Green synthesis of silver nanoparticles using Indian male fern (Dryopteris Cochleata), operational parameters, characterization and bioactivity on Naja naja venom neutralization

Abstract

Snakebite is considered as one of the acute severe medical problems across the world. Snake venoms composed of various group of toxins, enzymes and non-toxic enzymes. Phospholipases A₂ present in Naja naja snake venom plays a significant role in lipid signalling and contributes to different inflammation in the human body. Dryopteris cochleata rhizomes have antioxidant, antimicrobial property and used to treat lesions, gonorrhoea, sores, muscular pain, rheumatic and also useful in dog and snake bites. In this study, Indian male fern D. cochleata rhizomes have been used for green synthesis of silver nanoparticles with the aim to increase the bioactivity of plant extract and to evaluate N. naja snake venom inhibition activity of prepared nanoparticles. Green synthesized nanoparticles were characterized with the help of ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray powder diffraction and atomic force microscopy. Naja naja venom inhibition activity of nanoparticles was performed using in vitro phospholipases A₂ assay and tissue damage activity. The results showed that surface plasmon resonance maxima peaks of nanoparticles were observed at 424 nm. Average particle size was around 35 nm, with a spherical shape. Neutralization results exhibited that synthesized silver nanoparticles from D. cochleata decreased percentage of tissue damage, resulting in significant inhibition of phospholipase A₂ and N. naja snake venom. Results concluded that green synthesized silver nanoparticles from D. cochleata rhizome neutralize N. naja snake venom activity.

Graphical Abstract

Graphical Abstract.

Introduction

Snake envenomation is a serious problem of morbidity and mortality, especially in Asia. World Health Organization recognized snakebite as ‘Neglected Tropical Diseases’, but snakebite is an acute medical emergency in rural and tribal areas of India, Bangladesh, Nepal, etc. In India, ~35 000–50 000 deaths are supposed because of snakebite every year [1].

Naja naja snake venom contains different compounds, typically proteins, polypeptides and enzymes [phospholipase A₂ (PLA₂), hyaluronidase, L-amino acid oxidase, phosphodiesterase and acetylcholinesterase] and numerous other substances with poisonous and fatal properties [2]. PLA₂ catalyzes the hydrolysis of middle position (sn-2) of membrane glycerophospholipids to free arachidonic acid and metabolized to form numerous eicosanoids and related bioactive lipid mediators [3, 4]. Eicosanoids and platelet-activating factor are main mediators of inflammation and other pathophysiological conditions such as myotoxicity, necrosis, hypotension, haemolysis, anticoagulation, haemorrhage and plasma leakage [5].

Nanobiotechnology is a fast-developing field and has the unique potential to develop new therapeutic agents [6]. Silver nanoparticles (SNP) synthesized by green synthesis utilize medicinal plants or extracts and have little or no toxicity [7]. Secondary metabolite plays an essential role during the green synthesis of SNP by acting as reducing or capping agents [8–10]. Previous studies have revealed that metal nanoparticles are highly stable and applicable to snake venom inhibition [11–13].

Even these days, individuals depend on natural medicine systems for their major health care, including snakebites. Phytoconstituents present in traditional medicine act against various effects induced by snakebite. Therefore, the continuous search and identification of new compounds that may be useful as alternative or complementary therapies for snakebite envenomation is a relevant task [14, 15]. Indian male fern Dryopteris cochleata (D. Don) C. Chr. belongs to Dryopteridaceae family and generally known as Jatashankari (vernacular name) [16]. Dryopteris cochleata rhizome has been commonly used in epilepsy, leprosy, ulcers, wound healing, blood purification and also has antifungal property. Traditionally fresh paste or juice and fronds are applied externally at dog and snakebites [17, 18]. Our previous studies show that the extract of D. cochleata rhizomes has neutralized activity against toxic enzymes of snake venom [19]. In the present study, D. cochleata rhizomes have been used for the green synthesis of SNP, and the synthesized nanoparticles are evaluated for anti-snake venom (ASV) (N. naja) activity.

Methods

Lyophilized venom of N. naja (Indian cobra) was procured from Irula Snake Catchers’ Industrial Cooperative Society, Kanchipuram District Chennai, Tamil Nadu, India. Polyvalent antivenom (as standard drug) was procured from VINS Bioproducts Ltd, Hyderabad, India (Antiserum Snake Venom I.P.). The rhizomes of D. cochleata was collected from Amarkantak, Madhya Pradesh, India, and taxonomic identification of this plant was made by the Department of Botany, Vindhya Herbal Testing and Research Laboratory, Bhopal, Madhya Pradesh, India (Sample LRN No. VHTRL30101801ER). Silver nitrate, PLA₂, cetyltrimethyl ammonium bromide, lecithin were purchased from Sigma-Aldrich Laboratories, Merck India Ltd. Remaining all other reagents were of analytical grade.

Preparation of D. cochleata extracts

The rhizomes of D. cochleata were collected, washed, shade dried, grounded into coarse powder and extracted by absolute ethanol through Soxhlet apparatus. The obtained extract was then stored in a cool and dry place until further use [20].

Green synthesis of SNP

For the green synthesis of SNP, a stock solution of silver nitrate 1 M/ml (AgNO₃) and 1 mg/ml plant extract were prepared. The experimental mixture was observed for colour change depending on different parameter studied such as silver nitrate concentration (0.1–10.0 ml), extract concentration (0.1–5.0 ml), temperature (25–80°C) and time interval until the colour of the solution slowly turned from yellow to dark brown [6, 21]. The product was then centrifuged at 13 000 rpm for 20 min and dried and stored at 4°C for further use [8].

Characterization of SNP

Ultraviolet–visible spectral analysis

Ultraviolet–visible (UV–Vis) spectroscopy is a preliminary technique and simplest approach to confirm the green synthesis of SNP. UV–Vis absorption spectra of prepared SNP were measured at different silver nitrate concentrations, plant extract concentrations, temperature and time interval over a wavelength range of 340–700 nm using Microplate Spectrophotometer (Biotek: Epoch2) with deionized water as a reference [8].

Fourier-transform infrared analysis

Fourier-transform infrared (FTIR) spectra of extracts and SNP samples were analysed by FTIR spectroscopy (Shimadzu: Spectrum IRAffinity-1). The FTIR analysis was performed with KBr pellets and recorded in the range of 400–4000/cm. FTIR analysis was carried out to identify functional group involved in nanoparticles synthesis [22].

X-ray powder diffraction studies

The green synthesized SNP were characterized using powder X-ray diffraction (XRD) (Rigaku: MiniFlex) to confirm particles as silver and to identify the structural information. The lyophilized SNP coated on XRD grid were subjected to XRD analysis. The XRD pattern was scanned in the 2θ range from 20 to 80° with a step size of 0.04°/s.

Atomic force microscopy analysis

Atomic force microscopy (AFM) (NT-MDT: Solver Next) was used to analyse SNP. A thin film of synthesized nanoparticles was deposited on a glass plate and allowed to dry at room temperature. The spread thin-film glass plate was then scanned with the AFM (ND-MDT) [22].

PLA₂ enzyme neutralization assay

PLA₂ enzyme inhibition activity of green synthesized SNP was evaluated by minimum indirect haemolytic dose (MIHD) assay [23]. The PLA₂ inhibition ability of SNP was carried out by mixing 50 μl of nanoparticles in different concentration ranges from 0.1 to 0.4 mg/ml with 50 μl of 0.01 mg/ml PLA₂, and then the reaction mixture is incubated for 1 h at 37°C. About 10 μl of pre-incubated nanoparticles–PLA₂ mixture was added to agarose gels well (1% agarose gels mixed with 1.2% sheep erythrocytes, 1.2% egg yolk and 10 mM CaCl₂). Plates were incubated at 37°C for 20 h and haemolytic halo diameter was measured. Control wells contained PLA₂ (0.01 mg/ml) and compared with extract PLA₂ mixture and PLA₂ enzyme. Neutralization assay was performed in triplicate.

Naja naja venom inhibition assays

Naja naja venom inhibition activity was evaluated by the MIHD assay [23, 24]. The N. naja venom inhibition ability of D. cochleata SNP was carried out by mixing 50 μl of different concentration of SNP (0.1–0.4 mg/ml) with 50 μl of 0.05 mg/ml N. naja venom and then incubated for 1 h at 37°C. Pre-incubated nanoparticles–venom mixture of 10 μl was added to agarose gels well. Plates were incubated at 37°C for 20 h and haemolytic halo diameter was measured. Control wells contained only N. naja venom (0.05 mg/ml) and compared with antiserum snake venom (standard drug). Inhibition assay was performed in triplicate.

In vitro tissue damaging activity

In vitro tissue damaging activity was performed by a previously described method [25, 26] with some modifications. Fresh chicken heart, lungs and liver were collected from the chicken shop and washed with normal saline. About 150 ± 10 mg of fresh tissues pieces were pre-incubated with 1 ml of 0.2 M potassium phosphate buffer, pH 7.4, for 45 min at 37°C. Then tissues pieces were incubated with N. naja venom in a final volume of 1.25 ml in 0.2 M potassium phosphate buffer, pH 7.4, for 5 h at 37°C. After incubation, reaction mixtures were centrifuged for 5 min at 3000 rpm, and absorbance of the supernatant was read at 540 nm by UV–Vis spectrophotometer. The percentage of haemoglobin released was calculated with respect to tissues incubated with 0.1% Triton X-100 (100% activity). For neutralization assay, 20 μl of N. naja venom (10 μg) were pre-incubated with 20 μl of SNP of D. cochleata (1 mg/ml) and D. cochleata extract (10 mg/ml) for 60 min at 37°C. Then the reaction mixture was mixed with all fresh tissues pieces and analysed as per the above-described method. In vitro tissue damaging activity was performed in triplicate.

Results

Characterization of green synthesized SNP

Silver nitrate aqueous solution has a transparent colour, which changed into yellowish brown on mixing with D. cochleata extract and indicate biosynthesis of SNP.

UV–Vis spectral analysis

UV–Vis absorption spectrum of the reaction mixture at different silver nitrate concentration (0.1–10.0 mM) was recorded (Fig. 1A). About 2 mM silver nitrate produce sharp surface plasmon resonance (SPR) compared to other concentrations. Colour changed in the reaction mixture can also be observed visually. Effect of D. cochleata extract at various concentrations from 0.1 to 5 ml on nanoparticle synthesis was observed; 0.5 ml extract increases the absorbance intensity from 420 to 440 nm (Fig. 1B). Figure 1C and D revealed the spectra of nanoparticles obtained at different temperature and time interval.

Figure 1.

Characterization of green synthesized silver nanoparticles. Ultraviolet–visible absorption spectra of synthesized silver nanoparticles at different silver nitrate concentration (A), different volume of extract solution (B), different temperature (C) and different time intervals (D). Inset showing colour change of reaction mixture. Fourier-transform infrared spectroscopy spectra of synthesized silver nanoparticles and D. cochleata extract (E). X-ray diffraction patterns of synthesized silver nanoparticles (F).

FTIR analysis

FTIR spectroscopy performed in the frequency range of 400–4000/cm confirms the presence of different functional groups on SNP. In SNP, peaks are observed at 1440, 1605, 1711, 2852 and 2924/cm, respectively (Fig. 1E).

XRD studies

The XRD pattern shows a lattice planes structures of biosynthesized SNP. The XRD pattern of biosynthesized SNP revealed three intense peaks in the spectrum of 2θ value ranging from 20 to 80° (Fig. 1F).

AFM analysis

AFM is one of the foremost tools for imaging and shows surface morphology and size distribution of biosynthesized SNP. AFM also revealed a capability of 3D visualization and both quantitative and qualitative observation on various physical properties including surface texture, size, morphology and roughness. Particles size of SNP of D. cochleata was around 35 nm and the shapes of the particles are spherical (Fig. 2).

Figure 2.

Atomic force microscopy (AFM) analysis of biosynthesized silver nanoparticle. Grains of biosynthesized silver nanoparticle from D. cochleata extract observed by AFM for particles size distribution analysis (A), silver nanoparticle size distribution pattern (B) and shape of biosynthesized silver nanoparticles (C and D).

PLA₂ enzyme neutralization assay

We investigated the bioactivity of D. cochleata and its nanoparticles by testing PLA₂ enzyme neutralization ability. The petri dishes were assessed for an MIHD halo around each sample of D. cochleata extract and D. cochleata nanoparticles. Each halo was measured physically with a ruler. As shown in Fig. 3A, halo diameter of D. cochleata SNP (0.4 mg/ml) and D. cochleata extract (1 mg/ml) significantly decreased to 1.47 and 8.75 mm when compared with halo diameter 12.89 mm of PLA₂ (0.01 mg/ml).

Figure 3.

In vitro inhibition assays by minimum indirect haemolytic dose of D. cochleata synthesized silver nanoparticle and extracts. Phospholipase A₂ inhibition assays (A). Naja naja venom neutralization activity (B). Data represent three independent experiments and are presented as the mean SD (n = 3). ***P < 0.001 is significant compared with D. cochleata extract.

Naja naja venom neutralization assays

We further investigated the N. naja venom neutralization activity by indirect haemolysis method. The effect of different concentrations of biosynthesized SNP of D. cochleata on indirect haemolysis of erythrocyte membrane phospholipids by N. naja venom is shown in Fig. 3B.

In vitro tissue damaging activity

The D. cochleata extracted and biosynthesized SNP were evaluated through in vitro tissue damaging activity. Inhibition studies were performed by incubating the venom with D. cochleata extract and its SNP prior to testing (pre-incubation method). The results showed that SNP of D. cochleata significantly decreased the release of haemoglobin in the heart from 29.3 to 11.6%, lungs from 45.0 to 9.5% and liver from 37.2 to 8.4% (Fig. 4).

Figure 4.

In vitro tissue damaging activity (%Hb released by 10 μg N. naja venom) by biosynthesized silver nanoparticle from D. cochleata. Data represent three independent experiments and are presented as the mean SD (n = 3). ***P < 0.001 is significant value.

Discussion

Snakebite is a severe medical, economic and social problem in the various rural and tribal regions in India. Snakebites are usually treated by parenteral administration of horse- or sheep-derived polyclonal antivenoms (ASV). This successful approach, though, still presents certain drawbacks due to its high cost, lack of availability, a difficulty for rural patients to access ASV and rapid local tissue damaging effects of snake venoms [27]. Therefore, medicinal plant extracts develop a capable alternative treatment, either alone where antivenom immunotherapy is not carried out promptly or as a beneficial supplement [28]. It also stated that metal associate with whole-plant extracts would improve and potentiate the activity of the phytoconstituents [29]. Metal nanoparticles and nano-based bioproducts are currently applied in the area of biomedicine and also presented capable drug molecules against snakebites [11, 30].

SNP were synthesized by green synthesis approach using D. cochleata rhizome extract as reducing agents. The reaction mixture in the synthesis process was optimized to reach better yield, size and stability by UV–Vis absorption spectrum at different silver nitrate concentration, various concentrations of extract, different temperature and time interval.

Synthesized nanoparticles was finally prepared by adding 2 mM silver nitrate in 0.5 ml extract incubated for 150 min at 60°C showing SPR at 424 nm [8], which reflects successful preparation of nanoparticles [8]. Furthermore, the green synthesized SNP were conformed through FTIR, XRD and AFM analyses. In FTIR analysis, the peak showed at 1440/cm, which corresponds to O–H bending vibration in the carboxylic group and flavanones. The strong, intense peaks at 1605/cm are assigned to C=C stretching in the aromatic ring confirmed the presence of the aromatic group on the surface of nanoparticles [31]. Strong, intense peaks appearing at 1711/cm corresponds to C=O stretching present in the conjugated aldehyde. Broad band appeared at 2852/cm, which corresponds to O–H stretching in carboxylic acids, and 2924/cm is due to C–H stretching vibration in an alkane. XRD analyses show some Bragg reflections with 2θ values of 32.52, 37.56 and 46.26 sets of lattice planes were detected, which may be indexed to (111), (200) and (222) facets of silver, respectively. AFM revealed a capability of 3D visualization of SNP of D. cochleata was around 35 nm and the shapes of the particles are spherical.

The inhibition of N. naja snake venom and PLA₂ activity of biosynthesized SNP of D. cochleata was evaluated through MIHD assay and in vitro tissue damage activity. PLA₂ neutralization not only confirmed the successful association of plant extract but also showed that biosynthesized SNP have potent PLA₂ neutralizing activity than a whole-plant extract. Dryopteris cochleata SNP (0.1 mg/ml) decreased halo diameter from 16.26 to 5.72 mm, resulting in significant neutralization of N. naja venom. However, increasing the amount of D. cochleata SNP progressively increases N. naja venom neutralization in a concentration-dependent manner. Other studies have shown parallel results of effect of snake venoms to tissue damage. A detailed study on local tissue damaging venoms has been done against several plant extracts for the effective neutralization of Bitis snakes. This envenomation causes local damage, hypotension, coagulopathy, thrombocytopenia and spontaneous local bleeding, which may be fatal [32]. In normal human keratinocytes, the cytotoxic effect of Lachesis muta muta venom have been studied using in vitro methods to identify the cellular processes involved in in cellulo envenomation [33]. In vitro tissue damaging activity of N. naja venom was shown by haemorrhagic potential, the most common PLA₂ and protease of Indian venom have cause tissue (lung and liver) haemorrhage [25]. The venom releases its endogenous catabolic enzymes, which disrupt the extra cellular matrix to allow the envenomation [34]. In our study, the in vitro tissue damaging activity of the crude venom was found to vary with the tissue samples used in this experiment, where maximum damage in terms of haemoglobin release was observed in chicken lung tissue strips followed by liver and heart.

Conclusion

The results have given a new aspect in green SNP research, which may substantiate to be prevailing in the field of developing neutralizing antidote against snake bites. Our studies concluded that the activity of D. cochleata extract as antivenoms were increased to 10-fold when it was administrated in the form of D. cochleata nanoparticles. This study also explored to understand the practical applicability of SNP as an alternative therapy for neutralization of snake venom.

Funding

This work was supported by the Council of Scientific and Industrial Research, New Delhi, India (project number 02(0187)/13/EMR-II) for financial support.

Conflict of Interest

None declared.