Comparative assessment of green and chemically synthesized glutathione capped silver nanoparticles for antioxidant, mosquito larvicidal and eco-toxicological activities – Scientific Reports

Synthesis and characterization of AgNPs

This study was aimed for the development of stable, feasible and eco-friendly AgNPs, with good bioactivity. This was done in two ways—preparing AgNPs using clove, as a green approach, using plant material and GSH capping for surface modification of chemically synthesized AgNPs for making them effective, safe and stable. Synthesis of clove AgNPs was visualized through a distinct change in colour of AgNO₃ solution from colourless to dark brown with the addition of aqueous clove extract. Eugenol (4-allyl-2-methoxyphenol), which is the main compound found in clove extract, is responsible for the reduction of silver nitrate and formation of clove AgNPs. A proton is released from OH group of eugenol, creating negative charge on it. The release of two electrons from resonating structure of anionic form of eugenol in this process are responsible for the reduction of 2 Ag+ ions to 2 Ag0. Eugenol, also acts as a capping agent and stabilizes the formed AgNPs¹⁶.

For the nanoparticles prepared using sodium borohydride, colour change from transparent to yellow indicated the formation of AgNPs. Herein, sodium borohydride facilitated the reduction of AgNO₃ solution. While for the synthesis of GSH-capped AgNPs, dark yellow colour after addition of capping agent-GSH, indicated the completion of capping process. The surface of AgNPs is positively charged, while the acetyl (–COO) and thiol (–SH) group of GSH are negatively charged. So, the stabilization of [Ag(GSH)] is due to formation of van der walls forces between these opposite charges. Ionic surface stabilizing agent adsorbs on the surface of AgNPs and generates uniform charge on particle surface, this results in the formation of uniform and stable size particles, with no aggregation¹⁷.

UV–Vis spectrophotometric analysis

Synthesis of AgNPs was monitored using UV–Vis Spectral analysis. Spectral peaks of synthesized AgNPs are shown in Fig. 1. Spectral peak of green synthesized AgNPs using clove extract was recorded at 439.2, while borohydride AgNPs showed a distinct peak at 388 nm. The spectral peak of GSH-capped AgNPs was observed at 400.8 nm. Our spectral study for GSH- capped is in line with the earlier reported work¹⁸. AgNPs exhibit a characteristic surface plasmon resonance (SPR) band at or around 400 nm. Spectral peaks of all three types of AgNPs in our study were near or around 400 nm, confirming the formation of AgNPs.

DLS and zeta potential analysis

DLS-size distribution and zeta potential studies were done to examine the hydrodynamic size, type of charge and stability of synthesized AgNPs, as shown in Fig. 2. Average hydrodynamic size for clove AgNPs was 100.0 nm, while for chemically synthesized borohydride AgNPs, the size was found to be 136.4 nm. GSH-capped AgNPs size was found to be 173.3 nm, which was larger as compared to the size of borohydride AgNPs. The pdI values of clove, borohydride and GSH-capped AgNPs were measured as 0.36, 0.52 and 0.41 respectively indicating their polydisperse nature.

DLS- Particle size distribution and Zeta potential of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.

Stability of all three types of synthesized AgNPs was examined through zeta potential. For AgNPs to be stable the value of zeta potential should be around ± 30 mV. The values obtained for clove AgNPs, borohydride AgNPs and GSH-capped AgNPs were as follows: − 28.5 mV, − 18.6 mV and − 23.5 mV. All the values were negatively charged and sharp, showing the surface of AgNPs to be negatively charged and dispersed in medium. The negative value also shows high electrostatic repulsion among the nanoparticles, thus preventing agglomeration and making them highly stable. All three types of AgNPs exhibited high zeta potential, however, clove AgNPs were found to be most stable, with highest zeta potential value. Moreover, zeta potential value of GSH-capped AgNPs was higher compared to borohydride AgNPs clearly indicating the importance of capping in adding stability to AgNPs.

XRD analysis

Figure 3 shows the XRD data, which confirms the crystalline nature of the synthesized AgNPs. XRD data shows the diffraction peaks at 38.09°, 44.59° and 64° corresponding to (111), (200) and (220) planes of face-centred cubic crystal structure of metallic silver. This was found with all three types of synthesized AgNPs.

XRD diffraction spectra of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.

FE-SEM and EDX analysis

FE-SEM clearly shows the shape and morphology of synthesized AgNPs, as shown in Fig. 4. The clove AgNPs were roughly spherical and evenly distributed. Few rod-shaped individual particles were also seen in clove AgNPs, but majority of particles were spherical in shape (Fig. 4a). Borohydride AgNPs were roughly spherical and aggregated (Fig. 4b). While chemically capped AgNPs were spherical, evenly distributed with small size (Fig. 4c). EDX analysis revealed presence of silver in all three types of synthesized AgNPs, Ag element shows a characteristic signal peak at 3 keV, due to surface plasmon resonance, which indicates the presence of silver in the system¹⁹. Silver (Ag) representative signal peak was observed near 3 keV (Lα) in all the AgNPs under study. Figure 4 also shows the quantitative element details of different AgNPs. The presence of elements apart from main Ag element like Na, Ca and Mg in clove AgNPs could be accounted to secondary metabolites present in clove extract, which are very crucial for nanobiosynthesis of AgNPs. Similar results have been reported for AgNPs synthesized using Pedalium murex leaf, which also had presence of elements like O, C, K, Ca, Cl and Na. This may be accounted for metabolites and functional moieties present in Pedalium murex leaf extract used for preparing AgNPs²⁰. Apart from this presence of C and O element in borohydride and GSH-capped AgNPs could be accounted to the grid used in FE-SEM study.

FE-SEM and EDS-spectra confirming presence of silver in AgNPs of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.

TEM analysis

TEM was done to study in details, the size and distribution of particles. TEM micrographs and the corresponding particle size distribution histogram of AgNPs obtained by TEM images are presented in Fig. 5. TEM micrographs of clove AgNPs clearly showed spherical shape AgNPs, with size around 28 nm (Fig. 5a). Similar size range was also found in AgNPs prepared using extracts of Tectona grandis seeds extract²¹. Possible reasons for the similar size range could be accounted to similar concentrations of extract used in preparation of AgNPs and presence of various phytopotentials moieties in the prepared AgNPs, due to the use of green compounds. Chemically synthesized AgNPs were spherical measuring 7 nm (Fig. 5b) whereas, GSH-capped AgNPs revealed spherical particles with slight aggregation measuring around 36 nm (Fig. 5c). The comparison between the sizes of borohydride AgNPs and GSH-capped AgNPs, clearly shows the role of GSH capping in enhancing the particle size.

Transmission electron micrographs and size distribution histograms of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.

Results of particle size obtained from DLS studies were larger compared to their size revealed through TEM results. Our results found correlation with study on AgNPs prepared using Pedalium murex leaf extract, in which DLS size was 23 nm larger than the TEM size of prepared AgNPs²⁰. In another case, of Zirconia nanoparticles, the difference in size of nanoparticles given by DLS was higher by a factor of two to three compared to size analyzed for same nanoparticles by TEM technique²². Larger size found in DLS studies compared to TEM could be explained with the fact that TEM analysis delineates actual particle size while DLS provides hydrodynamic size representing a sum of particles, hydration sphere and surfactant agent shell coverage hence, relatively larger size of nanoparticles. Moreover, DLS is measured in solution form, with a solvent layer, and thus has tendency to aggregate, while for TEM analysis samples are dried, and thus provides actual size.

FTIR analysis

Figure 6 presents FTIR analysis which provides information about different functional groups and this spectral study is important for deciphering interactions resulting in reduction, adsorption and formation of AgNPs. Analysis was done in the spectral window of 500 to 4000 cm⁻¹. FTIR spectra of clove AgNPs was done to elucidate knowledge about the chemical changes occurring in functional moieties of phytopotentials of clove bud extract, upon being adsorbed on the AgNPs surface and their participation in bioreduction of silver metal. A small peak was recorded at nearly 3700 cm⁻¹ corresponding to O–H stretching of free hydroxyl groups of alcohol and phenols. Apart from this, a broad peak was also observed between 3550 and 3200 cm⁻¹ corresponding to intermolecular bonded O–H group of residual eugenol¹⁶. A small peak was observed at 2600 cm⁻¹ corresponding to S–H thiol group. Also, a tiny stretch found at 2150 cm⁻¹ which may be accounted for ketene group. From literature, it is evident that band at 1650 cm⁻¹ corresponds to C=O is not found in clove extract, while appearance of this band in clove AgNPs spectra is proof of involvement of eugenol in bioreduction process and formation of AgNPs²³. Thus, it indicates the formation of clove AgNPs capped with different bio moieties²⁴. The IR spectrum showed a peak at 1098 cm⁻¹ corresponding to C–O (carboxylic acid derivative) stretching, formed in eugenol after bioreduction of silver. A very sharp and intense peak was obtained at 650 cm⁻¹ corresponding to R-CH alkane functional group, which are abundantly found in clove extract. These observations confirmed the presence of eugenol and other functional moieties (mainly water-soluble flavonoids), which acts as reducing and stabilizing agent in the synthesis of clove AgNPs.

FTIR spectra of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.

In borohydride AgNPs, several peaks with high intensity were obtained because of interaction of chemical NaBH₄ with AgNO₃. Stretches of O–H were also recorded between 3700 and 3500 cm⁻¹ corresponding to free hydroxyl and H-bonded alcohol and phenol groups. A broad stretch was recorded between 3500 and 3300 cm⁻¹ corresponding to N–H aliphatic primary amine. Peaks recorded at 2800 cm⁻¹ corresponded to aliphatic C–H bending vibrations while a small, weak band was observed at 2550 cm⁻¹ upper side, corresponding to S–H thiol group. A Significant peak was detected at 2260 cm⁻¹, which could be attributed to C≡N nitrile stretching whereas, a small vibration was observed at 2100 cm⁻¹, corresponding to alkyne (C≡C) however, a sharp and strong peak was noticed at 1620 cm⁻¹, which could be assigned to C=C α, β unsaturated ketone. A very sharp and intense peak was also obtained at 1423 cm⁻¹ which could be accounted to be generated by the borohydride anion of NaBH₄, that remained partly bound to the AgNPs and corresponded to C–H bending. Moreover, stretch at 800 cm⁻¹ corresponds to aliphatic C-H bending vibrations of alkanes.

The interactions of AgNPs with GSH were confirmed by the absence of N–H and S–H stretching bands, which are otherwise present in pure GSH and found at 3350 cm⁻¹ and 2600 cm⁻¹ respectively¹¹. The absence of spectral peaks of S–H and N–H, suggest that glutathione is modified onto the surface of AgNPs by the thiol and amine groups from the cysteine moiety of Glutathione. These findings are supported by a previous study where a small spectral stretch resembled our FTIR analysis¹⁷ however, as different conditions such as heating and longer stirring hours were used by them, there were some deviations in the present spectra. Additionally in the study, a broad peak was observed between 3550 and 3200 cm⁻¹ corresponding to O–H stretching of intermolecular bonded alcohol. Also, a small stretch at 2140 cm⁻¹ was found, which can be assigned to alkyne (C≡C). Apart from this, a very sharp and intense peak was obtained at 1650 cm⁻¹ for C=O stretch representing α, β unsaturated aldehydes and ketones. One more distinct peak was also obtained at 1250 cm⁻¹ which represents C–N functional group of aliphatic amines. Vibration of C–H bond due to alkanes and aromatic generated two sharp peaks of same intensity at 870 cm⁻¹ and 750 cm⁻¹. A small band was also pointed at 890 cm⁻¹ representing C–H group trisubstituted bonding. The broad peaks from 639 to 500 cm⁻¹ are related to Ag-NPs bonding with sulphur from thiol groups of GSH molecules.

Antioxidant activity

Radical scavenging activity is a measure of scavenging of free radicals which are generated in the form of reactive oxygen species (ROS). DPPH is a free radical capable of accepting an electron or hydrogen radical to become a stable molecule and it has an absorption maximum at 517 nm. Radical Scavenging Activity (RSA) of different synthesized AgNPs viz., clove (green), NaBH₄ (uncapped) and GSH (capped) were evaluated. The radical scavenging activity was found to be concentration dependent.

Figure 7 depicts the RSA values of the synthesized AgNPs. Ascorbic acid was used as a standard (gave RSA value of 97.17% at 100 ppm conc.) Radical scavenging efficacy of clove AgNPs was found to be the highest among all the samples—58.42% to 74.11%. These results correlate with the other findings done on antioxidant activity of Leptadenia reticulata and Ananas comosus^25,26. The percent RSA of chemically synthesized AgNPs i.e., borohydride AgNPs ranged from 33.34 to 46.62%, which was lower than percent RSA value of AgNPs synthesized from clove, moreover the percent RSA values of GSH-capped AgNPs was found to be in the range of 33.89–58.78%, which were lower than the percent RSA values of clove AgNPs but higher than borohydride AgNPs. The decreasing order of radical scavenging activity was as follows: clove AgNPs > GSH-capped AgNPs > Borohydride AgNPs. Chemically synthesized NPs were found to have least percent RSA value (46.62%) among all samples, the reason for this may be accounted to the free ends of chemically synthesized AgNPs which generates free radicals. GSH-capped AgNPs were found to have increased antioxidant efficacy than borohydride AgNPs, which may be due to the capping which probably reduced the free ends of AgNPs resulting in less generation of free radicals. Free radical generation is associated with nanotoxicity and enhancing the antioxidant activity seems to be a promising solution to manipulate the surface properties of nanoparticles imparting stability through thiol rich moieties either chemically or green route.

DPPH Antioxidant assay at different concentrations of (A) Clove AgNPs (B) Borohydride AgNPs (C) GSH-capped AgNPs (AA) Ascorbic acid-used as a standard (values are mean ± S.D of three replicates).

Mosquito larvicidal bioactivity

Mosquito larvicidal potential of synthesized AgNPs was determined against III instar larvae of Dengue vector Aedes aegypti under laboratory conditions. Control group showed mortality only at 72 h. Mean percent mortality at different concentrations at 24 h, 48 h and 72 h of green, chemically synthesized as well as capped AgNPs was found out. Various mortality patterns were observed at different concentrations and are shown in Fig. 8. Clove mediated AgNPs were found to be very effective and exhibited high mean percent mortality even at 24 h.

Mean percent mortality at different concentrations at 24 h, 48 h and 72 h of (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs (values are mean ± S.D of four replicates).

At the lowest concentration, clove AgNPs showed mean percent mortality of 21% which reached up to 96% with the highest concentration and at 72 h this range increased from 45 to 98%. Borohydride AgNPs showed lower mean percent mortality as compared to that of clove AgNPs. Their mortality range was found to be between 11 and 73% at 24 h and at 72 h it extended from 23 to 98%. For GSH-capped AgNPs, after 24 h, the mortality was in the range of 1–68%, and then at 72 h it reached from 21 to 88%. Similar results are also reported by various other researchers on dengue vector^27,28.

Table 1 demonstrates the LC₅₀ and LC₉₀ values for different AgNPs at different time interval along with lower fiducial limits (LFL) and upper fiducial limits (UFL). Results of the mean percent mortality and LC values clearly indicates that, clove AgNPs being most effective (LC₅₀—4.9 ppm, LC₉₀—30.2 ppm) followed by GSH-capped (LC₅₀—20.13 ppm, LC₉₀—46.63 ppm) and borohydride AgNPs (LC₅₀—13.43 ppm, LC₉₀—160.19 ppm) after 24 h. Green nanoparticles were found more effective owing to the phytopotentials involved. On the other hand, nanoparticles are known to trigger free radical activity on the surface of the particles whereas, capping enhances their scavenging activity as free sites becomes enclosed and reduces the nanotoxicity making them suitable for benign application in various fields. Hitherto, this is the first study where three different types of AgNPs were synthesised using different routes and compared for their different activities and a way is figured out to make the AgNPs much stable and potent for larvicidal applications.

Acute toxicity of AgNPs to D. magna

Toxicity assessment of AgNPs is important to understand the risk associated with AgNPs to the environment, hence, 48 h toxicity was evaluated for all the three AgNPs and compared with controls. Percent survivability of D. magna neonates treated with various AgNPs, at the highest test concentration depicted 80% and 60% survivability with clove and GSH-capped AgNPs respectively, while, exposure with borohydride AgNPs resulted in 100% mortality. Figure 9 shows the range of percent survivability for all the three AgNPs i.e., clove AgNPs (90–80%), borohydride AgNPs (60–0%) and GSH-capped AgNPs (90–60%) at various test concentrations. Higher survivability percentage with clove AgNPs could be due to the phytopotentials of botanical extract, that posed least threat to the eco-toxicity model organism D. magna. On the other hand, higher mortalities on exposure with borohydride AgNPs could be assigned to the chemicals employed during their synthesis, which might have interacted with the cellular and sub-cellular components of D. magna and generated oxidative stress, resulting in lesser or no survivability of the test organism. Surprisingly, GSH-capped AgNPs demonstrated higher survivability which was comparable with the green clove AgNPs. It may be deduced from these findings that this moderate to low toxicity could be due to capping of the free ends of these AgNPs, culminating into stabilized AgNPs with least toxicity and safer action as hypothesized in this study. Further, this finds support with the antioxidant activity of GSH-capped AgNPs that was enhanced as compared to the chemically synthesized borohydride AgNPs. The swimming behaviour of D. magna neonates was also observed in all three AgNPs treatment groups and compared with that of controls. It was noticed that control and clove AgNPs treated D. magna neonates showed normal swimming behaviour both after 24 h and 48 h, while borohydride AgNPs treatment slowed down their movement after 24 h and further after 48 h concentrating mainly at bottom. GSH-capped AgNPs treated neonates showed somewhat transitional swimming behaviour between other two treatment groups.

Survival percentage of Daphnia magna neonates after 48 h acute toxicity bioassay at different concentrations (ppm) (A) Clove AgNPs, (B) Borohydride AgNPs, (C) GSH-capped AgNPs.

Morphological alterations in D. magna observed by phase-contrast microscopy

Morphological alterations of D. magna were observed and recorded after 48 h acute toxicity assay (Fig. 10) and compared with that of the control. Control specimen showed healthy neonate with no morphological alterations. D. magna following treatment with the clove AgNPs, not much deviation from the controls could be noticed. On the contrary, borohydride AgNPs treated neonates demonstrated various morphological alterations such as overall carapace and lining of internal structures became lighter giving the hyaline appearance as compared to other treatment groups. AgNPs accumulation at the hindgut region, internal fluid leakage from ephippium due to carapace rupturing, disrupted gut region and mouth parts were other major alterations apparently noticeable. Since, D. magna is a bottom level filter feeding organism and occupies lower level in food chain and trophic level, any form of nanoparticles accumulation in this organism, may results in transferring nanoparticles and its toxicity to higher level organisms in food chain too^29,30. Interestingly, GSH-capping to these AgNPs greatly reduced their toxicity as most of the structures were intact and slight pigmentation was found both in foregut and midgut. Major morphological alterations in borohydride AgNPs treated neonates, clearly depicts its higher toxicity as compared to other two AgNPs. The toxicity assay results clearly indicate the non-toxic nature of green AgNPs and also highlights the effectiveness of capping in imparting stability and safety to chemical AgNPs.

Morphological alterations in Daphnia magna neonates after 48 h acute toxicity bioassay observed under phase contrast microscope (a) Clove AgNPs, (b) Borohydride AgNPs, (c) GSH-capped AgNPs.