Green Synthesis of Silver Nanoparticles Using Spilanthes acmella Leaf Extract and its Antioxidant-Mediated Ameliorative Activity against Doxorubicin-Induced Toxicity in Dalton's Lymphoma Ascites (DLA)-Bearing Mice

Abstract

Green synthesis of metal nanoparticles is a rapidly growing research area in the field of nanotechnology because of their biomedical applications. This study describes the synthesis of silver nanoparticles (AgNPs) using Spilanthes acmella leaf extract and its ameliorative effects against doxorubicin-induced toxicity. The formation of AgNPs was confirmed by a ultraviolet-visible (UV-vis) spectrum that revealed an absorption band at 430 nm. A shift in the absorption bands in Fourier-transform infrared spectroscopy (FT-IR) confirmed the bioactive molecules of S. acmella leaf extract that acted as a reducing and capping agent. The spherical shape of AgNPs was confirmed by scanning electron microscope (SEM) analysis, and the presence of elemental silver was indicated by energy dispersive X-ray spectroscopy (EDS) analysis. X-ray diffraction (XRD) analysis revealed that the crystalline size of the synthesized AgNPs was 6.702 nm. Treatment of Dalton's lymphoma ascites (DLA) mice with 20 mg/kg of doxorubicin (DOX) significantly increased the activities of serum toxicity markers including aspartate amino-transferase (AST), alanine amino-transferase (ALT), and lactate dehydrogenase (LDH). However, compared to DOX alone treatment, the coadministration of DOX and AgNPs reduced AST, ALT, and LDH activities. DOX alone treatment reduced glutathione (GSH) contents and decreased the activities of glutathione-s-transferase (GST) and superoxide dismutase (SOD) in DLA mice. However, the administration of AgNPs to DOX-treated DLA mice increased GSH content and the activities of GST and SOD. Consistently, biosynthesized AgNPs were found to possess significantly higher free-radical scavenging activities when compared to the S. acmella leaf extract, as measured by ABTS, DPPH, and O₂ ^•- assays. The biosynthesized AgNPs also showed significant inhibitory activities against erythrocyte hemolysis and lipid peroxidation in the liver homogenate.

Figure 1

Visual identification of biosynthesized AgNPs as recorded at different time intervals: (a) initial, (b) 2 h, and (c) 4 h. The formation of a reddish-brown color revealed the formation of AgNPs in the reaction mixture.

Figure 2

UV–vis absorption spectra of biosynthesized AgNPs using S. acmella leaf extract at different time intervals.

Figure 3

FT-IR analysis of (a) silver nitrate, (b) S. acmella leaf extract (SAAE), and (c) biosynthesized AgNPs.

Figure 4

(a–c) SEM micrographs of biosynthesized AgNPs. Elemental mapping of oxygen (d), silver (e), and chlorine (f), and all three elements (g) for the inset region, and (h) EDS data of the area in the white box in panel (c). Scale bars: (a) 1 μm, (b) 100 μm, (c) 5 μm, and (d–g and inset) 1 μm.

Figure 5

(a–d) TEM micrograph showing the size of biosynthesized AgNPs. Scale bars: (a) 10 nm, (b) 20 nm, (c) 50 nm, and (d) 100 nm. (e) Histogram of the TEM image (100 nm).

Figure 6

XRD pattern of biosynthesized AgNPs using the S. acmella leaf extract.

Figure 7

Plots of log-doses of AgNPs, SAAE, and ASA against (a) DPPH, (b) ABTS, and (c) O₂^•– inhibition (%) for the calculation of IC₅₀. AgNPs: biosynthesized AgNPs using S. acmella leaf extract; SAAE: S. acmella leaf extract; ASA: ascorbic acid (standard).

Figure 8

IC₅₀ (μg/mL) of AgNPs, SAAE, and ASA for (a) DPPH, (b) ABTS, and (c) O₂^•–. AgNPs: biosynthesized AgNPs using S. acmella leaf extract; SAAE: S. acmella leaf extract; ASA: ascorbic acid (standard). Values are expressed as mean ± SEM, n = 3. Different letters indicate significant variation.

Figure 9

Reducing power of AgNPs, SAAE, and ASA at different concentrations. AgNPs: biosynthesized AgNPs using S. acmella leaf extract; SAAE: S. acmella leaf extract; ASA: ascorbic acid (standard). Values are expressed as mean ± SEM, n = 3. Different letters indicate significant variation.

Figure 10

(a) Antihemolytic and (b) lipid peroxidation inhibitory activities of biosynthesized AgNPs. Values are expressed as mean ± SEM, n = 3. Different letters indicate significant variation.

Figure 11

Effects of biosynthesized AgNPs on (a) glutathione level (GSH) (μmol/mg protein), (b) glutathione-s-transferase activity (GST) (unit/mg protein), and (c) superoxide dismutase activity (SOD) (unit/mg protein) in the liver and heart of mice. C: normal control; DLA: Dalton’s lymphoma ascites bearing mice without any treatment; DOX: DLA mice treated with 20 mg/kg of doxorubicin; DOX + AgNPs25, DOX + AgNPs50, and DOX + AgNPs100: DLA mice treated with 20 mg/kg of doxorubicin followed by biosynthesized AgNPs at the dose of 25, 50, and 100 mg/kg, respectively. AgNPs50: DLA mice treated with biosynthesized AgNPs (50 mg/kg). Means not sharing the same letter are significantly different.

Figure 12

Effects of biosynthesized AgNPs on lipid peroxidation (LPO) expressed in malondialdehyde (MDA) (nmol/mg protein) in (a) liver and (b) heart of DLA mice. C: normal control; DLA: Dalton’s lymphoma ascites bearing mice without any treatment; DOX: DLA mice treated with 20 mg/kg of doxorubicin; DOX + AgNPs25, DOX + AgNPs50, and DOX + AgNPs100: DLA mice treated with 20 mg/kg of doxorubicin followed by biosynthesized AgNPs at the dose of 25, 50, and 100 mg/kg, respectively. AgNPs50: DLA mice treated with biosynthesized AgNPs (50 mg/kg). Means not sharing the same letter are significantly different.