Comparative phytochemical profiling and bioactivities of wild Thymus species from Mount Ida (Kazdağı)

Technical Summary: Comparative Phytochemical Profiling and Bioactivities of Wild Thymus Species from Mount Ida (Kazdağı)

Problem Statement
Wild Thymus species growing in the Kazdağları region of Türkiye possess rich phytochemical profiles and biological effects that have not yet been fully characterized. While Thymus vulgaris and Thymus serpyllum are well-documented, there is a lack of integrated data regarding four specific wild species native to this biodiversity hotspot: Thymus bornmuelleri (Purple thyme), Thymus citriodorus (Lemon thyme), Thymus longicaulis (Mountain thyme), and Thymus sibthorpii (Ball thyme). This study addresses the gap in understanding the specific phytochemical composition and the corresponding antioxidant, antimicrobial, and anticancer potential of these specific local varieties.

Methodology
The study employed a multi-faceted analytical approach using dried aerial parts of the four Thymus species collected from a commercial market in Edremit, Balıkesir, in August 2024. Botanical identification was verified by a medical services lecturer due to the processed nature of the samples.

• Extraction: Samples were extracted using 80% dilute methanol via shaking for 2 hours.
• Phytochemical Analysis:
  • Total Phenolic Content (TPC) & Radical Scavenging Activity (RSA): Determined spectrophotometrically using the Folin-Ciocalteu method (expressed as Gallic Acid Equivalents) and DPPH assay (expressed as Trolox Equivalents), respectively.
  • Phenolic Compounds: Quantified using High-Performance Liquid Chromatography (HPLC) with a C18 column, focusing on compounds such as rosmarinic acid, catechin, and various phenolic acids.
  • Volatile Components: Identified and quantified using Gas Chromatography-Mass Spectrometry (GC-MS) coupled with Headspace Solid-Phase Microextraction (HS-SPME). A total of 143 volatile compounds were analyzed.
• Biological Activity Assays:
  • Antimicrobial Activity: Evaluated against Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Candida albicans (yeast) using the disk diffusion method at concentrations ranging from 10 to 100 µg/mL.
  • Cytotoxicity: Assessed against the HT29 human colorectal adenocarcinoma cell line using the WST-1 assay over a 72-hour period across a concentration gradient of 31.25 to 2000 µg/mL.
• Statistical Analysis: Data were analyzed using One-way ANOVA, Tukey post-hoc tests, and Principal Component Analysis (PCA) to determine variance and correlations between phytochemical profiles and biological activities.

Key Results

• Phytochemical Composition:

  • Phenolics: Thymus citriodorus (TC) exhibited the highest Total Phenolic Content (94.08 mg GAE/g DW) and Radical Scavenging Activity (440.05 mg TE/g DW). Rosmarinic acid was the dominant phenolic acid across all samples, ranging from 5.89 to 9.23 mg/g, with the highest concentration found in TC (9.229 mg/g).
  • Volatiles: A total of 143 volatile compounds were identified. Thymol was the primary volatile component, with the highest concentration in Thymus sibthorpii (TSB) at 31.12 mg/kg, followed by Thymus bornmuelleri (TBV) at 26.43 mg/kg. PCA indicated that 11 compounds, including alpha-pinene, o-cymene, and thymol, accounted for over 50% of the variance in the samples.

• Antimicrobial Activity:

  • All extracts demonstrated dose-dependent antimicrobial activity.
  • Against S. aureus: TBV and TSB showed the largest inhibition zones (10.2 mm and 10.0 mm, respectively) at 80 µg/mL.
  • Against P. aeruginosa: TLC demonstrated the strongest activity (10.2 mm), though overall inhibition was lower compared to Gram-positive bacteria, consistent with intrinsic resistance mechanisms.
  • Against C. albicans: TSB showed the highest inhibition (8.9 mm), though antifungal activity was generally moderate across all species.
  • The study noted a correlation between high thymol content and strong antibacterial activity against S. aureus.

• Cytotoxic Activity (HT29 Cells):

  • All four extracts reduced HT29 cell viability in a concentration-dependent manner.
  • TBV and TC exhibited the most potent cytotoxicity, reducing cell viability to approximately 11.37% and 10.15%, respectively, at the highest concentration (2000 µg/mL). Significant cytotoxicity for these species was observed at concentrations as low as 62.5 µg/mL (TBV) and 250 µg/mL (TC).
  • TSB showed intermediate activity, while TLC was the least cytotoxic, maintaining viability above 90% until concentrations reached 500 µg/mL.
  • The superior cytotoxicity of TBV and TC was attributed to the synergistic effects of their specific phytochemical profiles, including thymol, rosmarinic acid, carvotanacetone, and β-caryophyllene.

Significance and Claims
The authors claim this study represents the first integrated phytochemical characterization and multiple biological activity assessment of these four specific wild Thymus species from the Kazdağları region.

• Novelty: The paper highlights that no previous studies have reported the antimicrobial activity of methanolic extracts specifically for T. bornmuelleri, T. citriodorus, or T. sibthorpii.
• Resource Value: The findings establish these taxa as valuable phytochemical resources with potential applications in pharmaceuticals, food preservation, and nutraceuticals.
• Specific Contributions:
  • Identification of T. citriodorus as a superior source of antioxidants and rosmarinic acid.
  • Confirmation of T. bornmuelleri and T. sibthorpii as potent antibacterial agents, likely driven by high thymol content.
  • Demonstration of significant cytotoxic potential against colorectal cancer cells, particularly for T. bornmuelleri and T. citriodorus, suggesting a role for these species in future anticancer research.

The study concludes that while all four species possess beneficial biological effects, their specific phytochemical dominance varies, suggesting they should be prioritized differently depending on the intended application (e.g., antioxidant vs. antimicrobial vs. anticancer).