**Background:** Echinops macrochaetus is a medicinal plant endemic to Saudi Arabia, known for bioactive compounds such as thiophenes, flavonoids, and sesquiterpene glycosides. Zinc is an essential micronutrient for plant growth, but its bioavailability in soil is often poor. Zinc oxide nanoparticles (ZnO-NPs) offer a potential nano-fertilizer solution due to their enhanced absorption and elicitor effects on secondary metabolite production. This study aimed to evaluate the effects of green-synthesized ZnO-NPs on growth, antioxidant response, bioactive compound accumulation, and genome size in E. macrochaetus.
**Methods:** ZnO-NPs were synthesized using an aqueous leaf extract of Heliotropium bacciferum and characterized by UV-Vis spectroscopy (peak at 276 nm), FTIR (functional groups including O-H, C=C), TEM (size range 18.730–25.531 nm), and zeta potential (−17 mV). Four-week-old E. macrochaetus plants were treated with 0 (control), 10 (T1), 20 (T2), or 40 (T3) mg/L ZnO-NPs (100 mL per pot once per month) and irrigated with distilled water every 15 days. After 120 days, morphological traits, biomass, photosynthetic pigments, antioxidant enzymes (SOD, APX, GR), proline, TBARS, total protein, genome size (by flow cytometry using propidium iodide staining), and bioactive compounds (quercetin-3-β-D-glucoside, luteolin 7-rutinoside, p-coumaric acid by HPLC) were measured. All analyses were performed in triplicate, and data were analyzed by one-way ANOVA with Duncan's test (p < 0.05).
**Key Results:** The lowest ZnO-NP concentration (10 mg/L) produced the greatest improvements: leaf number (16.33 per plant), leaf length (17.03 cm), shoot length (8.87 cm), shoot fresh weight (4.09 g/plant), and total chlorophyll (273.11 µg/g FW vs. 238.08 µg/g FW in control). Carotenoid content also peaked at T1. Higher concentrations (20 and 40 mg/L) reduced chlorophyll and carotenoids below control levels. Antioxidant enzyme activities increased dose-dependently: SOD activity was highest at T3 (1.63 U/mg/min vs. control), APX at T3 (0.875 U/mg/min), and GR significantly increased at T2 and T3. Proline content rose sharply with concentration, reaching 485 µg/g FW at T3 vs. 89.77 µg/g FW in control. TBARS content was not significantly different at T1 (0.32 nM/g FW in control vs. not significant at T1) but increased significantly at T2 (0.66 nM/g FW) and T3 (1.19 nM/g FW). Total protein content was highest at T3 (166.53 mg/g FW vs. 89.66 mg/g FW in control). Genome size (2C DNA content) showed minor variation: control 2.28 pg, T1 2.27 pg, T2 2.25 pg, T3 2.24 pg. Bioactive compounds varied by plant part and treatment: quercetin-3-β-D-glucoside was highest in leaves at T1 (5.59 mg/g DW vs. 4.52 mg/g DW control); luteolin 7-rutinoside peaked in leaves at T2 (7.53 mg/g DW vs. 5.99 mg/g DW control); p-coumaric acid was highest in leaves at T2 (348.55 µg/g DW vs. 270.22 µg/g DW control). In roots, p-coumaric acid increased significantly at T1 (86.67 µg/g DW) and T2 (97.11 µg/g DW) compared to control (65.20 µg/g DW).
**Clinical Implications:** This study demonstrates that green-synthesized ZnO-NPs at low concentrations (10 mg/L) can act as effective nano-fertilizers to enhance growth, biomass, and bioactive compound accumulation in the medicinal plant E. macrochaetus. The minor changes in genome size suggest low genotoxicity at the tested doses. These findings support the use of biogenic ZnO-NPs as elicitors to improve the production of valuable secondary metabolites in medicinal plants, potentially increasing their therapeutic efficacy. Further research is needed to optimize concentrations and understand the mechanisms underlying differential compound accumulation in various plant organs.