles (AgNPs) are widely accepted as they are cost effective, bio‐compatible and eco‐friendly. The plant extracts contain various bio‐molecules, which are relentlessly busy in the redox reactions. These reactions act as bio‐reductants for metal ions or provide a platform to direct the formation of metallic nanoparticles in the solution after which the colour of solution changes. This is attributed to the surface plasmon resonance (SPR) phenomenon [6]. Owing to their distinctive properties, nanoparticles have a significant role in the field of medicine, biology, material science, physics and chemistry [7]. During last decade a number of research papers have been published on green AgNPs synthesised using various plants such as Azadirachta indica [8], Cymbopogon flexuosus [9], Aloe vera [10], Camellia sinensis [11], Jatropha curcas [12], Acalypha indica [13] as well as various other plant extracts [14].

B. laciniosa seeds have important medicinal properties as an Ayurvedic medicine but till date they have not been characterised in detail. Thus, in the present paper, we have synthesised the AgNPs using B. laciniosa seed extract and characterised them using optical absorption, photoluminescence, X‐ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The biological activity was also accessed to compare its anti‐microbial as well as cytotoxic potential in both aqueous extract as well as in the synthesised nanoparticles.

2 Material and methods

2.1 Material

The seeds of B. laciniosa were gifted by Divya Pharmacy, Haridwar, India and stored in ambient conditions for further study. The other solvents and chemicals were purchased from Sigma‐Aldrich and S.D. Fine Chemicals, India.

2.2 Methods

2.2.1 Preparation of extract

About 10 g of seeds were milled into a fine powder and boiled for 2 h in 100 ml of deionised water. The extract was filtered and refrigerated (4°C) for further experiments. In each and every step of the experiment, sterility conditions were maintained for the accuracy of the results.

2.2.2 Synthesis of AgNPs

In a typical reaction procedure, 5 ml of seed extract was added to 20 ml of 10⁻² M aqueous Ag nitrate (AgNO₃) solution, the mixture was heated at 80°C for 1 h. Complete reduction of AgNO₃ to Ag⁺ ions resulted in the colour change from yellowish to colloidal brown. The precipitate was collected by filtration, washed with deionised water several times and finally air dried at 60°C for 6 h.

2.2.3 Characterisation of nanoparticles

The prepared AgNPs were analysed and characterised using optical absorption (Shimadzu UVPC‐1601 spectrophotometer, Japan), photoluminescence spectrum (PerkinElmer LS‐55 luminance spectrophotometer, USA), crystalline metallic Ag was examined by XRD (Bruker D8 diffractometer, USA), scanning electron microscopy (SEM) (MIRA 3, TESCAN), TEM (TEM‐TECHANI‐20‐G2), FTIR (Agilent Technologies Cary 630) and Raman spectroscopy (Deitanu Rock Hound Nuspec 2.0, USA).

2.2.4 Anti‐microbial activity

Test micro‐organisms: The micro‐organism used for the test were Escherichia coli (MCC226), Pseudomonas aeruginosa (MCC2035), Staphylococcus aureus (MCC2408), Bacillus cereus (MCC2128), Aspergillus niger (MCC1074) and Candida albicans (MCC1151) procured from NCL (Pune, India).

Experimentation: Nutrient agar plates were prepared and swabbed with bacterial strains by using sterilised cotton swabs. Sample loaded discs were placed on the bacterial strain containing nutrient agar plates. The plates were allowed for diffusion for 30 min and incubated for 24 h. Both positive control (PC) (tetracycline 100 mg/ml) and negative control (distiled water) were placed along with the sample loaded disc. After 24 h, inhibition zones were measured around the sample discs.

2.2.5 Total antioxidant activity

The total antioxidant capacity of the plant extract/fraction was evaluated as described [15]. The diluted sample solution was mixed with 3.0 ml of the reagent solution (600 mM sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The reaction mixture was incubated at 95°C for 60 min and the absorbance was measured at 695 nm against a reagent blank.

2.2.6 Brine shrimp lethality assay

Brine shrimp (BS) eggs were obtained from the Lucky Extra^TM Artemia Cysts. The brine solution was prepared (3.8% sea salt solution) for hatching the shrimp eggs. The brine solution was put in a small plastic container with a partition for dark (covered) and light areas. The shrimp eggs were added to the dark side of the chamber. After 2 days, when the shrimp larvae (nauplii) were ready, brine and 15 BSs were introduced into each vial. The volume was adjusted to 5 ml with the brine and the vials were left uncovered under the lamp. The number of surviving shrimps was counted and recorded after 24 h [16].

The percentage mortality (%M) = number of dead nauplii/total number of nauplii × 100.

3 Result and discussion

3.1 Characterisation of AgNPs

The possible reason for bio‐reduction of Ag⁺ into metallic Ag might be its reaction with different bio‐molecules present in the plant extract (e.g. reducing sugars, proteins, terpenoids, phenolic compounds etc.) [17]. The colour of the reaction mixture changed from yellowish to colloidal brown due to excitation of SPR vibration of AgNPs [6] (Fig. 1).

Fig. 1.

Colour change after the formation of AgNPs

(a) B. laciniosa seed extract (b) After the formation of AgNPs

3.2 Optical absorption and photoluminescence spectrum

The optical absorption and photoluminescence spectrum of Ag nano‐crystals were observed using the aqueous seed extract of B. laciniosa (Fig. 2). Curve (a) shows intense absorption peak of Ag nano‐crystals at ∼420 nm with signs of surface plasmon states ∼ 460 nm. The peak positions of AgNPs in curve (b) exhibited a size dependent red shift but the surface state emission at wavelength ∼580 nm. The excitation wavelength for a AgNP with seed extract at 380 nm. The red shift indicates that the thin film of AgNPs consists of nano‐crystals with an average radius smaller than the Bohr excitation radius. It means that the sample has a relatively narrow size distribution of ∼15 ± 5 nm [18].

Fig. 2.

Optical absorption and photoluminescence spectrum

(a) Optical absorption spectrum (b) Photoluminescence spectrum of AgNPs prepared using the aqueous seed extract of B. laciniosa

3.3 Scanning electron microscopy analysis

Each of the colloidal solution containing AgNPs was centrifuged at 4000 rpm for 15 min, and the pellet was discarded and the supernatants were again centrifuged at 25,000 rpm for 30 min. This time, the supernatants were discarded and the final pellets were dissolved in 0.1 ml of deionised water. The pellet was mixed properly and carefully placed on a glass cover slip followed by air drying. The cover slip itself was used in Scanning electron microscopy analysis. The details regarding applied voltage, magnification used and size of the contents of the images were implanted on the images itself. The self‐organised AgNP thin film gave rise to another interesting feature with smooth surface cluster (Fig. 3). The average smallest cluster size was measured as ∼400 nm, which is in good agreement with XRD and optical absorption spectroscopy results.

Fig. 3.

SEM image of AgNP clusters synthesised using B. laciniosa

3.4 X‐ray diffraction analysis

X‐ray diffraction analysis of the prepared sample of AgNPs was done using a Bruker D8 diffractometer, Cu‐Kα X‐rays of wavelength λ  = 1.54056 Å and data was taken for the 2θ range of 35–70° with a step of 0.02°. The X ray diffraction patterns of synthesised AgNPs revealed a crystalline, face‐centred cubic geometry ( Fig. 4 ). The diffraction peaks obtained at 2θ  = 38.04° (111), 46.14° (200) and 63.13° (220) are identical with those reported for standard Ag metal (JCPDS, USA). Similarly, the diffraction pattern of AgNPs revealed the existence of peaks (111), (200) and (220) matched with the standard JCPDS data file # 04784 [19]. The synthesised AgNP had a normal size distribution of ∼15 ± 5 nm using Scherrer equation.

Fig. 4.

XRD patterns of AgNPs synthesised using B. laciniosa

3.5 Transmission electron microscopy

A typical dark and bright field image of nano‐crystals have been shown in Figs. 5 a and b. The average particle size of the nano‐crystals was estimated as ∼10 ± 2 nm, which is in good agreement with the XRD analysis results. The size distribution histogram of nano‐crystals has been shown in Fig. 5 c. The TEM image revealed the homogeneous spherical shape of Bryonia extract (BE) capped AgNPs. It could be possible because of the capping agent that these AgNPs are showing spherical and clustered shape geometry [20].

Fig. 5.

TEM image of AgNPs synthesised using B. laciniosa

(a) Dark field TEM image, (b) Bright field TEM image, (c) Particle size distribution histogram of 8 nm AgNPs

3.6 Fourier transform infrared spectroscopy

The FTIR measurement was studied to identify the possible bio‐molecules responsible as capping and reducing agent for the AgNPs synthesised by the extract (Fig. 6). The intense broad bands at 3324 and 2929 cm⁻¹ are probably due to the O–H and C–H stretching modes, respectively. The AgNP derivatised extract did not show any band at 1080 cm⁻¹, whereas the original extract showed the prominent band at the same frequency indicating that the AgNPs have stabilised through the C–O bond. Similarly, the absence of a peak at 1408 cm⁻¹ (C=C group) in the AgNP derivatised extract is probably due to the reduction of AgNO₃ to Ag [21].

Fig. 6.

Fourier transformation infrared spectra

(a) AgNPs derivatised B. laciniosa seed extract, (b) B. laciniosa seed extract

3.7 Raman spectroscopic analysis

As an important technique Raman spectroscopy (Deitanu Rock Hound Nuspec 2.0) can be used to study chemical identification, characterisation of molecular structures, effects of bonding, environment and stress of the compounds. In Raman spectrum of the AgNP derivatised with Bryonia seeds, four intense peaks centred at 2 ± 7, 332, 582 and 788 cm⁻¹ are evident (Fig. 7). Annealing at a high temperature induces dissolution of oxygen in the Ag. The peak at 560 cm⁻¹ can be assigned to dissolve atomic oxygen in AgNPs. The bands at 582 and 788 cm⁻¹ can be ascribed to the ν (Ag–O) vibrations for sub‐surface species and ν (O–O) mode for adsorbed molecular oxygen.

Fig. 7.

Raman Spectra of AgNPs synthesised by treating B. laciniosa seed extract with aqueous 10⁻² M AgNO₃ solution

Although, sub‐surface species might promote adsorption of molecular oxygen, it could also be a probable consequence of surface restructuring in AgNPs [19, 20, 22].

3.8 Anti‐microbial activity

Water extract and AgNPs of B. laciniosa seeds were analysed for anti‐microbial activity using different bacterial and fungal strains. However, no clear zone of inhibition was observed in either case. This suggests that B. laciniosa seed extract lacks anti‐microbial activity (Tables 1a and b). Similar results were obtained for seeds when researchers compared the anti‐microbial activity of various parts of B. laciniosa [23]. The results obtained were in good agreement with Minimum Inhibitory Concentration (MIC)/Minimum Bactericidal Concentration (MBC) tests, where the clear zone of inhibition was observed for AgNPs up to 10 nm size but not for larger nanoparticles [24]. Moreover, the release of Ag from large nanoparticles is stated to be lower in the solid culture media [25].

Table 1a.

Anti‐microbial activity of B. laciniosa seed extract.

Micro‐organism	Concentration (mg/ml)
	10		30		60		100
	BE (mm)	PC (mm)	BE (mm)	PC (mm)	BE (mm)	PC (mm)	BE (mm)	PC (mm)
E. coli	nil	31.9	nil	22.9	nil	26.0	nil	21.4
P. aeruginosa	nil	43.6	nil	45.6	nil	20.8	nil	37.4
S. aureus	nil	39.8	nil	38.2	nil	42.0	nil	35.2
B. cereus	nil	42.8	nil	40.7	nil	32.0	nil	41.8
A. niger	nil	42.5	nil	39.3	nil	32.6	nil	40.1
C. albicans	nil	16.6	nil	12.0	nil	9.2	nil	11.0

Table 1b.

Anti microbial activity of nanoparticles prepared using B. laciniosa seed extract.

Micro‐organism	Concentration (mg/ml)
	10		30		60		100
	NP (mm)	PC (mm)	NP (mm)	PC (mm)	NP (mm)	PC (mm)	NP (mm)	PC (mm)
E. coli	nil	39.5	nil	35.0	nil	37.0	nil	38.5
P. aeruginosa	nil	42.3	nil	37.5	nil	40.0	nil	25.3
S. aureus	nil	46.4	nil	42.9	nil	36.3	nil	38.0
B. cereus	nil	41.3	nil	41.0	nil	40.4	nil	35.6
A. niger	nil	36.6	nil	44.4	nil	51.3	nil	46.7
C. albicans	nil	9.5	nil	11.5	nil	8.9	nil	8.9

BE = Bryonia extract; PC = positive control (tetracycline 100 mg/ml); NP = nanoparticle.

3.9 Total antioxidant activity

The total antioxidant activity of B. laciniosa seed extract was increased from 2.087 ± 1.02 to 3.33 ± 0.22 mg by the formation of AgNPs. The increased antioxidant activity of nanoparticles as compared with the crude extract has been observed in a number of studies [26, 27]. AgNPs might act as electron donors, which transforms free radicals to the more stable product. Also, the reducing power of Ag nanoparticles is associated with the radical scavenging activity [28].

3.10 BS lethality assay

To study the pharmacological properties of B. laciniosa seed extract, BS lethality bioassay was performed which is based on the ability to kill laboratory cultured BS. In the present paper, different concentrations of B. laciniosa seed extract, as well as the synthesised nanoparticles, were used to access their cytotoxicity using BS lethality assay. The LC₅₀ values of water extract (1091 µg/ml) and the nanoparticles (592 µg/ml) were calculated using Probit analysis. The lower LC₅₀ of nanoparticles indicates that it is more cytotoxic than the crude extract. The criteria of toxicity followed was LC₅₀ values >1000 μg/ml (non‐toxic), ≥500≤1000 μg/ml (weak toxicity) and <500 μg/ml (toxic) [29, 30]. A significant lethality in BS assay is an indicator of effective cytotoxic constituents in the extract [31]. However, to explore the mechanism of cytotoxicity, various bio‐active constituents have to be identified.

4 Conclusion

We have synthesised the AgNPs using the aqueous seed extract of B. laciniosa and characterised them using various techniques. All the analyses (optical absorption and photoluminescence spectrum, SEM, XRD, TEM, FTIR and Raman) confirmed the formation of AgNPs. The optical absorption showed an intense peak of Ag nano‐crystals at 420 nm with signs of surface plasmon states near 460 nm. The self‐organised AgNP thin film has indicated the smooth surface cluster having an average smallest cluster size of 400 nm. The XRD analysis further confirmed the size to be ∼15 ± 5 nm. TEM images revealed homogeneous ball shaped ∼10 ± 2 nm BE capped AgNPs. The Raman spectroscopy indicated that the bands at 582 and 788 cm⁻¹ are due to the stretching of ν (Ag–O) vibrations for sub‐surface species and stretching ν (O–O) mode for adsorbed molecular oxygen, which endorses the formation of AgNPs. The biological studies indicated that both Bryonia seed extract and the nanoparticles lack anti‐microbial activity; however, the nanoparticles had better cytotoxicity and total antioxidant activity. The LC₅₀ value of water extract and the nanoparticles were found to be 1091 and 592 µg/ml, respectively. The lower LC₅₀ of nanoparticles indicates that it is more cytotoxic than the crude extract. This will be advantageous if it is ingested as a drug, because it may not harm microbial flora of the gut. In addition, the better total antioxidant activity and cytotoxicity of the nanoparticles to seed extract suggest an important role of nanoparticles in anticancer drug development.