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Article

Assessment of the Antioxidant Activity of Lyophilized Cistus creticus Extract as a Functional Fortifier in Yogurt: A Cellular and Non-Cellular Evaluation

by
Panoraia Bousdouni
1,
Eleni Dalaka
2,
Aikaterini Kandyliari
1,3,
Vasileios Gkalpinos
4,
Nikolaos Parisis
4,
Andreas G. Tzakos
4,
Georgios Theodorou
2,
Maria Kapsokefalou
3,* and
Antonios E. Koutelidakis
1,*
1
Laboratory of Nutrition and Public Health, Department of Food Science and Nutrition, University of the Aegean, 81400 Myrina, Greece
2
Laboratory of Animal Breeding and Husbandry, Department of Animal Science, Agricultural University of Athens, 11855 Athens, Greece
3
Laboratory of Food Chemistry, Department of Food Science and Human Nutrition, Agricultural University of Athens, 11855 Athens, Greece
4
Laboratory of Organic Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
*
Authors to whom correspondence should be addressed.
Oxygen 2025, 5(3), 17; https://doi.org/10.3390/oxygen5030017
Submission received: 14 May 2025 / Revised: 22 August 2025 / Accepted: 25 August 2025 / Published: 28 August 2025
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Abstract

Experimental evidence indicates that aqueous extracts of the Cistus genus have significant antioxidant properties, suggesting their potential as food fortifiers. In the present study, the antioxidant capacity and total phenolic content of lyophilized Cistus creticus extract were examined before and after in vitro digestion. Three concentrations of Cistus creticus extract were evaluated before and after in vitro gastrointestinal digestion, along with yogurt products fortified with these extracts, examined after digestion. Biochemical and cellular analyses were performed to assess these properties. The results showed statistically significant differences in total antioxidant capacity and total phenolic content, with values increasing from the lowest to the highest concentration studied, for both the lyophilized extracts and fortified yogurts after digestion. Additionally, cellular antioxidant activity after digestion was concentration-dependent (p < 0.05) within the range 25–500 mg/mL for both the extracts and fortified yogurts. In conclusion, based on the high phenolic content and the increased antioxidant capacity observed in epithelial cells, 250 mg of extract per 200 g of yogurt was proposed as the optimal fortification dose.

1. Introduction

The genus Cistus L., belonging to the Cistaceae family, comprises approximately 20 species with pink or white flowers, is primarily distributed in the Mediterranean region, and typically grows in degraded coastal areas with infertile soil [1]. The main Cistus species found in the Mediterranean basin are C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii [2]. Numerous studies on Cistus species have been published, particularly focusing on their volatile constituents, suggesting the presence of many compounds with antimicrobial and antifungal properties [3]. Moreover, Cistus species are rich in diterpenes and polyphenols (flavonoids, tannins, and phenolic acids), which are associated with their aromatic and medicinal properties [4]. Currently, its use as a medicinal plant is vestigial; however, its phytochemical composition, particularly the combination of terpenoids and polyphenols, makes it a promising species with many pharmacological activities [5]. Experimental evidence indicates interesting antioxidant properties of aqueous extracts of the Cistus genus that may explain their antioxidant activity, suggesting their potential as a means of treating human diseases in which oxygen-free radical production occurs, such as ischemic injury, inflammation, and cancer [6].
Natural antioxidants, including polyphenols, tocopherols, ascorbic acid, rosemary extracts, lycopene, and certain flavonoids, are being considered as alternatives to synthetic additives in foods [7]. Antioxidants in food systems play a crucial role in preserving nutrient levels, texture, color, taste, freshness, functionality, and aroma due to their high stability and low volatility [8]. Concurrently, antioxidants also have a positive impact on human health, acting against oxidative stress, fighting free radicals, and/or enhancing the effectiveness of the organism’s natural antioxidant systems [9]. Antioxidant activity can be assessed in a number of different ways, including biochemical measurements and cellular model systems [10]. While biochemical assays are useful for the initial screening of antioxidants based on their intrinsic activity, model systems are commonly utilized to further evaluate antioxidant potential in specific applications, such as food preservation or reducing oxidative damage in biological systems and the body [11]. Biochemical antioxidant assays can be categorized into two main types: those based on hydrogen atom transfer (HAT) reactions and those based on electron transfer (ET) to probe molecules. Tests based on HAT include the Oxygen Radical Absorption Capacity (ORAC) test, the Hydroxyl Radical Antioxidant Capacity (HORAC) test, the Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) test, and the Total Oxyradical Scavenging Capacity (TOSC) test. Tests based on ET include the Cupric Reducing Antioxidant Power (CUPRAC) test, the Ferric Reducing Antioxidant Power (FRAP) test, and the Folin–Ciocalteu test. Mixed tests, including the transfer of both a hydrogen atom and an electron, include the 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test, and the [2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl] (DPPH) test [12].
In terms of antioxidant testing, cell-based assays offer a practical and efficient means of estimating the antioxidant potential of functional foods, bridging the gap between in vitro chemical assays and in vivo studies [13]. A quantitative cellular antioxidant activity (CAA) assay has been proposed to study the bioavailability of dietary antioxidants, as it takes into account several additional factors, including cellular uptake and metabolism [14]. A quantitative CAA assay proposed by Wolfe et al., originally conceived and reported by Wang and Joseph [15], uses human hepatocarcinoma HepG2 cells to measure the antioxidant activity of phytochemicals, food extracts, and dietary supplements [16]. Initially, the assay utilized PC12 cells from rat adrenal glands, while HepG2 cells, derived from human hepatocellular carcinoma, were commonly reported in previous studies. Although HepG2 cells, which are of human origin, represent an improvement over PC12 cells, they are not ideal for assessing the effectiveness of dietary antioxidants [17,18]. Therefore, there has been a logical shift towards using human colorectal adenocarcinoma (Caco-2) cells, which share similar morphology, marker enzymes, microvillar structure, tight junctions, and permeability with small intestinal epithelial cells [19].
The metabolism of bioactive compounds is a key factor in creating an innovative functional product. Currently, one of the most popular methods for developing functional foods is the enrichment of conventional foods that are familiar to consumers [20]. Fermented foods are highly favored by consumers due to their beneficial lactic acid bacteria and probiotics, which offer various health benefits [21]. In recent years, there has been a gradual decline in the consumption of traditional milk and a rise in the intake of yogurts, fermented milk products, and dairy-based functional foods [22]. A recent study examining the enrichment of yogurt products with natural antioxidants showed that sensorial analysis, including visual evaluation of color and consistency, as well as fruit aroma, taste, and consistency, was encouraging, suggesting potential market success [23]. While several studies have examined the role of natural antioxidants in dairy products as food preservatives, data on their effects on human health are limited [24].
The form in which plant extracts are incorporated into foods is extremely important for the preservation of bioactive ingredients. Lyophilization, or freeze-drying, is a dehydration method that preserves perishable materials and makes them easier to transport; inhibiting enzymatic and bacterial activity increases the stability of heat-sensitive components and maintains the purity of the compound [25]. In addition, by reducing the oxygen concentration under vacuum, lyophilization minimizes oxidative changes in metabolites. Despite its benefits, this method is more expensive than other plant extract drying techniques due to the higher costs associated with equipment, materials, and operation [26]. However, this method enables the incorporation of concentrated doses of vitamins, antioxidants, and other beneficial compounds into functional foods, enhancing their efficacy in promoting health and well-being; furthermore, lyophilized extracts contribute to a longer shelf life and improved quality of functional foods, making them more appealing to consumers looking for nutritious options [27].
Freeze-dried extracts have been successfully applied in several food systems. Lyophilized blueberry extracts have been incorporated into snack bars to provide high levels of antioxidants, particularly anthocyanins, to increase the antioxidant capacity of the final product, which has significant health benefits in terms of heart health and inflammation reduction [28]. Similarly, lyophilized green tea extracts are added to energy drinks and dietary supplements for their potent polyphenols, which have been shown to aid weight management and reduce the risk of chronic disease [29]. Lyophilized extracts have been successfully used in dairy products to improve the organoleptic characteristics and antioxidant content of the final product; however, there is insufficient data on the bioavailability of the bioactive compounds of fortification [30].
The aim of the present study was to evaluate the antioxidant activity of proposed concentrations of lyophilized Cistus creticus extract as a fortifier of conventional yogurt using a range of cellular and non-cellular assays, to develop a functional product of high nutritional value.

2. Materials and Methods

2.1. Chemicals and Reagents

The chemicals and enzymes used were all of high purity or analytical grade. 2,20-azobis(2-methyl-propionamidine) dihydrochloride (AAPH), 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ), fluorescein sodium salt (FL), ferric chloride (FeCl3), ferric sulfate, sodium 1,2-naphthoquinone-4-sulfonate (folin ciocalteau), sodium carbonate, quercetin, trypsin, 20,70-Dichlorofluorescein diacetate (DCFH-DA), pepsin, pancreatin, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), and bile acids were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The 96-well transparent flat-bottom plates were purchased from Kisker Biotech (Steinfurt, Germany), while the 96-well cell culture transparent flat-bottom plates were purchased from SPL Life Sciences (Pocheon, Republic of Korea). DMEM, RPMI 1640, L-glutamine, sodium pyruvate, non-essential amino acids, and penicillin–streptomycin were purchased from Biosera (Cholet, France). Fetal bovine serum (FBS) was purchased from Gibco ThermoFisher Scientific (Waltham, MA, USA). Phosphate-buffered saline (PBS) was purchased from Takara Bio (Shiga, Japan).

2.2. Collection and Preparation of Samples

2.2.1. Collection and Characterization of Plant Material

Cistus creticus plant material was collected from the Epirus region in Greece. A voucher specimen of the plant was stored in the Epirus Herbs herbarium at the Department of Chemistry, University of Ioannina, Greece, with the voucher code 0267.
A customized database comprising flavonoids previously identified in various Cistus species was established and utilized as a reference for metabolite annotation in the most active plant extract. This in-house structural library was compiled from literature-reported compounds and served as a comparison tool for compound identification, which was based on high-resolution ESI-QToF-MS data, characteristic fragmentation behavior, and retention time alignment via UPLC, against both authenticated standards and entries in the proprietary Cistus metabolite collection. Before chromatographic analysis, the extract was diluted appropriately and passed through a 0.22 μm regenerated cellulose (RC) syringe filter to remove particulates. The separation of phytochemical constituents was carried out using an ACQUITY iClass Plus UPLC system (Waters, Manchester, UK) fitted with a column oven maintained at 45 °C and a cooled autosampler at 6 °C. Chromatographic separation was achieved using an Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.7 μm particle size). The elution was performed at a flow rate of 0.4 mL/min using a binary solvent system: solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The gradient program began with 1% B (0.00–2.00 min), ramped linearly to 100% B (2.00–24.00 min), held isocratically at 100% B (24.00–26.00 min), followed by a rapid return to 1% B (26.00–26.01 min) and re-equilibration at 1% B for 30.00 min. A sample volume of 2 μL was injected for each run. The UPLC system was hyphenated to a Xevo G2-XS QToF mass spectrometer (Waters, Manchester, UK) operating with an electrospray ionization (ESI) source in negative mode. Ion source conditions were optimized as follows: capillary voltage at 1.0 kV, cone voltage at 40 V, source temperature set to 120 °C, desolvation temperature at 550 °C, cone gas flow at 20 L/h, and desolvation gas flow at 1000 L/h. Lock mass correction was applied using leucine-enkephalin (5 ng/mL), infused at a rate of 10 μL/min to provide a reference ion at m/z 554.2620. Data were acquired in MSE mode, employing two scan functions: a low-energy function at 4 eV and a high-energy function with a collision energy ramp from 25 to 45 eV. Both functions operated over a mass range of m/z 50–1800 with a scan time of 0.2 s, enabling the simultaneous capture of both precursor and product ion spectra. Instrument control and data acquisition were performed using MassLynx software (v4.2, SCN 1029), while subsequent data analysis and compound annotation were executed in the UNIFI platform (v1.9.4.053). Structural assignment of detected metabolites was based on a comprehensive evaluation of exact mass, MS/MS fragmentation patterns, and retention time matching against standard compounds and the curated Cistus phytochemical library.
The composition of bioactive compounds in Cistus creticus extract, as identified through the experimental methodology described above and used in the yogurt formulation, is presented in Table 1.

2.2.2. Preparation of Plant Extracts

Fresh plant material, including leaves and stems, underwent initial treatment to eliminate moisture content. This was accomplished by lyophilizing the material at 0.1 mbar and 25 °C for 48 h. The dried material was then processed into a fine powder using a commercial blender. Water was subsequently added to the resulting powder at 10 mL/g of powder, and the borosilicate bottle containing the resulting slurry was placed in an ultrasonic bath set to 200 W and 30 °C for 1 h. The bottles were shaken vigorously every 15 min. The resulting suspension was filtered through a glass funnel equipped with a sintered glass filter (porosity: 4), and the collected filtrate was then processed to remove the solvent using a rotary evaporator. The solution was frozen and lyophilized at 0.01 mbar at 30 °C until all the volatiles were sublimed. The residue was then collected and stored in a borosilicate vial sealed at −30 °C.
Three concentrations of the Cistus creticus extract (CCE) were evaluated: 50 mg (CCE1), 250 mg (CCE2), and 500 mg (CCE3).
The same concentrations were used for the fortification of 2% fat cow yogurt purchased from the market; specifically, three functional yogurt products were produced and evaluated: 50 mg (CCY1), 250 mg (CCY2), and 500 mg (CCY3).

2.3. In Vitro Digestion

The in vitro gastrointestinal (GI) digestion assay was conducted according to the method proposed by Kapsokefalou et al., with some modifications [31]. Briefly, the samples were adjusted to pH 2.8 with 1 M HCL in 6-well plates; then, 2 mL of each extract was added to each well and mixed with 0.1 mL of human pepsin. The plates were then placed in a shaking incubator for 2 h at 37 °C. After incubation, a dialysis membrane was placed in well rings, and piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer reagent was added to each well to neutralize the mixture at pH 6.3. The role of the dialysis membrane was to separate high-molecular-weight peptides from lyophilized extracts and functional yogurts to obtain a small fraction (Spectra/Por Dialysis Membrane, Standard RC Tubing, MWCO: 6–8 kDa). Another incubation was performed (0.5 h, 37 °C), and then a mixture of pancreatin–bile acids (0.5 mL) was added to each well and incubated for 2 h at 37 °C. The digested samples were centrifuged for 15 min at 4000 rpm at 4 °C.
The corresponding fractions obtained after digestion were named CAE (1, 2, or 3)-D-P and CAY (1, 2, or 3)-D-P for the fractions of digestate containing peptides with molecular weights below 6 kDa, while the fractions of digestate containing peptides with molecular weights between 6 and 8 kDa were named CAE (1, 2, or 3)-D-R and CAY (1, 2, or 3)-D-R.

2.4. Biochemical Analysis

2.4.1. Determination of Total Antioxidant Capacity (TAC) Using Ferric Reducing Antioxidant Power Assay

The total antioxidant capacity of the food extracts was determined using the ferric reducing antioxidant power (FRAP) assay, as described by Benzie & Strain (1996) [32]. This method was based on the conversion of the TPTZ-Fe3+ complex to TPTZ-Fe2+, and the absorbance was measured at 595 nm using a microplate reader (EPOCH 2, BioTek Instruments, Inc, Winooski, United States). Higher absorbance values indicated greater antioxidant capacity to reduce the TPTZ-Fe3+ complex to TPTZ-Fe2+. The antioxidant activity was quantified using a standard FeSO4 curve, with the FRAP assay results expressed in mmol of Fe2+ per liter of sample extract. The analysis was performed in triplicate.

2.4.2. Determination of Total Phenolic Content (TPC) Using Folin–Ciocalteau Method

The total phenolic content of the sample extracts was assessed using the Folin–Ciocalteu method [33]. This technique measures the reductive capacity of the Folin–Ciocalteu reagent in an alkaline environment, with absorbance recorded at 765 nm using a microplate reader (BioTek, EPOCH 2). The total phenolic content was quantified using a standard gallic acid (GAE) curve, and results were expressed in milligrams of GAE per gram of dried food sample. The analysis was performed in triplicate.

2.5. Cellular Assays

2.5.1. Culture and Viability of the Caco-2 Cell Line

Caco2 cells were kindly gifted by Dimitris Kletsas (National Center for Scientific Research “Demokritos,” Greece). The cells were cultured in DMEM supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin, 10 U/mL of L-glutamine, 100 µM of non-essential amino acids, 1 mM of sodium pyruvate, and 10% (v/v) FBS. Cells were incubated at 37 °C in humidified air containing 5% CO2. Cells were passaged with trypsin before reaching confluency.

2.5.2. Cellular Antioxidant Activity (CAA) Assay

The CAA assay measures the overall oxidative status by tracking the decomposition of DCFH-DA within cells and its subsequent oxidation by reactive oxygen species (ROS) into the fluorescent compound DCF [34]. Intracellular reactive oxygen species (ROS) levels were determined in the Caco-2 cells as described by Piccolomini et al. [35] and adapted by Feng et al. [36], with some modifications. Briefly, cells were seeded at 0.5 × 105 cells/well in 96-well plates for 24 h. The cells were then washed with PBS and treated with 50 µL of the corresponding fractions, namely CCE-D-P, CCY-D-P, CCE-D-R, and CY-D-R, along with 50 µL of DCFH-DA (final concentration 50 μΜ) for 0.5 h. Subsequently, the cells were washed twice with PBS and treated with 100 µL of AAPH (final concentration 500 μΜ). Fluorescence was recorded immediately at 485 nm/535 nm at 37 °C every 1 min for a total of 60 min using the VICTOR 2030 multilabel counter (Perkin Elmer, Waltham, MA, USA). CAA was measured in at least quadruplicate, and the experiment was repeated five times. The results are expressed as % of ROS generation compared to untreated cells.

2.6. Statistical Analysis

The experimental results are reported as means ± standard error of means (SEMs) of at least two biological replicates. All data were tested for normality using the Kolmogorov–Smirnov test and transformed into logarithmic or normalized form where necessary until the data were normally distributed [37]. Subsequently, all data generated were compared using one-way ANOVA followed by Duncan’s post hoc test. Differences between means were considered significant at p < 0.05. Statistical analysis was performed using the SPSS for Windows statistical package program, version 22.0.0. Graphs were generated using the GraphPad Prism 8 program.

3. Results and Discussion

Several parameters, such as the structural diversity of antioxidant compounds, food matrix composition, the presence of various nutrients, and the proportions of dietary antioxidants in standard diets, significantly influence antioxidant bioavailability and absorption [38]. The present study evaluated three concentrations of lyophilized extract before and after gastrointestinal digestion. Moreover, the same concentrations were examined after the incorporation of Cistus creticus extract into yogurt products to evaluate the concentration of fortification of the final products. The antioxidant capacity and total phenolic content of the samples were measured using the FRAP and Folin–Ciocalteu methods, as previously described. Additionally, the cellular antioxidant activity of the samples was evaluated using the CAA assay in the Caco-2 cell line to comprehensively assess the total antioxidant activity and determine the bioavailability of the functional ingredients.

3.1. Assessment of Antioxidant Activity of Samples Before and After In Vitro Digestion Using FRAP and Folin–Ciocalteau Biochemical Assays

According to the results presented in Table 2, total antioxidant capacity and total phenolic content, expressed as FRAP and Folin–Ciocalteu values, showed wide, statistically significant variations between the samples. The total antioxidant capacity ranged from 50.0 g mmol Fe2+/L to 471.1 g mmol Fe2+/L before in vitro digestion, in accordance with the increase in sample concentration. Total phenolic content concentrations range from 199.5 mg GAE/g to 1446.2 mg GAE/g.
The Cistus genus comprises approximately 20 species across three subgenera, though data on many species are limited. A comparative study of Cistus species revealed that their polyphenolic composition is similar to that of members of the Cistus subgenus, such as C. creticus, C. crispus, and C. incanus, but differs significantly from species in the Halimioides and Leucocistus subgenera [39]. Cistus incanus infusions contain a variety of phenolic compounds, including phenolic acids, flavonoids, and flavan-3-ol derivatives, all of which have potent antioxidant activity [40]. According to a recent study, aqueous ethanolic extracts of Cistus incanus contained high phenolic content, reaching a value of 363.61 ± 2.29 mg GAE/g of extract [41]. In contrast, Bernacka et al. and Ziarno et al. found lower phenolic contents in water infusions prepared from C. incanus leaves [42,43]. Concerning the antioxidant capacity, Gori et al. found that the ethyl acetate fraction of C. incanus, enriched with phenolic compounds, especially flavonols, had a higher DPPH scavenging activity compared to the aqueous fraction enriched with tannins [44].
Evaluation of samples after in vitro gastrointestinal digestion revealed statistically significant (p < 0.05) reductions in antioxidant capacity and total phenolics compared to the initial extracts. Permeable (D-P) and non-permeable (D-R) aliquots differed significantly (p < 0.05) in terms of bioactive compounds as follows: CCE1-D-P < CCE2-D-P < CCE3-D-P and CCE1-D-R < CCE2-D-R < CCE3-D-R. Consistent with our results, several studies have reported lower FRAP and total phenolic values after in vitro gastrointestinal digestion [45,46]. A recent research by Costa et al., focusing on freeze-dried grape pomace extract, found that phenolic compounds were dramatically reduced after in vitro gastrointestinal digestion, with only 5.69% of the initial total phenolic content reaching the intestine [47]. These results are in line with literature estimates, which report that 90% of polyphenols are digested before they can be absorbed by the gut [48]. A recent study of aqueous extracts from Turkish Cistus species found that these extracts are rich in phenolic compounds, exhibiting strong antioxidant and anti-diabetic properties; however, in vitro human digestion simulation revealed that digestion significantly reduces their bioactivity, including total phenolic content, antioxidant activity, and diabetes-related enzyme inhibition [49]. The decrease in total antioxidant capacity and total phenolic content after in vitro gastrointestinal digestion is due to the degradation, transformation, and interactions of phenolic compounds under the conditions present in the digestive system [50]. Specifically, digestive enzymes such as pepsin and pancreatin may break down polyphenolic compounds into smaller, less active fragments. This degradation reduces both the total phenolic content and antioxidant capacity, because smaller fragments may have lower bioactivity [51]. Moreover, the stability of phenolic compounds is significantly influenced by the pH conditions encountered during digestion, which reduces their antioxidant activity, particularly affecting compounds such as anthocyanins, which lose stability as they move through the digestive tract [52].
The food matrix can also influence the bioavailability of bioactive compounds through various mechanisms, including physical inclusion, chemical interaction, and the influence of matrix composition on digestive and absorption processes [53]. Depending on the specific interactions between the compounds and the other food components, the matrix can either increase or decrease the bioavailability of bioactive compounds [54,55,56]. Yogurt is one of the most popular and healthy dairy products, and has been exploited as a delivery matrix for phenolic compounds [57]. The incorporation of bioactive compounds into yogurt products has been studied, and the results after in vitro digestion showed that the yogurt matrix has a protective effect on the stability of phenolic compounds, improving their release in the gastrointestinal tract.
In the present study, the total antioxidant capacity and total phenolic content of the samples, CCY before and after digestion, were evaluated after incorporation into yogurt at the corresponding concentrations. According to the results presented in Table 3, the values of total antioxidant capacity were statistically significantly different, with increasing values from the lowest to the highest concentrations studied, for both permeable and non-permeable aliquots. Evaluation of total phenolic concentration showed a statistically significant difference between the lowest concentration (CCY1-D-P) and the two highest concentrations of the permeable samples (CCY2-D-P and CCY3-D-P), whereas for the non-permeable samples, there was a significant difference for three samples. This differentiation may indicate a potential food matrix effect, suggesting that the composition and structure of the sample matrix can influence the extractability or retention of phenolic compounds. Results of a recent study contradict these findings by showing a significant increase in antioxidant activity in fortified yogurt at the end of gastrointestinal tract digestion, which was higher than that in plain yogurt [58]. In parallel, a study of herbal extracts incorporated into yogurt revealed a loss of several phenolic compounds, as well as the formation of new ones during storage. Despite these changes, functional yogurts maintained higher antioxidant activity compared to plain yogurt over 21 days of storage [59].
As foods fortified with plant extracts become increasingly popular, there is a greater need to monitor their quality and safety. Analyzing polyphenols presents challenges due to the complexity of food matrices, interactions with other ingredients, and the formation of new compounds during processing [60]. Therefore, more advanced methodologies beyond standard biochemical analyses are required.

3.2. Assessment of Cellular Antioxidant Activity of Samples After In Vitro Digestion

As stated above, the study of antioxidants is influenced by several factors, and it is therefore essential to consider the composition of the system, the type of oxidizable substrate, the method used to accelerate oxidation, the techniques used to evaluate oxidation, and the measurement of antioxidant activity [61]. Cellular antioxidant activity assays address these issues and can yield reliable results, emphasizing the importance of using a more biologically relevant model, as highlighted by the discrepancies between pure chemistry assays and cell culture-based results [16]. The CAA assay is a highly biologically relevant method that reflects the absorption, metabolism, and distribution of antioxidants at the cellular level. Regarding the type of cell, Caco-2 can mimic the absorption of nutrients due to its ability to differentiate into cells that resemble the human small intestinal epithelium [62].
Experimental evidence suggests that most herbs and spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage. Additionally, research shows that cellular antioxidant assays have been used to evaluate several herbal extracts [63]. According to the results in Figure 1a, the highest cellular antioxidant activity for permeable digested aliquots was observed at 25 mg/mL, whereas for non-permeable digested aliquots, the highest CAA was detected at 500 mg/mL. This might be attributed to the ability of dietary polyphenols to act as pro-oxidants in higher concentrations [64,65]. A study of a similar design by Aherne et al. investigated the effects of four plant extracts—rosemary, oregano, sage, and echinacea—on the antioxidant capacity of the Caco-2 cell line. The results showed that sage was the only extract that positively influenced the antioxidant status of the cells. Additionally, sage, oregano, and rosemary provided protection against H2O2-induced DNA damage, while sage alone protected against H2O2-induced cytotoxicity [66]. It is important to note that the above study did not involve lyophilized extracts. Moving on to the evaluation of herbal extracts, Liu and Huang demonstrated that black tea extract plays a significant antioxidant role against AAPH-generated peroxyl radicals by preventing the oxidation of DCFH and membrane lipid, as well as decreasing DCF formation in HepG2 cells [67].
Moreover, our results demonstrated that the cellular antioxidant activity was concentration-dependent (p < 0.05) at concentrations of 25–500 mg/mL for both permeable and non-permeable aliquots. Consistent with our findings, a recent study by Guo et al. examined the bioaccessibility of digested berry extracts in a Caco-2 cell culture model and found that, while the extracellular antioxidant activity of the digesta was lower than that of the extracts, the cellular antioxidant activity (CAA) effects of the berry extracts were significantly enhanced by digestion [68]. Several studies have referred to enhanced CAA of samples by the gastrointestinal process, which has been commonly attributed to the possible formation of new compounds with improved antioxidant activity as a result of the pH shift in each digestive phase [69,70].
Results from studies on CAA determination in food and feed components after in vitro digestion using cell-based assays are limited and mostly focused on non-dairy products. Specifically, CAA assays have been implemented in several food matrices, such as fruits [71], vegetables [72], and whole grain varieties [73]. To date, there have been a limited number of studies evaluating CAA in eukaryotic cells for dairy compounds after in vitro digestion. Corrochano et al. evaluated the antioxidant capacity of whey proteins after in vitro gastrointestinal digestion, and results showed that samples effectively inhibited ROS and stimulated antioxidant enzymes in HT29 cells [74]. A similar study by Dalaka et al. showed an increase in the antioxidant capacity of whey proteins after simulated digestion, while the samples appeared to enhance the antioxidant capacity of HT29 cells [75].
The current study investigated yogurt fortified with different concentrations of Cistus creticus, and the CAA results are shown in Figure 1b. Our findings indicated that cellular antioxidant activity was concentration-dependent (p < 0.05) at concentrations ranging from 25 to 500 mg/mL. Interestingly, the lowest fortification level, which exhibited the highest antioxidant activity in the extract evaluation, did not show any CAA when incorporated into the yogurt. The intermediate concentration demonstrated the highest CAA levels among the permeable fractions. This might be due to hormesis, where low doses of a compound can trigger beneficial cellular responses while higher doses can be harmful, highlighting how phenolics might act differently at varying concentrations [76]. Moreover, elevated concentrations (e.g., 500 mg/mL) may lead to a plateau or decline in cellular antioxidant activity (CAA), potentially indicative of pro-oxidant behavior exhibited by phenolic compounds under such conditions [77]. In addition, high concentrations of phenolic compounds can lead to the formation or aggregation of complexes, thereby reducing the availability of active compounds for antioxidant activity [78]. A corresponding study with yogurt enriched with cacao improved the integrity of Caco-2 cells by strengthening tight junctions and had a predominant hypoglycaemic effect in rats [79]. Moreover, data from the literature indicate that intestinal digestion of yogurt was found to negatively affect inflammation-induced barrier dysfunction in Caco-2 cells. However, yogurt fortified with quercetin, marshmallow root, maitake mushroom, and licorice root demonstrated significantly higher Transepithelial Electrical Resistance (TEER) values compared to the control yogurt, indicating enhanced barrier integrity [80].

4. Conclusions

The present study evaluated three different concentrations of Cistus creticus extract before and after in vitro gastrointestinal digestion. Yogurt products fortified with the respective extract concentrations were also evaluated. Samples were evaluated via multiple analyses, biochemical and cellular, to determine antioxidant capacity and total phenolic content, as one class is insufficient to control relevant factors affecting the bioactive compounds studied. In conclusion, the present study has shown that CCY3-D-P has a high total phenolic content and increases the antioxidant capacity of epithelial cells; therefore, it is recommended as a concentration for fortification of functional yogurt products. Further research is needed to evaluate the sensory properties of the novel yogurt products and justify their in vivo health effects. Validation of the results could be achieved through nutritional interventions, such as clinical trials evaluating the effect of the proposed functional product on plasma antioxidant capacity and other relevant biomarkers.

Author Contributions

Conceptualization, P.B., A.K. and M.K.; Data curation, E.D., A.K., G.T. and M.K.; Formal analysis, P.B., V.G., A.G.T. and G.T.; Funding acquisition, A.E.K.; Investigation, P.B., E.D., A.K., V.G., A.G.T. and G.T.; Methodology, P.B., E.D., N.P., A.K., G.T., M.K. and A.E.K.; Project administration, G.T., M.K. and A.E.K.; Resources, P.B., E.D., V.G., A.G.T. and G.T.; Software, P.B. and E.D.; Supervision, M.K. and A.E.K.; Validation, P.B., A.K., N.P., G.T. and A.E.K.; Visualization, M.K. and A.E.K.; Writing—original draft, P.B., E.D., A.K., V.G., A.G.T. and G.T.; Writing—review and editing, M.K. and A.E.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research is part of the project, “Infrastructure of Microbiome Applications in Food Systems-FoodBiomes”, which is co-funded by the European Regional Development Fund (ERDF) under the Operational Program “Competitiveness, Entrepreneurship and Innovation-EPANEK 2014–2020”, Call 111 “Support for Regional Excellence”.

Data Availability Statement

The data are available upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Oxygen 05 00017 g001
Figure 1. Effect of (a) Cistus creticus extract and (b) yogurt supplemented with C. creticus on cellular antioxidant activity (CAA) % in Caco-2 cells (seeded at 5 × 104 cells/well) after 30 min of exposure to corresponding digested fractions [namely CCE-D-P and CCE-D-R for (a) and CCY-D-P and CCY-D-R for (b)]. Cell experiments were performed in quadruplicate five times independently. Values are presented as mean ± SEM. Columns with different letters differ significantly (p < 0.05). ns = non significant.
Figure 1. Effect of (a) Cistus creticus extract and (b) yogurt supplemented with C. creticus on cellular antioxidant activity (CAA) % in Caco-2 cells (seeded at 5 × 104 cells/well) after 30 min of exposure to corresponding digested fractions [namely CCE-D-P and CCE-D-R for (a) and CCY-D-P and CCY-D-R for (b)]. Cell experiments were performed in quadruplicate five times independently. Values are presented as mean ± SEM. Columns with different letters differ significantly (p < 0.05). ns = non significant.
Oxygen 05 00017 g001
Table 1. Cistus creticus extract compounds used as functional fortifiers in yogurt.
Table 1. Cistus creticus extract compounds used as functional fortifiers in yogurt.
NoCompound NameNeutral Mass (Da)Observed m/z (Da)Mass Error (ppm)ResponseFormula
1Myricitrin464.09548463.08820.1432,381C21H20O12
2Myricetin 3-galactoside480.09039479.08330.3237,079C21H20O13
3Gallocatechin306.07395305.0664−0.9197,753C15H14O7
4Quercitrin (Quercetin 3-O-rhamnoside)448.10056447.093−0.6130,032C21H20O11
5Prodelphinidin B *610.13226609.1248−0.3104,051C30H26O14
6Myricetin 3-arabinoside450.07983449.07260.294,787C20H18O12
7Catechin290.07904289.07180.057,668C15H14O6
8Quercetin 3-O-glucoside464.09548463.088−0.442,322C21H20O12
9Rutin (Quercetin-3-rutinoside)610.15338609.14630.337,512C27H30O16
10Guaijaverin (Quercetin 3-arabinoside)434.08491433.0776−0.133,038C20H18O11
11Tiliroside *594.13734593.13030.426,725C30H26O13
12Prodelphinidin B *610.13226609.1248−0.413,886C30H26O14
13Tiliroside *594.13734593.13142.213,192C30H26O13
14Quercetin 3-O-sophoroside626.1483625.14150.712,606C27H30O17
15Prodelphinidin C *898.19564897.18920.910,305C45H38O20
16Avicularin (Quercetin 3-arabinfuranoside)434.08491433.0780.79610C20H18O11
17Tiliroside *594.13734593.13040.58128C30H26O13
18Prodelphinidin B *610.13226609.1248−0.37044C30H26O14
19Prodelphinidin B *610.13226609.12591.66198C30H26O14
20Luteolin-7-O-glucoside448.10056447.09411.96055C21H20O11
* Isomer.
Table 2. Total antioxidant capacity and total phenolic content of three different concentrations of lyophilized Cistus creticus extracts.
Table 2. Total antioxidant capacity and total phenolic content of three different concentrations of lyophilized Cistus creticus extracts.
TAC (Fe2+/L)TPC (mg GAE/g)
Before DigestionCCE150.0 (8.6) a199.5 (11.8) a
CCE2413.6 (13.1) b1278.3 (43.7) b
CCE3471.1 (23.7) c1446.2 (160.5) c
After Digestion
(<6 kDa)		CCE1-D-P46.1 (3.1) a58.9 (9.8) a
CCE2-D-P134.8 (26.8) b119.0 (8.3) b
CCE3-D-P223.4 (36.0) c175.5 (9.6) c
After Digestion
(6–8 kDa)		CCE1-D-R56.5 (20.4) a75.4 (12.0) a
CCE2-D-R243.4 (9.1) b392.9 (25.9) b
CCE3-D-R334.1 (39.6) c921.3 (86.9) c
Data are presented as mean ± SD. Different letters in the same column indicate significant differences (p < 0.05) between the samples.
Table 3. Total antioxidant capacity and total phenolic content in functional yogurt enriched with three different concentrations of Cistus creticus lyophilized extract.
Table 3. Total antioxidant capacity and total phenolic content in functional yogurt enriched with three different concentrations of Cistus creticus lyophilized extract.
TAC (Fe2+/L)TPC (mg GAE/g)
After Digestion
(<6 kDa)		CCY1-D-P4.5 (1.4) a16.9 (3.5) a
CCY2-D-P18.9 (5.7) b48.5 (13.4) b
CCY3-D-P27.9 (5.2) c43.9 (2.4) b
After Digestion
(6–8 kDa)		CCY1-D-R9.2 (2.9) a34.6 (3.6) a
CCY2-D-R22.5 (8.3) b89.5 (4.3) b
CCY3-D-R38.5 (3.3) c368.5 (34.8) c
Data are presented as mean ± SD. Different letters in the same column indicate significant differences (p < 0.05) between the samples.
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Bousdouni, P.; Dalaka, E.; Kandyliari, A.; Gkalpinos, V.; Parisis, N.; Tzakos, A.G.; Theodorou, G.; Kapsokefalou, M.; Koutelidakis, A.E. Assessment of the Antioxidant Activity of Lyophilized Cistus creticus Extract as a Functional Fortifier in Yogurt: A Cellular and Non-Cellular Evaluation. Oxygen 2025, 5, 17. https://doi.org/10.3390/oxygen5030017

AMA Style

Bousdouni P, Dalaka E, Kandyliari A, Gkalpinos V, Parisis N, Tzakos AG, Theodorou G, Kapsokefalou M, Koutelidakis AE. Assessment of the Antioxidant Activity of Lyophilized Cistus creticus Extract as a Functional Fortifier in Yogurt: A Cellular and Non-Cellular Evaluation. Oxygen. 2025; 5(3):17. https://doi.org/10.3390/oxygen5030017

Chicago/Turabian Style

Bousdouni, Panoraia, Eleni Dalaka, Aikaterini Kandyliari, Vasileios Gkalpinos, Nikolaos Parisis, Andreas G. Tzakos, Georgios Theodorou, Maria Kapsokefalou, and Antonios E. Koutelidakis. 2025. "Assessment of the Antioxidant Activity of Lyophilized Cistus creticus Extract as a Functional Fortifier in Yogurt: A Cellular and Non-Cellular Evaluation" Oxygen 5, no. 3: 17. https://doi.org/10.3390/oxygen5030017

APA Style

Bousdouni, P., Dalaka, E., Kandyliari, A., Gkalpinos, V., Parisis, N., Tzakos, A. G., Theodorou, G., Kapsokefalou, M., & Koutelidakis, A. E. (2025). Assessment of the Antioxidant Activity of Lyophilized Cistus creticus Extract as a Functional Fortifier in Yogurt: A Cellular and Non-Cellular Evaluation. Oxygen, 5(3), 17. https://doi.org/10.3390/oxygen5030017

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