Green Synthesis of Silver Nanoparticles using Crassula Ovata and Bryophyllum Pinnatum Leaf Extracts: Characterisation and Comparative Antioxidant, Cytotoxic and Anti-Inflammatory Activity of the Individual and Combined Systems

Abstract

Two members of the Crassulaceae with different secondary-metabolite profiles were used as reducing and capping systems for biogenic silver nanoparticles, and the resulting particles were carried through three unrelated in vitro endpoints under one protocol. Ethanolic leaf extracts of Crassula ovata and Bryophyllum pinnatum were reacted with 1 mM silver nitrate at a 1:9 extract-to-salt ratio (pH 8.0, 60 °C, 2 h, 24 h dark maturation) to give CO-AgNPs and BP-AgNPs. A 1:1 (w/w) physical mixture of the two purified powders (MIX-AgNPs) was carried as a combination arm. Both batches gave a single surface plasmon resonance band, at 422.77 ± 0.35 nm for CO-AgNPs and 427.20 ± 0.40 nm for BP-AgNPs, with no corresponding band in the solvent, salt or extract controls. Hydrodynamic diameter was 83.83 ± 1.25 nm and 75.57 ± 1.25 nm, polydispersity index 0.294 ± 0.011 and 0.252 ± 0.008, and zeta potential −28.37 ± 0.75 mV and −32.57 ± 0.80 mV. Diffraction peaks indexed to face-centred cubic silver, with mean crystallite sizes of 29.75 ± 3.70 nm and 23.87 ± 2.29 nm; energy-dispersive X-ray analysis returned 67.15 wt% and 70.18 wt% silver with carbon and oxygen signals consistent with an adsorbed phytochemical layer. Cumulative silver-equivalent release over 24 h in phosphate-buffered saline pH 7.4 reached 84.10 ± 2.55% and 88.60 ± 2.60%. The potency order was the same on every endpoint: MIX-AgNPs > BP-AgNPs > CO-AgNPs > BP-EXT > CO-EXT. DPPH IC50 fell from 106.13 µg/mL for the C. ovata extract to 34.79 µg/mL for MIX-AgNPs, against 10.60 µg/mL for ascorbic acid. At 24 h the MTT IC50 for MIX-AgNPs was 65.68 µg/mL (MCF-7), 76.60 µg/mL (HCT116) and 89.00 µg/mL (A549), three- to six-fold below the parent extracts. In lipopolysaccharide-stimulated RAW 264.7 macrophages held at or above 80% viability, MIX-AgNPs lowered TNF-α by 75.07 ± 2.06% and IL-6 by 75.05 ± 2.33% at 50 µg/mL. After Holm adjustment the mixture was not consistently better than BP-AgNPs at matched concentrations, so the combination result is reported as enhanced activity and not as synergy.