DNA Fingerprint Profile of Zizania spp. Plant, Monitoring Its Leaves with Screening of Their Biological Activity: Antimicrobial, Antioxidant and Cytotoxicity

Abstract

This study presents an integrated approach combining molecular, phytochemical, and biological analyses to characterize a newly discovered Zizania specimen from the northern Nile Delta, Egypt. Genetic fingerprinting using RAPD and ISSR markers revealed 85% band-sharing similarity with Zizania texana (Z. texana), though distinct morphological and genetic traits suggested potential intraspecific variation. Phytochemical profiling identified high concentrations of bioactive compounds, including quercetin (42.1 µg/mL), β-caryophyllene (11.21%), and gallic acid (23.4 µg/mL), which are pertinent and correlated with robust biological activities. The ethanolic leaf extract exhibited significant antioxidant capacity (IC₅₀ = 38.6 µg/mL in DPPH assay), potent antimicrobial effects against Candida albicans (C. albicans) (IC₅₀ = 4.9 ± 0.6 µg/mL), and dose-dependent cytotoxicity against cancer cell lines. MCF-7 has the lowest IC₅₀ (28.3 ± 1.5 µg/mL), indicating the highest potency among the tested cell lines. In contrast, HepG2 demonstrates moderate sensitivity (IC₅₀ = 31.4 ± 1.8 µg/mL), while A549 shows the highest IC₅₀ value (36.9 ± 2.0 µg/mL), indicating greater resistance. These findings underscore the taxonomic novelty of the specimen and its potential as a source of natural antioxidants, antimicrobials, and anticancer agents. The study highlights the importance of interdisciplinary approaches in resolving taxonomic uncertainties and unlocking the medicinal value of understudied aquatic plants.

Keywords: Zizania spp.; antimicrobial activity; antioxidant activity; cytotoxicity; phytochemical profiling.

Figure 1

DNA fingerprinting, phytochemical profiling, DPPH assay, antimicrobial, antioxidant, and cytotoxic activities of Zizania spp. leaves analysis.

Figure 2

ISSR/RAPD Profiling of Zizania Species.

Figure 3

Color-coded multiple sequence alignment (MSA) of rbcL gene regions from Zizania spp. compared to a reference sequence. Red-highlighted bases indicate nucleotide mismatches, while white cells represent matches or gaps.

Figure 4

Band presence/absence matrix for Zizania species (L2–L11), including unknown specimens (L10, L11). Unique bands (e.g., 1200 bp in Z. latifolia) and shared markers (e.g., 750 bp in Z. texana and unknowns) highlight interspecific divergence and intraspecific cohesion. Taxonomic classification of unknowns as Z. texana is supported by 85% band-sharing similarity.

Figure 5

Hierarchical clustering of Zizania specimens based on Jaccard genetic distances. Z. texana (L8–L9) and unknowns (L10–L11) form a cohesive clade (distance = 0.2), while Z. latifolia (L2–L3) clusters distantly (distance = 0.4). Bootstrap values (577–10558) indicate moderate node support.

Figure 6

Principal Component Analysis of Genetic Divergence in Zizania Species.

Figure 7

(Figures (1–4)). (1): Habitat morphology and ecology of an emergent aquatic grass (Poaceae). (2): Mature stands in freshwater wetlands (water depth: 20–40 cm) with linear-lanceolate leaves (≤80 cm) emerging vertically. (3): Dimorphic panicles (≤45 cm) in reproductive phase: female spikelets (pubescent lemma/palea) above male counterparts (anther-bearing florets) in shallow lake margins. (4): Riparian ecotone colonization, demonstrating adaptation to fluctuating hydrology (0–50 cm depth).

Figure 8

The gas chromatography (GC) analysis of Zizania leaf extract. Peaks reveals a complex phytochemical profile dominated by bioactive terpenes, fatty acids, and hydrocarbons.

Figure 9

HPLC Profile of Zizania Extract.

Figure 10

Antimicrobial activity of ethanolic Zizania leaf extracts (5–20 µg/mL) against E. coli (Panel 1), S. aureus (Panel 2), and C. albicans (Panel 3), with comparative dose–response analysis (Panel 4). Inhibition zones (mm) were measured via agar disc diffusion assays. Columns in Panel 4 represent mean inhibition zones ± SD (n = 3). Asterisks denote statistically significant differences between microbial responses (p < 0.05, one-way ANOVA with Tukey’s post hoc test). S. aureus exhibited the highest sensitivity (max inhibition: 14.0 mm at 20 µg/mL), followed by E. coli (12.5 mm) and C. albicans (10.0 mm). Extract batch identifier: Kinole. All experiments were conducted in triplicate under standardized conditions.

Figure 11

Dose–response curve showing the antimicrobial activity of Zizania leaf extract, with inhibition zones increasing dose-dependently. C. albicans showed the highest sensitivity (IC₅₀ = 4.9 ± 0.6 µg/mL), followed by E. coli and S. aureus.

Figure 12

DPPH Dose-response Curve of Zizania Extract.

Figure 13

Dose-response curves of Zizania extract against MCF-7, HepG2, and A549 cell lines. Data points represent mean ± SD (n = 3).