Phytochemical Investigation and Biological Activities of Ophioglossum gramineum Leaf Extracts

1. Botanical Overview

2. Phytochemical Composition

2.1 Major Phytochemical Classes Reported in Ophioglossum spp.

2.2 Standard Qualitative Screening Methods Used for Leaf Extracts

• Tannins: Gelatin-salt test (white precipitate)
• Polyphenols: Ferric chloride test (blue-black coloration)
• Flavonoids: Shinoda test / aluminum chloride colorimetric
• Alkaloids: Mayer's, Dragendorff's, Wagner's reagents
• Saponins: Foam test, Liebermann-Burchard test
• Terpenoids / Steroids: Salkowski test
• Glycosides: Keller-Kiliani test
• Reducing sugars: Benedict's/Fehling's test

3. Antioxidant Activity

Methods

• DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay - most widely used; IC50 values reported
• NBT (Nitroblue tetrazolium) superoxide anion scavenging
• Lipid peroxidation inhibition (TBARS/MDA)
• FRAP (Ferric Reducing Antioxidant Power)
• Total phenolic content (TPC) by Folin-Ciocalteu

Key Findings Across the Genus

• O. vulgatum ethyl acetate fraction at 250 µg/mL scavenged 97.28% DPPH free radicals in the 2025 study (PMID: 40143123)
• O. thermale EtOAc fraction outperformed green tea (EGCG standard) in DPPH, NBT, and lipid peroxidation assays; it also inhibited UVB-induced ROS generation in human dermal fibroblasts (PMID: 22419423)
• Antioxidant mechanisms are attributed primarily to flavonoids and high phenolic content, via hydrogen atom donation and electron transfer
• The Nrf2/HO-1 signaling axis is implicated in intracellular antioxidant defense modulation

4. Anticancer Activity

In Vitro Approaches Used

• MTT/CCK-8 cell viability assays on cancer cell lines (HeLa, MCF-7, HepG2, A549)
• Colony formation assays
• Apoptosis detection (Annexin V/PI staining, flow cytometry)
• Cell migration/wound scratch assays

Relevant Findings in the Genus

• Flavonoids from Ophioglossum spp. - particularly ginkgetin (biflavonoid) - have demonstrated cytotoxic activity via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in non-small cell lung cancer (PMID: 25 in SAJB review, DOI:10.1016/j.sajb.2024.10.040)
• Quercetin derivatives and kaempferol glycosides promote apoptosis through mitochondrial pathways and cell cycle arrest (G2/M checkpoint)
• Phenolic compounds in general modulate cancer cell signaling through PI3K/Akt pathway inhibition and induction of caspase-dependent apoptosis
• Homoflavonoids (pedunculosumosides) isolated from O. pedunculosum showed anti-HBV activity (IC50 70.5 µM for blocking HBsAg secretion) (PMID: 21401115)

5. Antimicrobial Activity

Methods

• Agar disc diffusion (Kirby-Bauer method) - inhibition zone diameter (mm)
• Minimum Inhibitory Concentration (MIC) by broth microdilution
• Minimum Bactericidal Concentration (MBC)
• Resazurin-based assay for antibiotic-resistant strains

Organisms Tested and Results

• Peroxy fatty acids from O. thermale showed direct antibacterial activity (Fitoterapia, DOI: 10.1016/j.fitote.2015...)
• Flavonoids and tannins disrupt membrane integrity and inhibit bacterial enzyme systems
• Saponins cause membrane permeabilization
• Antimicrobial mechanisms include: DNA/RNA damage, disruption of the peptidoglycan layer, protein denaturation, and oxidative stress induction

6. Extraction Methods and Solvent Selection

• Maceration (48-72 h) or Soxhlet extraction (reflux)
• Rotary evaporation at 40-50°C for concentration
• Sequential fractionation (hexane → chloroform → ethyl acetate → methanol → water) for compound class separation

7. Summary of Key Signaling Pathways

Phytoconstituents (flavonoids, phenolics)
          |
          ├─→ ANTIOXIDANT: Nrf2/HO-1 ↑ → Oxidative stress ↓
          |
          ├─→ ANTI-INFLAMMATORY: TLR4/MYD88 ↓, NF-κB ↓, MAPK ↓
          |
          ├─→ ANTICANCER: PI3K/Akt ↓ → Apoptosis ↑, Cell cycle arrest
          |           Ferroptosis (Nrf2/HO-1 axis disruption)
          |
          └─→ ANTIMICROBIAL: Membrane disruption, enzyme inhibition

8. Research Gaps and Future Directions

1. Species-specific studies on O. gramineum remain very limited - most data is extrapolated from O. vulgatum, O. thermale, and O. pedunculosum. Dedicated phytochemical profiling (LC-MS, GC-MS, NMR) of O. gramineum leaf extracts is needed.
2. In vivo anticancer validation of crude extracts and isolated compounds is largely absent.
3. Isolation and structure elucidation of novel compounds unique to O. gramineum has not been published in peer-reviewed literature to date.
4. Bioavailability and toxicity studies (acute, sub-chronic) are required before any clinical translation.
5. A 2024 comprehensive review on Ophioglossum ethno-pharmacology and phytochemistry (Yousaf et al., South African Journal of Botany, DOI: 10.1016/j.sajb.2024.10.040) is the most current authoritative reference and should be consulted as a primary source for genus-level data.

Key References

1. Feng S et al. (2025). Phytochemical Composition and Skin-Friendly Activities of the Ethyl Acetate Fraction in Ophioglossum vulgatum Linn. Pharmaceuticals, 18(3):345. PMID: 40143123
2. Zhang X et al. (2012). In vitro antioxidant and in vivo anti-inflammatory activities of Ophioglossum thermale. Am J Chin Med, 40:279. PMID: 22419423
3. Clericuzio M et al. (2012). Flavonoid oligoglycosides from Ophioglossum vulgatum L. having wound healing properties. Planta Med, 78:1639. PMID: 22936389
4. Wan CX et al. (2011). Homoflavonoid glucosides from Ophioglossum pedunculosum and their anti-HBV activity. J Nat Prod, 74:881. PMID: 21401115
5. Yousaf AM et al. (2024). A comprehensive review on ethno-pharmacological and phytochemical properties of selected species of genus Ophioglossum. S Afr J Bot, 538-548. DOI: 10.1016/j.sajb.2024.10.040
6. Zhu X et al. (2025). Anti-inflammatory properties of ophioglonin derived from Ophioglossum vulgatum L. via inactivating NF-κB and MAPK signaling. FEBS Open Bio. PMID: 39455284