J. Microbiol. Infect. Dis., (2026), Vol. 16(2): 107–117

Research Article

10.5455/JMID.2026.v16.i2.7

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Antioxidant activity of saffron extracts: A comparative study of phenolic and flavonoid content

Zamzam Ali Basher Alshreef^1* and Mahjoubah Salih Altayyib Munayr²

¹Department of Analytical Chemistry, Faculty of Nursing, Fezzan University, Murzuq, Libya

²Chemical Engineering Department, Faculty of Engineering, Sebha University, Sebha, Libya

*Corresponding Author: Zamzam Ali Basher Alshreef. Department of Analytical Chemistry, Faculty of Nursing, Fezzan University, Murzuq, Libya. Email: zam.alshreef [at] fezzanu.edu.ly

Submitted: 01/12/2025 Revised: 05/05/2026 Accepted: 17/05/2026 Published: 06/06/2026

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ABSTRACT

Background: Crocus sativus L., globally recognized as saffron, transcends its traditional culinary utility to emerge as a cornerstone of contemporary pharmacological research. Recent scientific inquiry has pivoted toward its intricate profile of bioactive secondary metabolites, which are increasingly scrutinized for their robust therapeutic potential and their role as pivotal agents in neutralizing oxidative stress.

Aim: The primary objective of this investigation was to systematically evaluate the antioxidant efficacy of saffron stigma extracts while providing a rigorous quantitative assessment of total phenolic content (TPC) and total flavonoid content (TFC) as a function of diverse solvent polarities.

Methods: Crocus sativus L. stigmas were subjected to solid-liquid extraction employing a polarity-based gradient consisting of four solvent systems: aqueous methanol (50% v/v), aqueous propanol (50% v/v), ethyl acetate, and chloroform. The radical scavenging potential of the resulting extracts was evaluated via the 2,2-diphenyl-1-picrylhydrazyl assay. Furthermore, the quantitative determination of TPC and TFC was conducted using the Folin–Ciocalteu and aluminum chloride colorimetric protocols, respectively.

Results: The results indicated that the extraction efficiency of bioactive metabolites from C. sativus L. was significantly influenced by solvent polarity. The 50% methanolic extract demonstrated the highest phytochemical recovery, yielding a maximum mean TPC of 4,822.6 ± 145.2^a µg GAE/g and a TFC of 2,210.0 ± 88.4^a µg QE/g, with an extraction yield of 1.50%. In stark contrast, the chloroform extract exhibited the lowest extraction yields, with 1,411.9 ± 56.5 d µg GAE/g for TPC and 271.1 ± 10.8 µg QE/g for flavonoid content, alongside the minimum overall yield (0.53%). This quantitative superiority was directly reflected in the antioxidant capacity of the extracts. Specifically, the 50% methanolic extract displayed the most potent radical scavenging activity, characterized by the minimum IC₅₀ value of 6.98 ± 0.3^a µg/ml. Conversely, the chloroform extract demonstrated the weakest antioxidant potential, recording the maximum IC₅₀ value of 10.20 ± 0.6^c µg/ml. These findings substantiate a robust linear correlation between the recovery of polar phenolic fractions and the resultant antioxidant potency, indicating that phenolic concentration is a primary determinant of radical scavenging efficiency.

Conclusion: These findings validate the intrinsic dependency of antioxidant potency on phenolic density, demonstrating that the scavenging efficiency is a direct function of the successful recovery of polar secondary metabolites during the extraction process.

Keywords: Crocus sativus L., DPPH assay, Total phenolic content, Total flavonoid content, Solvent polarity, IC₅₀.

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Introduction

Crocus sativus L., commonly known as saffron, is a perennial, stemless geophyte belonging to the Iridaceae family (Pitsikas, 2016). It is extensively cultivated in arid and semi-arid regions, with Iran serving as the global epicenter of production. Significant cultivation also occurs in Spain, Greece, Turkey, France, Italy, Egypt, Switzerland, Morocco, Azerbaijan, India, Pakistan, China, the United Arab Emirates, New Zealand, Japan, and Australia (Abdullaev, 1993; Abdullaev and Espinosa-Aguirre, 2004; Abdullaev et al., 2007; Zarinkamar et al., 2011). Valued for its unique organoleptic profile, saffron is characterized by a pungent flavor and a distinctive honey-like aroma. Etymologically, “saffron” is derived from the Arabic “zafaran,” meaning “yellow,” which reflects the high concentration of carotenoid pigments in the stigmas that confer the spice’s characteristic golden-yellow hue (Bostan et al., 2015). The anatomical architecture of the C. sativus flower is illustrated in Figure 1. Recent investigations in Iran have underscored the critical influence of extraction methodologies and solvent systems on the antimicrobial efficacy of saffron petals (p ≤ 0.01). Notably, ethanolic extracts obtained via ultrasonic spore infusion demonstrated potent inhibitory activity against Staphylococcus epidermidis, with an efficacy comparable to the reference antibiotic rifampin (Seyyedeh and Mansureh, 2025). These findings highlight the viability of repurposing saffron floral bio-residues as sustainable precursors for low-cost, natural antimicrobial agents. Contemporary literature further identifies C. sativus by-products as rich reservoirs of bioactive metabolites, including polyphenols, flavonoids, and carotenoids (Lahmass et al., 2018). These antioxidants hold significant potential for integration into diverse industrial sectors, including food science, cosmetics, and pharmacology. Among floral bio-residues, tepals (undifferentiated petals and sepals) have emerged as promising candidates for high-value applications due to their abundance of flavonols and anthocyanins. Specifically, saffron tepals represent one of the richest natural sources of kaempferol and its glycosidic derivatives (Mottaghipisheh et al., 2020; Stelluti et al., 2021; Khadfy et al., 2023), a profile that underpins their documented anti-inflammatory, antinociceptive, and antioxidant properties (Cardone et al., 2020). The global shift toward plant-based medicine is driven by minimal adverse effects and high patient compliance (Ning et al., 2021; Savchenko et al., 2022; Gonfa et al., 2023; Liu et al., 2023; Matera et al., 2023; Nemaa et al., 2023). According to the World Health Organization, over 20,000 medicinal plant species contribute to approximately 50% of modern pharmaceuticals (Vaou et al., 2021). Key phytochemicals—including phenolic compounds, saponins, proanthocyanidins, nitrogenous compounds, alkaloids, and terpenoids—exhibit significant pharmacological properties (Vernarelli and Lambert, 2017; Lim et al., 2019; Ginwala et al., 2019; Alam et al., 2020; Li et al., 2023; Yoon et al., 2023). Contemporary research increasingly focuses on these bioactive constituents due to their diverse biological applications, particularly in mitigating oxidative stress-associated conditions (Forni et al., 2019; Guan et al., 2021; He et al., 2023; Muscolo et al., 2024).

Among saffron’s constituents, safranal has demonstrated a broad pharmacological spectrum, including anxiolytic, anticonvulsant, and anticancer activities, as well as the attenuation of subacute diazinon toxicity (Hosseinzadeh and Ghenaati, 2006; Maggi et al., 2011). The antioxidant and free-radical scavenging activities of saffron are primarily attributed to its phenolic and flavonoid profiles, with gallic acid and pyrogallol identified as major representative compounds. These metabolites serve as critical inhibitors of various oxidizing molecules (Serrano-Díaz et al., 2012; Rahaiee et al., 2015). Comparative assessments using DPPH and FRAP assays indicate that methanolic extracts of saffron stigmas possess superior antioxidant potency relative to aqueous or ethanolic counterparts, an effect directly correlated with total phenolic (TPC) and flavonoid (TFC) concentrations (Papandreou et al., 2006; Karimi et al., 2010). A recent study by (Seyyedeh and Mansureh, 2025) explored the valorization of C. sativus petals as a sustainable source of antimicrobial agents. The researchers evaluated various classical and modern extraction techniques, concluding that solvent type and extraction method significantly influenced both yield and bioactivity (Pleq 0.01). Notably, ethanolic extracts obtained via ultrasonic-assisted extraction and shaking maceration exhibited potent inhibitory effects against Staphylococcus epidermidis, with an inhibition zone (~23 mm) comparable to the reference antibiotic rifampin. These findings highlight the potential of repurposing saffron bio-waste into high-value, cost-effective natural pharmaceuticals. In a recent invivo study (Ahmet et al., 2024), the antioxidant efficacy of C. sativus and its derivatives (safranal and crocin) was evaluated against carbon tetrachloride (CCl4)-induced toxicity. The results established that saffron administration significantly countered the depletion of fat-soluble vitamins (A, D, E, and K) and inhibited the formation of protein carbonyls (PCO), a hallmark of oxidative protein damage (p < 0.01). This hepatoprotective effect is attributed to the ability of saffron’s polyphenols to stabilize cellular membranes and neutralize reactive free radicals, thereby preventing the oxidative chain reactions triggered by chemical pollutants.

Fig. 1. Structure of Crocus sativus flower.

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Materials and Methods

Materials

All chemicals and reagents utilized in this study were of analytical grade and used without further purification. The following reagents were procured from Sigma-Aldrich (Merck, Germany): antioxidant standards and assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH, 98%), Gallic acid standard (98%), and Ascorbic acid (98%). Colorimetric reagents: Folin–Ciocalteu reagent and Sodium carbonate (Na₂CO₃, 98%). Extraction solvents: Methanol (95%), Propanol (99.9%), Ethyl acetate (99.5%), and Chloroform (99%). Other reagents: Potassium persulfate (99%). Double-distilled water was employed throughout all experimental procedures and solution preparations. All experiments were repeated four times.

Preparation of plant extracts

Preparation of C. sativus methanol extract

To maximize the extraction surface area, dried C. sativus stigmas were pulverized into a fine, homogeneous powder. A 5 g aliquot of the ground material was subjected to cold maceration in 100 ml of a 50% (v/v) aqueous-methanol solution. The extraction was conducted over a 24 to 48 hours period at ambient temperature (25°C ± 2°C) under constant agitation using an orbital shaker (SO1, Stuart Scientific, England) to ensure the efficient recovery of bioactive constituents. Subsequently, the suspension was clarified by filtration through Whatman No. 1 filter paper. The resulting filtrate was concentrated to dryness via vacuum evaporation under reduced pressure using a rotary evaporator. The final weight of the crude extract was recorded at 0.0751 g, representing an extraction yield of 1.50% (w/w). The obtained extract was stored in an amber glass vial at 4°C to maintain phytochemical stability for subsequent analytical and biological assessments.

Extraction of C. sativus using aqueous propanol system

To enhance the solvent-to-solid contact, dehydrated C. sativus stigmas were pulverized into a fine, homogeneous powder. A 5.0 g aliquot of the powdered biomass was subjected to maceration in 100 ml of a 50% (v/v) aqueous propanol solution for 24 to 48 hours at ambient temperature (25°C ± 2°C). Constant orbital agitation was applied to ensure maximal mass transfer of bioactive constituents. Subsequently, the suspension was clarified via filtration through Whatman No. 1 filter paper. The solvent was eliminated under reduced pressure using a rotary evaporator (maintained at 40°C) to obtain the concentrated crude extract. The final weight of the resultant extract was recorded at 0.0440 g, corresponding to an extraction yield of 0.88% (w/w). The sample was stored in amber glass vials at 4°C to prevent photo-degradation and maintain phytochemical stability until further characterization.

Preparation of C. sativus ethyl acetate extract

To optimize the extraction efficiency, 5 g of finely ground C. sativus stigmas were macerated in 100 ml of ethyl acetate. The extraction was carried out at ambient temperature (25°C ± 2°C) for a duration of 24 to 48 hours under periodic orbital agitation to facilitate the diffusion of semi-polar metabolites. Following the incubation period, the mixture was clarified by filtration through Whatman No. 1 filter paper. The solvent was subsequently removed using a rotary evaporator at a controlled temperature of 40°C under reduced pressure. The final weight of the crude extract was 0.0304 g, corresponding to an extraction yield of 0.61% (w/w). The extract was preserved in dark amber glass vials and stored at 4°C to ensure the stability of the bioactive constituents for subsequent phytochemical and biological analyses.

Preparation of C. sativus chloroform extract

To maximize the extraction efficiency, dehydrated C. sativus stigmas were pulverized into a fine powder. A 5 g aliquot of the resultant powder was subjected to cold maceration in 100 ml of analytical-grade chloroform for 24 to 48 hours at ambient temperature (25°C ± 2°C). To facilitate the optimal diffusion of lipophilic constituents, the mixture was maintained under periodic orbital agitation. Subsequently, the suspension was clarified via filtration through Whatman No. 1 filter paper to remove insoluble residues. The solvent was then eliminated under reduced pressure using a rotary evaporator to obtain the concentrated crude extract. The final weight of the extract was 0.0263 g, representing an extraction yield of 0.53% (w/w). The extract was preserved in amber glass vials and stored at 4°C to maintain its phytochemical stability for subsequent analytical assessments.

DPPH radical scavenging assay

The antioxidant capacity of the C. sativus extracts was evaluated using the DPPH radical scavenging assay, based on the methodology described by Singh et al. (2018) with minor modifications. Briefly, a 0.1 mM methanolic solution of DPPH was prepared. Aliquots of the extracts at various concentration gradients were mixed with an equal volume of the DPPH solution. The reaction mixture was subsequently incubated in the dark at ambient temperature for 30 minutes to facilitate stable radical-antioxidant interaction. The absorbance of the resulting solutions was measured at λ=517 nm using a UV-Vis spectrophotometer. Ascorbic acid served as the positive reference standard, while pure methanol was utilized as the blank. The radical scavenging activity, expressed as the percentage of inhibition, was calculated according to the following equation:

where A_C represents the absorbance of the DPPH radical solution without the sample, and A_S denotes the absorbance of the DPPH solution in the presence of the extract.

Determination of IC₅₀ value

The antioxidant potency of the C. sativus extracts was quantified by determining the half-maximal inhibitory concentration (MIC) (IC₅₀), defined as the extract concentration required to scavenge 50% of the initial DPPH free radicals. The radical scavenging activity (RSA) was established by assessing the absorbance of the sample (As) relative to the control (Ac). To derive the IC₅₀ values, the inhibition percentages were plotted against the corresponding extract concentrations. The values were calculated using non-linear regression analysis (or linear interpolation from the dose-response curve). For comparative assessment, ascorbic acid was utilized as the benchmark reference standard. In this context, a lower IC₅₀ value signifies a superior antioxidant capacity, as it reflects the minimum concentration necessary to achieve significant radical neutralization.

TPC in C. sativus

The TPC of the C. sativus. extracts was quantified using the Folin–Ciocalteu colorimetric method, as described by Nurhanan and Rosli (2014) . Gallic acid was utilized as the reference standard to establish a calibration curve across a concentration range of 5–50 µg/ml. The resulting curve exhibited a high coefficient of determination (R (Abdullaev and Espinosa-Aguirre, 2004) > 0.99), confirming the linearity, accuracy, and reproducibility of the analytical procedure within the specified range. The TPC values are expressed as micrograms of gallic acid equivalents (µg GAE) per gram of dry weight. The adoption of gallic acid as a benchmark ensures standardized results, facilitating a robust comparative analysis with existing literature on diverse botanical matrices.

TFC in C. sativus L

The TFC of the C. sativus extracts was determined using the aluminum chloride colorimetric assay, following the protocol described by Sadh et al. (2018) with minor modifications. Briefly, a 2 ml aliquot of the extract was sequentially mixed with 0.2 ml of 5% (w/v) sodium nitrite (NaNO₂) and 0.2 ml of 10% (w/v) aluminum chloride (AlCl₃). After an incubation period of 6 minutes, 2 ml of 1 M sodium hydroxide (NaOH) was added to the mixture. The final volume of the reaction mixture was adjusted using distilled water. The absorbance was subsequently measured at λ=510 nm using a UV-Vis spectrophotometer. Quercetin was employed as the standard for the calibration curve (R (Abdullaev and Espinosa-Aguirre, 2004) > 0.99). The flavonoid concentration was calculated and expressed as micrograms of quercetin equivalents per gram of extract µg QE/g).

Statistical analysis

All experiments were performed in quadruplicate (n=4) to ensure reproducibility. Data are presented as Mean ± SD. Statistical significance was evaluated using One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. All calculations and linear regressions (R²) were performed using GraphPad Prism (Version 9.0) or SPSS (Version 26.0). A p-value of <0.05 was considered statistically significant.

Ethical approval

Not needed for this study.

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Results

Antioxidant activities in saffron extract

The antioxidant capacity of C. sativus L. stigmas was evaluated across four solvent systems using the DPPH assay. As shown in Figure 2, the ascorbic acid standard exhibited a robust inhibition range (45.2%–98.2%). All extracts displayed a significant dose-dependent scavenging trajectory (Table 1). Aqueous methanolic extract (50%): Demonstrated the highest potency with an inhibition range of 38.15%–94.13% and a minimum IC₅₀ of 6.98 ± 0.3^a µg/ml (Fig. 3). Aqueous propanolic extract (50%): followed with an inhibition range of 32.40%–86.45% and an IC₅₀ of 8.50 ± 0.4^bµg/ml (Fig. 4). Ethyl acetate extract: recorded an inhibition range of 25.60%–72.10% with an IC₅₀ of 9.94 ± 0.5^c µg/ml (Fig. 5). Chloroform extract: exhibited the lowest activity 18.20%–54.30% with the highest IC₅₀ of 10.20 ± 0.6^c µg/ml (Fig. 6).

Fig. 2. DPPH radical scavenging activity of ascorbic acid as a positive reference standard.

Table 1. Presents the extraction yield, TPC, TFC, and antioxidant activity of C. sativus stigmas.

Fig. 3. Dose-response relationship of Crocus sativus L. methanolic extract on DPPH radical scavenging activity.

Fig. 4. Dose-response relationship of Crocus sativus L. propanolic extract on DPPH radical scavenging activity.

Total phenolic compounds (TPC)

The recovery of polyphenolic constituents from C. sativus L. was significantly influenced by the solvent's physicochemical properties, specifically the polarity and dielectric constant. The results are detailed in the following subsections.

TPC in methanolic extract (50% v/v)

To optimize the recovery of polyphenolic constituents from C. sativus L., a binary aqueous-methanolic system (50% v/v) was strategically employed. As presented in Table 1, the TPC was quantified using a gallic acid calibration curve, which demonstrated excellent linearity (R (Abdullaev and Espinosa-Aguirre, 2004) > 0.99). The TPC values exhibited a consistent distribution, ranging from 4,650 to 4,985 µg GAE/g, with a mean concentration of 4,822.6 ±145.2 µg GAE/g. Statistical analysis (ANOVA, p < 0.05) revealed that this hydro-alcoholic matrix yielded significantly higher phenolic concentrations compared to the less polar solvents, which is indicated by the distinct superscript letter (^a).

Fig. 5. Dose-response relationship of Crocus sativus L. ethyl acetate extract on DPPH radical scavenging activity.

Fig. 6. Dose-response relationship of Crocus sativus L. chloroform extract on DPPH radical scavenging activity.

TPC in propanol extract of C. sativus L

Under identical experimental conditions, the aqueous propanol system yielded a mean TPC of 3,670.6 ± 110.1 µg GAE/g, with observed values ranging from 3,520 to 3,815 µg GAE/g. According to the data summarized in Table 1, this yield was significantly lower (p < 0.05) than that of the methanolic extract, which is indicated by the distinct superscript letter (ᵇ).

TPC in ethyl acetate extract of C. sativus L

The TPC for the ethyl acetate fraction was established at 2,661.9 ± 93.2 µg GAE/g, with values exhibiting a precise distribution ranging from 2,540 to 2,795 µg GAE/g. As illustrated in Table 1, this yield represents a significant decrease (p < 0.05) compared to the aqueous-alcoholic systems, as indicated by the distinct superscript letter (ᶜ).

TPC in chloroform extract of C. sativus L

The chloroform extract exhibited the lowest phenolic recovery among the tested solvents, with values ranging from 1,355 to 1,468 µg GAE/g and a mean concentration of 1,411.9 ± 56.5 µg GAE/g. As shown in Table 1, this yield was significantly lower (p < 0.05) than all other fractions, as indicated by the superscript letter (ᵈ).

TFC of C. sativus L

The TFC was quantitatively assessed across various solvent systems to evaluate their respective extraction efficiencies for the bioactive flavonoid fractions of C. sativus L. (saffron). Quantification was achieved through a spectrophotometric assay, and the results were normalized against standard equivalents to ensure analytical comparability. The following subsections detail the flavonoid yields and their correlation with solvent physicochemical properties.

Determination of (TFC) in C. sativus L. of methanolic extract

The flavonoid recovery in the aqueous methanolic extract demonstrated high analytical consistency, with values ranging from 2,125 to 2,305 µg QE/g. As detailed in Table 1, the mean TFC was established at 2,210.0 ± 88.4 µg QE/g. Statistical evaluation (One-way ANOVA) confirmed that this yield was significantly higher (p < 0.05) than all other solvent systems, as indicated by the superscript letter (ᵃ).

Quantification of TFC in C. sativus L. of propanol extract

The TFC in the propanol extracts exhibited a consistent distribution, with values ranging from 545 to 618 µg QE/g. As illustrated in Table 1, the mean TFC was precisely established at 580.0 ± 37.0 µg QE/g. Statistical analysis (One-way ANOVA) indicated that this yield was significantly lower (p < 0.05) than that of the methanolic extract, as denoted by the superscript letter (ᵇ).

Analysis of (TFC) in ethyl acetate extract of C. sativus

The TFC of the C. sativus L. ethyl acetate extracts exhibited a precise concentration ranging from 352 to 388 µg QE/g. As summarized in Table 1, statistical evaluation established the mean TFC at 370.2 ± 14.8 µg QE/g. According to the One-way ANOVA and Tukey’s post-hoc test, this yield was significantly lower (p < 0.05) than the aqueous-alcoholic systems, as indicated by the superscript letter (ᶜ).

TFC in chloroform extracts and comprehensive discussion

The chloroform extracts of C. sativus L. exhibited the lowest flavonoid recovery among the tested solvents, with values ranging from 260 to 282 µg QE/g. As shown in Table 1, statistical analysis established the mean TFC at 271.1 ± 10.8 µg QE/g, representing a significant reduction (p < 0.05) compared to all other fractions, as indicated by the superscript letter (ᵈ).

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Discussion

The pronounced antioxidant superiority of the aqueous methanolic extract is fundamentally governed by the “solvent-analyte” interaction dynamics. Saffron’s primary bioactive profile is dominated by crocins (glycosyl esters of crocetin) and kaempferol glycosides. These molecules possess multiple hydroxyl groups and sugar moieties that confer high molecular polarity. The 50% methanolic system, characterized by a high dielectric constant, facilitates optimal mass transfer through hydrogen bonding and dipole-dipole interactions, ensuring the comprehensive recovery of these polar antioxidants. The mechanism of action is primarily driven by hydrogen atom transfer. Crocins, with their extensive conjugated double-bond system (polyene chain), effectively delocalize the unpaired electron of the DPPH radical, leading to its stabilization. In contrast, the significant decline in efficacy observed in ethyl acetate and chloroform extracts is attributed to their inability to solvate glycosylated structures. In these non-polar media, the antioxidant activity is likely restricted to a minor fraction of lipophilic aglycones or methylated flavonoids, which are present in trace amounts compared to the polar crocins. Statistical Rigor: Data precision was confirmed by non-linear regression analysis of the sigmoidal dose-response curves, yielding coefficients of determination (R (Abdullaev and Espinosa-Aguirre, 2004) > 0.997) across all experimental sets. The IC₅₀ values are reported as Mean ± SD for n=4 replicates. Statistical significance (p < 0.05) was established using One-way ANOVA followed by Tukey’s post-hoc test, ensuring that the reported potency hierarchy is scientifically robust and reproducible. The selection of the DPPH assay as the primary method for evaluating antioxidant capacity is based on its high stability, simplicity, and its specific ability to assess the hydrogen-donating potential of saffron’s bioactive metabolites, such as crocin and kaempferol glycosides. While DPPH provides a robust initial screening of radical scavenging efficiency, it is acknowledged that this assay primarily reflects one facet of antioxidant mechanisms. Future studies will incorporate complementary orthogonal assays, such as ferric reducing antioxidant power (FRAP) and (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS), , to provide a more comprehensive characterization of the multi-mechanistic antioxidant profile of C. sativus extracts. As illustrated in Figure 7, a dual-axis comparative profiling was established to evaluate the relationship between maximal radical scavenging percentages and the calculated IC₅₀ values. The figure clearly depicts a perfect inverse correlation; the 50% methanolic extract attained the highest inhibition zenith (94.13%) corresponding to the most significant potency dip in IC₅₀ (6.98 ± 0.3^a µg g/ml). This synchronized visualization effectively highlights the “potency-concentration” trade-off across the solvent polarity gradient, confirming that the polar fractions are the primary drivers of saffron’s antioxidant efficacy.

Fig. 7. Comparative profiling of antioxidant scavenging activity and IC₅₀ values of Crocus sativus L. extracts across different solvent systems. Note: Statistical significance (p < 0.05) is indicated by lowercase letters for inhibition percentages and italicized letters for IC₅₀ values across different solvent systems.

These findings highlight the critical influence of solvent polarity and synergistic interactions; specifically, the aqueous fraction promotes the swelling of the plant cellular matrix and enhances membrane permeability, while the methanolic component, characterized by a high dielectric constant, ensures the efficient solvation of antioxidant-rich metabolites through hydrogen bonding. The low relative standard deviation (RSD=3.01%) and the narrow confidence intervals confirm the high analytical precision of the procedure and the stability of the phenolic compounds within this polar system. These results underscore that the majority of saffron’s bioactive polyphenols are hydrophilic, necessitating a high-polarity medium for comprehensive recovery.

Despite the water-induced swelling of the plant matrix, which facilitates solvent penetration, the lower yield reflects propanol’s reduced affinity for highly glycosylated polyphenols (such as crocin) compared to the methanolic system. This outcome aligns with the “like-dissolves-like” principle, as the moderate relative polarity of propanol (0.617) favors the recovery of semi-polar aglycones rather than highly polar glycosides. The analytical consistency of this extraction was confirmed by a low relative standard deviation (RSD=3.00%), demonstrating high precision across the replicates (n=4) and ensuring the reliability of the comparative assessment between the alcoholic solvent systems.

This relatively lower phenolic recovery is scientifically attributed to the solvent's intermediate polarity (relative polarity=0.229). Ethyl acetate acts as a selective medium that primarily targets lipophilic phenolic fractions and flavonoids in their aglycone forms. However, it exhibits restricted efficiency in recovering the highly polar glycosylated polyphenols (e.g., crocin) that dominate the C. sativus phytochemical profile. Consequently, while ethyl acetate provides a lower total yield, it serves to concentrate specific semi-polar antioxidant constituents that exhibit limited solubility in purely aqueous or highly polar phases. The analytical rigor of this extraction was confirmed by a low relative standard deviation (RSD=3.50%), which underscores the consistency and reliability of the process across replicates (n=4) for these specific phenolic subclasses.

A comparative assessment across the four solvent systems reveals a distinct hierarchy in extraction efficiency that directly correlates with the solvent polarity gradient. The mean TPC yields followed the descending order: Methanol > Propanol > Ethyl Acetate > Chloroform. The superior performance of the 50% aqueous methanolic system is attributed to its high dielectric constant; while the aqueous fraction promotes the swelling of the plant matrix, the polar organic component ensures the efficient solvation of hydrophilic phenolics through hydrogen bonding. In contrast, the significantly lower yield obtained with chloroform (Relative Polarity=0.259) underscores its inefficiency in solvating the predominantly polar phytochemicals of C. sativus. This observed pattern confirms that the physicochemical properties of the solvent, specifically polarity and dielectric constant are the primary determinants of phenolic solubility. These findings validate the use of high-polarity binary solvent systems as the most effective strategy for the comprehensive recovery of antioxidant-rich fractions from saffron. The analytical precision was further confirmed by a low relative standard deviation (RSD=4.00%) across the replicates (n=4). A previous study (Ana et al., 2023 ) reported that the ultrasound-assisted extraction (UAE)-MAE synergistic approach, using a 50:50 methanol-water system, was the most effective for C. sativus extraction, yielding a peak TPC of 31,560 µg GAE/g and superior antioxidant activity (83.24%).

This superior yield is primarily attributed to the protic nature of the methanol-water system, which facilitates the formation of strong hydrogen bonds with the hydroxyl groups of flavonoids. Specifically, the predominant flavonoids in saffron, such as kaempferol glycosides, possess sugar moieties that significantly increase their molecular polarity. The high dielectric constant of the binary solvent ensures the stabilization of these glycosylated structures during the extraction process. The analytical precision was verified by a low relative standard deviation (RSD=4.00%), confirming the efficacy of the methanolic matrix in recovering these polar metabolites across the replicates (n=4). These findings further validate that the primary antioxidant-rich flavonoid fractions in C. sativus are predominantly hydrophilic in nature.

This reduction in yield, relative to the methanolic system, is scientifically attributed to the decreased dielectric constant and lower relative polarity (0.617) of propanol. Unlike methanol, propanol exhibits a reduced affinity for highly glycosylated flavonoid structures, such as kaempferol glucosides, which dominate the phytochemical profile of C. sativus stigmas. The extraction of these compounds is energetically favored in more hydrophilic, protic solvents that can effectively solvate the sugar moieties through dipole-dipole interactions. These findings confirm that the primary flavonoid fraction in the analyzed samples is predominantly polar, thereby limiting its solubility in less polar propanolic media. The analytical reliability was validated by a relative standard deviation (RSD=6.38%) across the four replicates (n=4), reflecting the high sensitivity of flavonoid solubility to solvent polarity gradients.

Ethyl acetate, characterized as a semi-polar aprotic solvent, demonstrated a significantly lower recovery compared to the aqueous-methanolic system. This efficiency gap is primarily attributed to its inability to facilitate the necessary hydrogen-bonding networks required to solubilize highly polar flavonoid glycosides, the predominant polyphenolic forms in saffron. While ethyl acetate exhibits a higher affinity for lipophilic flavonoid aglycones or methylated derivatives, its restricted dielectric constant limits the extraction of glycosylated structures. Consequently, the lower recovery observed here provides further evidence that the flavonoid profile of the analyzed saffron is primarily composed of polar, sugar-bound derivatives, which remain poorly soluble in an aprotic organic matrix. The analytical consistency was validated by a low relative standard deviation (RSD=4.00%) across the four replicates (n=4), reflecting the selective nature of this solvent system and the high reproducibility of the results.

Chloroform, a non-polar solvent with a low dielectric constant, is inefficient in solvating the vast majority of saffron flavonoids, such as glycosylated kaempferol and quercetin derivatives. These molecules possess high molecular polarity due to their sugar moieties and multiple hydroxyl groups. The trace amounts detected likely represent a minor fraction of lipophilic flavonoid aglycones or methylated flavones. This study underscores the critical influence of solvent polarity on the mass transfer and extraction efficiency of bioactive secondary metabolites from C. sativus stigmas. Our findings demonstrate that the 50% aqueous methanolic system is the most potent medium for recovering phenolic and flavonoid fractions, which directly correlates with its superior antioxidant capacity (IC₅₀=6.98 ± 0.3 µg/ml). This confirms that saffron’s primary antioxidants are predominantly polar glycosylated constituents, exhibiting maximum solubility in protic polar solvents. While this research provides robust quantitative data across four distinct solvent systems, future investigations should incorporate a broader range of solvents and orthogonal antioxidant assays (e.g., FRAP and ABTS) to further elucidate the plant's therapeutic potential. These findings are consistent with previous literature. For instance, Ghizlane et al. (2023) reported significant variability in C. sativus quality, with TPC values ranging from 31,620 to 62,710 µg GAE/g and crocin content ranging from 137,440 to 228,390 µg/g. The low relative standard deviation (RSD=4.00%) across the replicates (n=4) validates the analytical precision and the reliability of these findings. A prior study demonstrated that ethanol was the optimal solvent for C. sativus anthers, yielding the highest TPC (7,290 µg GAE/g) and TFC (3,770 µgQE/g). The ethanolic extracts exhibited superior antioxidant activity (88%), with RP-HPLC identifying gallic acid and quercetin as the primary bioactive constituents. Similarly, Huda et al. (2023) demonstrated that ethanol was the optimal solvent for C. sativus anthers, yielding the highest TPC 7,290 µg GAE/g) and TFC (3,770 µgQE/g). The ethanolic extracts exhibited superior antioxidant activity (88%), with RP-HPLC analysis identifying gallic acid and quercetin as the primary bioactive constituents. Building upon the advancements in green extraction methodologies discussed previously, Chaimae et al. (2025) further optimized the recovery of bioactive polyphenols from both the stigmas and corm residues of C. sativus L. using UAE. While earlier investigations primarily focused on conventional solvent extraction, this study implemented a response surface methodology (RSM) based on a Box-Behnken design to fine-tune extraction temperature, duration, and solid-to-liquid ratios. For saffron stigmas, the established optimal conditions (50°C for 15 minutes) yielded an exceptional TPC of 118,550 μg GAE/g and a superior antioxidant potency with an IC₅₀ of 23 μg/ml. In contrast, corm residues exhibited a TPC of 21,180 μg GAE/g and a significantly higher IC₅₀ (1,020 μg/ml). These findings corroborate the results reported by Chaimae et al. (2025) regarding the impact of operational parameters on metabolite yield, yet Slimani’s work stands out by validating a robust Quadratic Model tailored for industrial-scale scalability. In addition to the aforementioned studies, Elgudayem et al. (2023) investigated the bioprocessing and functional characterization of Polygonum equisetiforme roots as a promising source of high-quality proteins, unsaturated fatty acids, and bioactive molecules. This study focused on optimizing the ultrasound-assisted aqueous extraction (UAE) of phenolic compounds by employing a Box-Behnken design integrated with statistical modeling. The researchers systematically evaluated the synergistic interactions between three independent operational variables: extraction temperature (30°C–70°C), UAE duration (1–9 minutes), and a liquid-to-solid ratio ranging from 35,000 to 45,000 μl/g to maximize both phenolic yield and DPPH RSA. The mathematical modeling identified the optimal extraction parameters at 50°C for 5 minutes with a liquid-to-solid ratio of 40,000 μgl/g. Under these optimized conditions, the TPC achieved a peak value of 45,321 μg GAE/g of dry weight, while the antioxidant capacity reached 120,354 μg mol Trolox/g of dry weight. Comprehensive biochemical characterization via fourier-transform infrared spectroscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy corroborated that the roots are exceptionally rich in water-soluble phenols, high-potency anthocyanins, and condensed tannins. Furthermore, the extracts exhibited a robust correlation between their phytochemical profile and biological efficacy, specifically within FRAP and ABTS scavenging assays. Antimicrobial evaluations demonstrated significant inhibitory potency against various pathogenic bacteria, where the lowest minimum inhibitory concentration was recorded against Staphylococcus aureus, followed by Bacillus species. The study concluded that the integration of UAE with RSM provides a highly efficient, sustainable, and scalable framework for the recovery of potent bioactive ingredients for pharmaceutical and nutraceutical applications.

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Acknowledgments

We extend our deepest appreciation for biodiversity to both the Faculty of Science at Sebha University and the Sebha Central Laboratory.

Funding

None.

Authors’ contributions

Mahjoubah Salih Altayyib Munayr: Collected the data, contributed data or analysis tools, performed the analysis and interpretation of data, and drafting the manuscript/revising for important intellectual context. Zamzam Ali Basher Alshreef: Conceived & design of the study, collected the data, and drafting the manuscript/revising for important intellectual context. Both authors approved of the final version of the manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

All data were provided in the manuscript.

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References

Abdullaev, F., Ortega, C.H. and Miranda, P.R. 2007. HPLC quantification of major active components from 11 different saffron (Crocus sativus L.) sources. Food. Chem. 100(3), 1126–1131.

Abdullaev, F.I. and Espinosa-Aguirre, J.J. 2004. Biomedical properties of saffron and its potential use in cancer therapy and chemoprevention trials. Cancer. Detection. Prevention. 28(6), 426–432.

Abdullaev, F.I. 1993. Biological effects of saffron. Biofactors 4(2), 83–86.

Ahmet, B.A., Damla, Y.D., Damla, E.S., Gokhan, O.G., Ibrahim, A. and Irfan, B. 2024. Protective effects of saffron, safranal and crocin administration on vitamins (A, D, E, K) and protein carbonyl levels against CCl4 induced oxidative damage in rats. Marmara Med. J. 37(3), 344–352.

Alam, W., Khan, H., Shah, M.A., Cauli, O. and Saso, L. 2020. Kaempferol as a dietary anti-inflammatory agent: current therapeutic standing. Molecules 25(18), 4073.

Ana, B.M., Juan, J.V., Francisca, P.L., María, E.C., María, T.G., María, J.F., Antonio, C., Josefa, H.-R. and Marino, B.A. 2023. Antioxidant activity, total phenolic and flavonoid contents in floral saffron bio-residues. Processes 11, 1400; doi: 10.3390/pr11051400

Bostan, H.B., Mehri, S. and Hosseinzadeh, H. 2015. Toxicology effects of saffron and its constituents: a review. Iranian J. Basic Med. Sci. 20(2), 113–121.

Cardone, L., Castronuovo, D., Perniola, M., Cicco, N. and Candido, V. 2020. Saffron (Crocus sativus L.), the king of spices: an overview. Sci. Horticult. 272, 109560.

Chaimae, S., Mouhcine, F., Chaimae, R., Laila, E., Meryem, B., Hyeong-Moo, S., John, P.G., Abderrahim, L., Mahmoud, M.A., Khalid, M. and Mourad, A.M. Extraction of natural antioxidants from Moroccan saffron (Crocus sativus L.) using ultrasound-assisted extraction: an optimization approach with box-behnken design. Ultrason. Sonochem. 120(2025), 107448.

Elgudayem., Aldiyab, A., Aziz., Omran., Ebru., Saghrouchni., Var, I., Md, A., SabaghA, E., Sakran, M. and Ahmed, C. 2023. Box-Behnken design based optimization of phenolic extractions from Polygonum equisetiforme roots linked to its antioxidant and antibacterial efficiencies. Front. Sustain. Food Syst. 7, 1141207; doi:10.3389/fsufs.2023.1141207

Forni, C., Facchiano, F., Bartoli, M., Pieretti, S., Facchiano, A., D’Arcangelo, D., Norelli, S., Valle, G., Nisini, R., Beninati, S., Tabolacci, C. and Jadeja, R.N. 2019. Beneficial role of phytochemicals on oxidative stress and age-related diseases. Biomed. Res. Int. 2019, 8748253.

Ghizlane, K., Lahcen, H., Nadia, N., Karim., Said, E., MarieL, F. and Hafida. 2023. Aromatic potential, quality and antioxidant activity of saffron grown in Morocco. Flavour Fragr. J. 38, 13–26.

Ginwala, R., Bhavsar, R., Chigbu, D.G.I., Jain, P. and Khan, Z.K. 2019. Potential role of flavonoids in treating chronic inflammatory diseases with a special focus on the anti-inflammatory activity of apigenin. Antioxidants 8(2), 35.

Gonfa, Y.H., Tessema, F.B., Bachheti, A., Rai, N., Tadesse, M.G., Nasser Singab, A., Chaubey, K.K. and Bachheti, R.K. 2023. Anti-inflammatory activity of phytochemicals from medicinal plants and their nanoparticles: a review. Curr. Res. Biotechnol. 6(6), 100152.

Guan, R., Van Le, Q., Yang, H., Zhang, D., Gu, H., Yang, Y., Sonne, C., Lam, S.S., Zhong, J., Jianguang, Z., Liu, R. and Peng, W. 2021. A review of dietary phytochemicals and their relation to oxidative stress and human diseases. Chemosphere 271, 129499.

He, W.J., Lv, C.H., Chen, Z., Shi, M., Zeng, C.X., Hou, D.X. and Qin, S. 2023. The regulatory effect of phytochemicals on chronic diseases by targeting Nrf2-ARE signaling pathway. Antioxidants 12(2), 236.

Hosseinzadeh, H. and Ghenaati, J. 2006. Evaluation of the antitussive effect of stigma and petals of saffron (Crocus sativus) and its components, safranal and crocin in guinea pigs. Fitoterapia 77(6), 446–448.

Huda., Ali., Virginia, D.A. and ThiresiaT, T. 2023. Extraction of phenolic and flavonoid compounds and evaluation of their antioxidant activity in saffron anthers (their antioxidant activity in saffron anthers (Crocus sativus L.),. Not Sci. Biol. 15(4), 11640.

Karimi, E., Oskoueian, E., Hendra, R. and Jaafar, H.Z.E. 2010. Evaluation of Crocus sativus L. stigma phenolic and flavonoid compounds and its antioxidant activity. Molecules 15(9), 6244–6256.

Khadfy, Z., Atifi, H., Mamouni, R., Jadouali, S.M., Chartier, A., Nehmé, R., Karra, Y. and Tahiri, A. 2023. Nutraceutical and cosmetic applications of bioactive compounds of saffron (Crocus sativus L.) stigmas and its by-products. South. Afr. J. Botany. 163, 250–261.

Lahmass, I., Ouahhoud, S., Elmansuri, M., Sabouni, A., Elyoubi, M., Benabbas, R., Choukri, M. and Saalaoui, E. 2018. Determination of antioxidant properties of six by-products of Crocus sativus L. (Saffron) plant products. Waste. Biomass. Valorization. 9, 1349–1357.

Li, K., Hu, W., Yang, Y., Wen, H., Li, W. and Wang, B. 2023. Anti-inflammation of hydrogenated isoflavones in LPS-stimulated RAW264.7 cells via inhibition of NF-κB and MAPK signaling pathways. Mol. Immunol. 153, 126–134.

Lim, H., Heo, M.Y. and Kim, H.P. 2019. Flavonoids: broad spectrum agents on chronic inflammation. Biomolecules. &. Therapeutics. 27(3), 241–253.

Liu, H.M., Cheng, M.Y., Xun, M.H., Zhao, Z.W., Zhang, Y., Tang, W., Cheng, J., Ni, J. and Wang, W. 2023. Possible mechanisms of oxidative stress-induced skin cellular senescence, inflammation, and cancer and the therapeutic potential of plant polyphenols. Int. J. Mol. Sci. 24(4), 3755.

Maggi, L., Sánchez, A.M., Carmona, M., Kanakis, C.D., Anastasaki, E., Tarantilis, P.A., Polissiou, M.G. and Alonso, G.L. 2011. Rapid determination of safranal in the quality control of saffron spice (Crocus sativus L.). Food. Chem. 127(1), 369–373.

Matera, R., Lucchi, E. and Valgimigli, L. 2023. Plant essential oils as healthy functional ingredients of nutraceuticals and diet supplements: a review. Molecules 28(2), 901.

Mottaghipisheh, J., Mahmoodi Sourestani, M., Kiss, T., Horváth, A., Tóth, B., Ayanmanesh, M., Khamushi, A. and Csupor, D. 2020. Comprehensive chemotaxonomic analysis of saffron crocus tepal and stamen samples, as raw materials with potential antidepressant activity. J. Pharm. Biomed. Anal. 184, 113183.

Muscolo, A., Mariateresa, O., Giulio, T. and Mariateresa, R. 2024. Oxidative stress: the role of antioxidant phytochemicals in the prevention and treatment of diseases. Int. J. Mol. Sci. 25(6), 3264.

Nema, N., Rajan, N., Babu, M., Sabu, S., Khamborkar, S., Sarojam, S., Sajan, L., Peter, A., Chacko, B. and Jacob, V. 2023. Polyphenols. In Plant polyphenols as nutraceuticals and their antioxidant potentials. New York, NY: Wiley, pp: 21–44.

Ning, P., Yang, G., Hu, L., Sun, J., Shi, L., Zhou, Y., Wang, Z. and Yang, J. 2021. Recent advances in the valorization of plant biomass. Biotechnol. For Biofuels 14(1), 102.

Nurhanan, A.R. and Rosli, W.I.W. 2014. Nutritional compositions and antioxidative capacity of the silk obtained from immature and mature corn. J. King Saud Univ. Sci. 26(2), 119–127.

Papandreou, M.A., Kanakis, C.D., Polissiou, M.G., Efthimiopoulos, S., Cordopatis, P., Margarity, M. and Lamari, F.N. 2006. Inhibitory activity on amyloid-beta aggregation and antioxidant properties of Crocus sativus stigmas extract and its crocin constituents. J. Agricult. Food. Chem. 54(23), 8762–8768.

Pitsikas, N. 2016. Constituents of saffron (Crocus sativus L.) as potential candidates for the treatment of anxiety disorders and schizophrenia. Molecules 21(3), 303.

Rahaiee, S., Moini, S., Hashemi, M. and Shojaosadati, S.A. 2015. Evaluation of antioxidant activities of bioactive compounds and various extracts obtained from saffron (Crocus sativus L.): a review. J. Food Sci. Technol. 52(4), 1881–1888.

Sadh, P.K., Chawla, P. and Duhan, J.S. 2018. Fermentation approach on phenolic, antioxidants and functional properties of peanut press cake. Food Biosci. 22, 113–120.

Savchenko, T., Degtyaryov, E., Radzyukevich, Y. and Buryak, V. 2022. Therapeutic potential of plant oxylipins. Int. J. Mol. Sci. 23(23), 14627.

Serrano-Díaz, J., Sánchez, A.M., Maggi, L., Martínez-Tomé, M., García-Diz, L., Murcia, M.A. and Alonso, G.L. 2012. Increasing the applications of Crocus sativus flowers as natural antioxidants. J. Food. Sci. 77(11), C1162–C1168.

Seyyedeh, R. H., and Mansureh, G. (2025). Saffron petal waste as a source of natural antimicrobials: comparative analysis of extraction methods. Research 7, 418. https://doi.org/10.1007/s42452-025-06883-9

Singh, J., Kaur, S. and Rasane, P. 2018. Evaluation of the nutritional and quality characteristics of black carrot fortified instant noodles. Curr. Nutr. &. Food. Sci. 14, 1–8.

Stelluti, S., Caser, M., Demasi, S. and Scariot, V. 2021. Sustainable processing of floral bio-residues of saffron (Crocus sativus L.) for valuable biorefinery products. Plants 10(3), 523.

Vaou, N., Stavropoulou, E., Voidarou, C., Tsigalou, C. and Bezirtzoglou, E. 2021. Towards advances in medicinal plant antimicrobial activity: a review study on challenges and future perspectives. Microorganisms 9(10), 2041.

Vernarelli, J.A. and Lambert, J.D. 2017. Flavonoid intake is inversely associated with obesity and C-reactive protein, a marker for inflammation, in US adults. Nutr. &. Diabetes 7(5), 276.

Yoon, J.H., Kim, M.Y. and Cho, J.Y. 2023. Apigenin: a therapeutic agent for treatment of skin inflammatory diseases and cancer. Int. J. Mol. Sci. 24(2), 1498.

Zarinkamar, F., Tajik, S. and Soleimanpour, S. 2011. Effects of altitude on anatomy and concentration of crocin, picrocrocin and safranal in Crocus sativus L. Austral. J. Crop Sci. 5(7), 831–838.