Method Article

Impact of Different Extraction Methods on the In Vitro Bioactivity Profiles of Terfezia Claveryi

DOI:

10.3791/69950

⸱

March 20th, 2026

 ,  ,  ,  , 
1Department of Biophysics, Department of Basic Medical Sciences, Faculty of Medicine, Kirsehir Ahi Evran University, 2Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Kirsehir Ahi Evran University, 3Department of Physiology, Gulhane Faculty of Medicine, University of Health Sciences, 4Department of Medical Services and Techniques, Medical Laboratory Techniques Program, Vocational School of Health Services, Kirsehir Ahi Evran University, 5Department of Field Crops, Faculty of Agriculture, Kirsehir Ahi Evran University

In This Article

Summary

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The in vitro bioactivity of Terfezia claveryi extracts depended on the biophysical extraction method and solvent polarity. The water extract from maceration-assisted extraction (MAE) exhibited the strongest antioxidant activity, whereas the Soxhlet extraction (SE) extract induced greater cytotoxicity in SH-SY5Y cells. These results demonstrate clear method-dependent differences in the in vitro bioactivity of Terfezia claveryi extracts.

Abstract

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Terfezia claveryi is an edible desert truffle traditionally consumed in arid regions. In recent years, it has attracted increasing scientific interest due to its potential antioxidant, antimicrobial, and therapeutic properties, suggesting possible applications in food and health-related fields. However, despite growing evidence of its biological potential, the influence of different extraction strategies on the recovery of bioactive compounds and the resulting in vitro bioactivity profile of Terfezia claveryi has not yet been clarified in the literature. In this study, the in vitro bioactivity of Terfezia claveryi extracts obtained using different extraction methods and solvents with varying polarity was evaluated. Extraction yield was determined, and the obtained extracts were assessed for antioxidant, antimicrobial, antibiofilm, and cytotoxic activities using established testing systems. Antioxidant capacity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) tests, while cytotoxic effects were investigated in SH-SY5Y neuroblastoma cells. Extraction yield varied depending on both method and solvent polarity, with the highest yield obtained from the aqueous extract prepared by the ultrasound-assisted extraction (UAE) method (18.40%). It was determined that antioxidant activity showed significant differences depending on the extraction method, and aqueous extracts obtained by the maceration-assisted extraction (MAE) method exhibited a profile rich in phenolic compounds along with the strongest radical scavenging capacity. LC-MS/MS analysis of this extract revealed the presence of nine phenolic compounds; quinic acid and chlorogenic acid were identified as the dominant components. In contrast, Soxhlet-derived extracts induced greater cytotoxic effects in SH-SY5Y cells. Antimicrobial and antibiofilm responses displayed relatively limited variation among extraction methods, indicating a weaker dependence on extraction strategy for these activities. Overall, these findings demonstrate that the biophysical extraction method and solvent selection are critical determinants of the in vitro bioactivity profile of Terfezia claveryi extracts, with pronounced effects observed for antioxidant and cytotoxic activities.

Introduction

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The use of various mushroom species to improve health and to prevent and treat diseases dates back to ancient times1,2,3. Macrofungi are generally classified into two groups: Epigeous (mushrooms) and hypogeous (truffles)4. Known as the desert truffle, Terfezia claveryi (T. claveryi) is a world-renowned and edible truffle species belonging to the genus Terfezia5. T. claveryi is commonly distributed in arid and semi-arid regions where Helianthemum species are prevalent....

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Protocol

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NOTE: The overall experimental workflow for the preparation and analysis of T. claveryi extracts is schematically summarized in Figure 1. All materials used in the study are described in the Table of Materials.

1. Fungal material authentication and preparation

  1. Collection and processing of material
    NOTE: Fresh T.claveryi truffles were collected in May 2023 from steppe and sandy, uncultivated (non-agricultural) lands in Kırsehir Province, Central Anatolia, Türkiye. Sampling was carried out at three distinct locations: Dedeli Village (38°54′15.8″....

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Results

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Molecular identification of T. claveryi
PCR amplification of the ITS region was performed using the universal primers ITS1 and ITS4 for molecular identification of T. claveryi. The resulting sequences were analyzed and compared against reference sequences in the NCBI database using the BLAST algorithm, allowing definitive species identification (Supplementary file 1).

Extraction efficiency
Extraction efficiencies vari.......

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Discussion

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The present study establishes a comparative and standardized extraction–bioactivity platform for T. claveryi, demonstrating that the extraction strategy is a decisive determinant of both chemical composition and biological functionality. The primary methodological strength of this protocol lies in the strict control of extraction parameters, particularly the fixed solid–liquid ratio, standardized solvent volume, identical post-extraction processing, and uniform stock solution preparation prior to bio.......

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Disclosures

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The authors declare no competing interests. All data supporting the findings of this study are available within the main manuscript and the supplementary information files. The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgements

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The authors declared that this study has received no financial support.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
96-well polystyrene microtiter platesNest Scientific701001Cell culture / assay plates
ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))Sigma-AldrichA3219Cation radical scavenging assay
Acetone (Analytical grade)Sigma-Aldrich179124Extraction solvent
Acetonitrile (LC-MS grade)Sigma-Aldrich34851Organic mobile phase
Acinetobacter baumanniiATCCManassas, VA, USAATCC 17978
Triple Quad LC-MS/MS with 1290 Infinity UPLC systemAgilent Technologies6460Quantitative analysis
Analytical column ( C18, 4.6 × 100 mm, 3.5 μm)Agilent Technologies (Zorbax)959990-902Reversed-phase column
Antioxidant standards (Ascorbic acid / Trolox)Sigma-AldrichA7506 / 238813Positive controls
Applied Biosystems 3730xl DNA AnalyzersApplied Biosystems 3730xl DNA AnalyzersDNA Sequencing System
Applied Biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing KitApplied Biosystems 4337455Sanger Sequencing Kit
Bacillus cereusATCCManassas, VA, USAATCC 14579
Bioinformatics Analysis, BioEdit software, Algorithm used: CAP contig assemblyIbis BiosciencesNASequence Assembly Software
Cellulose extraction thimbleFisherbrand™ 11724043Used for Soxhlet extraction
Dimethylsulfoxide (DMSO)Merck102952Solvent
DNA isolation kit (Plant&Fungi DNA isolation kit (Poland) )EurX GeneMATRIXE3595DNA isolation 
DPPH (2,2-diphenyl-1-picrylhydrazyl)Sigma-AldrichD9132Radical scavenging assay
Dulbecco's Modified Eagle MediumCapricorn, DMEM-HA
Enterobacter aerogenesATCCManassas, VA, USAATCC 13048
Enterococcus faecalisATCCManassas, VA, USAATCC 29212
Escherichia coliATCCManassas, VA, USAATCC 25922
EthanolMerck100983Solvent
Ethyl acetateMerck109623Extraction Solvent
Fetal Bovine Serum (FBS)Capricorn ScientificFBS-16A
BigDyeTerminator v3.1 Cycle Sequencing KitThermoFisher4337455Sequencing Kit
Folin-Ciocalteu reagentSigma-Aldrich 47641Total phenolic content
Formic acidSigma-Aldrich 33015Mobile phase additive
Gallic acidCarlo Erba406335Calibration curve
Gene MATRIX Plant&Fungi DNA isolation kitEurX E3595DNA isolation kit
GraphPad Prism (v9)Data visualization and statistical analysisN/AStatistical analysis
Heating mantleElectrothermal EME30500Used to maintain solvent reflux
HighPrep™ PCR Clean-upSystemMAGBIOAC-60005
IceLaboratory grade—Temperature control during UAE
Klebsiella pneumoniaeATCCManassas, VA, USAATCC 13883
L-glutamineCapricorn ScientificGermany
Listeria monocytogenesATCCManassas, VA, USAATCC 19115
LyophilizerScientzN/AFreeze-drying of extracts
Magnetic bead–based PCR clean-up systemMAGBIOAC-60005PCR
MethanolMerck106009Extraction solvent
n-HexaneMerck103701Extraction solvent
Penicillin/StreptomycinCapricorn ScientificGermany
Pseudomonas aeruginosaATCCManassas, VA, USAATCC 27853
Filter paper (No. 541)Cytiva1541-125 VIn the extract filtration process
Rotary evaporatorBuchi R10011100V101Solvent removal
Shigella dysenteriaeATCCManassas, VA, USAATCC 13313
Sodium carbonateMerck106392Total phenolic analysis
SPECTROstar Nano Microplate Reader BMG LabtechAbsorbance measurements
Staphylococcus aureusATCCManassas, VA, USAATCC 25923
Staphylococcus epidermidisATCCManassas, VA, USAATCC 12228
Statistical analysis software (SPSS, v23)Data analysisNA
Thermo Scientific Nanodrop 2000 Thermo Fisher ScientificND-2000
Tryptone Soy Agar (TSA)MerckGermany—
Tryptone Soy Broth (TSB)MerckGermany—
Ultrasonic homogenizerScientzSCIENTZ-650LUltrasound-assisted extraction

References

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  1. Wasser, S. P., Weis, A. L. Medicinal properties of substances occurring in higher basidiomycetes mushrooms: current perspectives (Review). Int. J. Med. Mushrooms. 1 (1), 31-62 (1999).
  2. Wasser, S. P.

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Tags

Terfezia ClaveryiExtraction MethodsIn Vitro BioactivityAntioxidant ActivityAntimicrobial ActivityCytotoxic ActivityUltrasound Assisted ExtractionMaceration Assisted ExtractionPhenolic CompoundsLC MS MS Analysis

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