Phenolic Profile and Antioxidant Activity of Extracts from Aerial Parts of Thymus vulgaris L. and Sideritis scardica Griseb.

Abstract

Herbal extracts have various biological properties, including antioxidant activity (AAC). This activity is mainly associated with presence of polyphenolic compounds, and depends on the type and origin of the raw material, the chemical profile, and the extraction method (including the type of solvent used). The aim of the study was to evaluate the chemical compound content and antioxidant activity in selected plants from the Lamiaceae family, depending on the solvent applied. Additionally, the study aimed to determine whether there are correlations between the content of biological compounds and antioxidant activity. The extracts were prepared from Thymus vulgaris L. and Sideritis scardica Griseb. using ethanol, methanol, ethyl acetate, and acetone as solvents. The obtained extracts were assessed for total polyphenols content (TPC), flavonoids, and phenolic acids, as well as antioxidant potential, using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing (FRAP) assays. The plants studied showed significant TPC and significant AAC, with the sideritis extracts exhibiting both high TPC (162.38 mg GA/g) and high AAC-DPPH (86.39%). Our study showed a significant positive correlation between antioxidant activity (DPPH) and antioxidant activity (FRAP) for acetone extracts (r = 0.664808), and strong negative correlations for the other extracts. It was also confirmed that selecting the appropriate solvent is essential for the efficient extraction of phenolic compounds, which are strongly linked to biological activity. As a result, extracts of T. vulgaris and S. scardica represent a potential source of antioxidants that could be used in the prevention of various diseases or in the food industry as preservatives.

1. Introduction

Plants have been used for their medicinal properties throughout history. Originally, they were harnessed by humans in their simplest, unrefined form (leaves, roots, fruits, seeds), and later in the form of herbal remedies (brews, infusions, macerates, tinctures, extracts). Traditional plant-based remedies, which offer both curative and preventive benefits, are attracting increased attention. Sourced from various species of medicinal plants, they are utilized both in traditional medicine practices and in the development of novel medicines, nutraceuticals, dietary supplements, and pharmaceutical products. The International Union for the Conservation of Nature (IUCN) and the Worldwide Fund for Nature (WWF) list 50,000–80,000 plant species that are used medicinally worldwide. Herbal medicines are estimated to account for up to 25% of total consumption in countries such as the United States and United Kingdom, in contrast with India and China, where that proportion can be as high as 80% [1]. The first step in processing medicinal plants into value-added bioresources involves creating herbal preparations (extracts) through a variety of methods, ranging from the simple and traditional to the modern and advanced. Extraction is a process where the active constituents or secondary metabolites of a plant (such as alkaloids, flavonoids, terpenes, saponins, steroids, or glycosides) are separated from the neutral or inactive ingredients using an appropriate solvent and a standardized extraction protocol. The solvents commonly used to make medicinal plant extracts can be polar (e.g., water and alcohols), intermediate polar (e.g., acetone and dichloromethane) or non-polar (e.g., n-hexane, ether, and chloroform) [2]. The preparation of medicinal plants for experimental purposes is an initial step and a key part of achieving reliable research results, producing high-quality extracts, and ultimately manufacturing effective medicinal products.

The family Lamiaceae encompasses a wide array of plants with biological and medicinal applications. These species feature a distinctive, pleasant fragrance, a rich chemical composition, and a wide range of exhibited bioactivities, including antioxidant activity [3,4,5,6]. Plant-derived antioxidants play an important role in preventing free radical generation and can help counteract various disorders, including cancer, aging, cardiovascular disease, cataracts, immune system suppression, and brain disorders [7]. The antioxidant activity of a plant is correlated with its phenolic compounds content, including phenolic acids and flavonoids [2,8,9,10]. Plants of the genus Thymus and Sideritis are known to contain such phenolic compounds, including rosmarinic acid, quercetin, caffeic acid, luteolin, and apigenin—substances with an established capacity to scavenge free radicals [11,12,13,14]. Species included in the genera Thymus and Sideritis from Lamiaceae family used worldwide, mostly due to their potential health benefits. There are about 250 species recorded within the genus Thymus, distributed throughout the Mediterranean area, Europe, Greenland, Asia, Australia, and America, that are used in pharmaceutical industries, cosmetics, and foods. The genus Sideritis includes 150 species that are widely distributed in the Mediterranean region. Many of them are used as traditional remedies due to their anti-inflammatory, spasmolitic, antiulcer, analgesic, and gastroprotective properties [4,14]. Thyme (T. vulgaris L.), appears in many different areas worldwide and is the most commercially cultivated species in the genus Thymus. In Europe, the total number of T. vulgaris specimens is estimated to be 202 [5]. Thyme has valuable aromatic and medicinal properties. Among the bioactive components of thyme, polyphenols constitute an important group [5,10,14]. One of the species of the genus Sideritis is S. scardica Griseb (known as mountain tea or Greek tea), a source rich in natural antioxidants and polyphenolic compounds with particularly beneficial effects for human health. This species can be found at Central Balkan Peninsula, in the southwest regions of Albania, in the northeastern regions of Greece, in the central and western regions of North Macedonia, in the southern regions of Bulgaria, and in the European part of Turkey. Sideritis’ active compounds have been shown to inhibit free radical-induced cytotoxicity and lipid peroxidation, acting as inhibitors of cell proliferation. Thus, they may protect against chronic and neurodegenerative diseases such as atherosclerosis, cancer, and Alzheimer’s disease [12].

Antioxidant capacity can be quantified by a variety of assays, each with its own specific mechanism of action—hydrogen atom transfer, single-electron transfer, targeted reactive oxygen species (ROS) scavenging, and others. These methods differ in terms of antioxidant mode of action, type of substrate, oxidation initiator, the expression achieved, and ease of use [15]. Comparative research on antioxidant assays strongly suggests that the assorted accepted methods vary significantly, which is why antioxidant activity (AAC) should be tested using more than one method [16]. Madhuranga and Samarakoon [17] point to three major methods—DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) assay, FRAP assay (ferric-reducing antioxidant power), and H₂O₂ assay (hydrogen peroxide free radical scavenging activity)—as reliable, straightforward, and inexpensive. Dontha [18] adds that the DPPH is chiefly used for its in vitro antioxidant activity testing, whereas lipid peroxidation is used in vivo, while also stating that ethanol is the preferred solvent for extraction. DPPH is a very simple and quick method for the manual analysis of antioxidant content. It can be used for both solid or liquid samples and is not specific to a single antioxidant, so it can detect the overall antioxidant capacity of a sample [15,18]. FRAP, on the other hand, has some limitations, especially when it comes to measuring at non-physiological pH values (pH 3.6). Furthermore, it cannot detect inert phenolic compounds and thiols [18]. Nevertheless, FRAP is low-cost, sensitive, and reproducible, being compatible with a wide range of biological materials [15]. Thus, it is best to use the DPPH in conjunction with various other useful methods, such as FRAP and ORAC, to obtain the most robust and accurate antioxidant activity measurement. Choosing the right assay or combination of assays is essential to accurately quantify antioxidant activity, and thus correctly assess the potential of an antioxidant as a health-promoting and/or preservative agent. In our study, we focused on analyzing the phenolic profile and conducting an antioxidant activity evaluation using two spectrophotometric methods (DPPH and FRAP) of Thymus vulgaris L. and Sideritis scardica Griseb. extracts, using aceton (ACT), methanol (MeOH), ethanol 70% (EtOH-H₂O), and ethyl acetate (EtOAc) as solvents. We aimed to identify which extraction method is the most effective and environmentally friendly. To the best of our knowledge, this study is the first attempt to compare and evaluate the chemical composition and antioxidant activity of thyme and sideritis extracts.

2. Materials and Methods

2.1. Extracts Preparation

The study was conducted on plant material from Thymus vulgaris L. and Sideritis scardica Griseb. aerial parts. The thyme variety Słoneczko from Polish seed breeding was selected for the study (Institute of Medicinal Plants and Products in Poznań, Poznań, Poland). Certified seeds came from the breeder. The aerial parts of thyme were collected from annual (non-flowering) plants grown in the field on the experimental plots of the Department of Vegetable and Herb Crops, and dried at 35 °C. In turn, sideritis was purchased from an herbal store (country of origin Türkiye, manufacturer Astron, Henryków, Poland). The desiccated plant material (Figure S1) was ground in a laboratory mill, and the target amount was weighed out for extract preparation. Extraction efficiency was calculated as the ratio of the amount of thick extract obtained to the mass of the sample that was used to prepare the extract.

2.1.1. Acetone Extract

The ACT extract was prepared by weighing out 20 g of ground thyme/sideritis aerial parts, transferring it to a flat-bottomed flask, and adding 200 mL of ACT solvent (pure analysis p.a.; ChemPur, Piekary Śląskie, Poland). The flasks were placed over a water bath (Heating Bath HB4 Basik; IKA-Werke GmbH & Co.; Staufen, Germany) under a reflux condenser, and heated for 30 min once brought to a boil. After 30 min, the extract was filtered into an Erlenmeyer flask through medium qualitative filter paper. The filter paper was then put in a flask, with 200 mL of the solvent being added to the sideritis flask and 100 mL of the solvent being added to the thyme flask. Again, the flask with the contents was heated for 30 min over a water bath. The extract was filtered again, and the entire process was repeated. The resultant filtrates were combined and then evaporated in a rotary evaporator (Rotary Evaporator 05-ST; IKA-Werke GmbH & Co.; Staufen, Germany) to a thick extract, which served as the sample for further testing.

2.1.2. Methanol Extract

The MeOH extract was prepared by weighing out 20 g of ground Thyme/Sideritis aerial parts, transferring it to a flat-bottomed flask, and adding 200 mL of MeOH solvent (99.8%; p.a., ChemPur, Piekary Śląskie, Poland). The flasks were placed over a water bath (Heating Bath HB4 Basik; IKA-Werke GmbH & Co.; Staufen, Germany) under a reflux condenser and heated for 30 min once brought to a boil. After 30 min, the extract was filtered into an Erlenmeyer flask through medium qualitative filter paper. The filter paper was then put in a flask, with 200 mL of the solvent added to the sideritis flask and 100 mL of the solvent to the thyme flask. Again, the flask with the contents was heated for 30 min over a water bath. The extract was filtered, and the entire process was repeated. The resultant filtrates were combined and evaporated in a rotary evaporator (Rotary Evaporator 05-ST; IKA-Werke GmbH & Co.; Staufen, Germany) to a thick extract, which served as the sample for further testing.

2.1.3. Ethanol–Water Extract

A 70% (v/v, Stanlab, Lublin, Poland) ethanol-in-water solution was used to prepare the extract. A total of 20 g of the plant material was ground and transferred to a flat-bottomed flask, with 200 mL of the solvent then being added. The flasks were placed over a water bath (Heating Bath HB4 Basik; IKA-Werke GmbH & Co.; Staufen, Germany) under a reflux condenser and heated for 30 min. Afterwards, the extract was filtered through medium qualitative filter paper. The filter paper was then put in a flask, with 200 mL of the solvent added to the sideritis flask and 100 mL of the solvent to the thyme flask. The material was again heated for 30 min in the flasks and then filtered. The entire process was then repeated. The resultant extracts were combined and evaporated in a rotary evaporator (Rotary Evaporator 05-ST; IKA-Werke GmbH & Co.; Staufen, Germany) to a thick extract, which served as the sample for further testing.

2.1.4. Ethyl Acetate Extract

The extract was prepared by weighing out 20 g of ground plant material of each type, transferring it to a flat-bottomed flask, and adding 200 mL of EtOAc solvent (99.9% p.a., ChemPur, Piekary Śląskie, Poland). The flasks were placed over a water bath (Heating Bath HB4 Basik; IKA-Werke GmbH & Co.; Staufen, Germany) under a reflux condenser and heated for 30 min once brought to a boil. After 30 min, the extract was filtered into an Erlenmeyer flask through medium qualitative filter paper. The filter paper was then put in a flask, with 200 mL of the solvent added to the sideritis flask and 100 mL of the solvent to the thyme flask. Again, the flask with the contents was heated for 30 min over a water bath. The extract was filtered, and the entire process was repeated. The resultant filtrates were combined and then evaporated in a rotary evaporator (Rotary Evaporator 05-ST; IKA-Werke GmbH & Co.; Staufen, Germany) to a thick extract, which served as the sample for further testing.

2.2. Flavonoid Content Determination

The total flavonoids content (TFC) was determined in extract obtained from plant material by a spectrophotometric method using a Hitachi U-2900 UV-Vis model spectrophotometer, (Hitachi High-Tech Corporation, Ibaraki, Japan). The analysis was performed according to the methodology given in Polish Pharmacopoeia V [19]. Extraction was performed with a mixture of solvents, acetone (Chempur, Piekary Śląskie, Poland), hydrochloric acid (250 g L⁻¹, Chempur, Piekary Śląskie, Poland), and methenamine (5 g L⁻¹, Merck, Poznań, Poland). To determine the total flavonoids content, about 0.5 g extract was used and extracted once. The analysis was performed in triplicate. The absorbance of the solutions was measured at a wavelength of 425 nm using the reference solution. Total flavonoids content (mg/g extract) was expressed in terms of quercetin (QE), assuming absorbability a_1cm^1% = 714, according to the following formula:

X = (A × k₁)/m

A—absorption of the solution of the research;

k₁—convection factor for quercetin; k₁ = 8.75;

m—the sample with the raw material in g [20].

2.3. Phenolic Acid Determination

The content of total phenolic acids (TPAC) was determined using the spectrophotometric method according to the methodology provided in Polish Pharmacopoeia X [21] using a Hitachi U-2900 UV-Vis model spectrophotometer (Hitachi High-Tech Corporation, Ibaraki, Japan). In order to determine the total phenolic acids in the tested extracts, samples of approximately 0.5 g of all tested extracts were prepared and 20 mL of hot distilled water was added and left for 12 h in a refrigerator. Then, they were filtered into measuring flasks and filled with water to a volume of 100 mL. Then, 1 mL of solution was taken from the prepared extracts, and 1 mL of distilled water, 1 mL of 0.5 M hydrochloric acid, and 1 mL of Arnova reagent were added. After 6 min, 1 mL of 1 M NaOH and 5 mL of distilled water were added. The analysis was performed in triplicate. The obtained mixture was transferred to cuvettes and absorbance was measured at a wavelength of 490 nm using a reference mixture of solvents without extract. TFAC was expressed in mg/g of extract, calculated as caffeic acid (CA), using the following formula:

X = (A × k₂)/m

A—absorbance of the tested solution;

K₂—conversion factor for caffeic acid (K₂ = 3.5087);

m—raw material weight (g).

2.4. Total Polyphenol Content Determination

The Folin–Ciocalteu method, with slight modifications, was used to determine the sum of phenol content (total polyphenols content (TPC)) in the extracts tested [22,23]. Measurements were made using a Hitachi U-2900 UV-Vis model spectrophotometer (Hitachi High-Tech Corporation, Ibaraki, Japan). Total polyphenols content was determined spectrophotometrically and absorbance was measured at 765 nm using the reagent mixture without the extract as a reference., For the determination, the about 0.5 g of prepared extracts and 50 mL of methanol (Chempur, Piekary Śląskie, Poland) were used. The research material prepared in this way was maintained for 30 min under reflux on a water bath. Then, 6 mL of distilled water and 0.5 mL of the Folin–Ciocalteu reagent (Chempur, Piekary Śląskie, Poland) were added to 0.1 mL of the methanol extracts tested, and the whole mixture was mixed and allowed to stand for 3 min. After this time, saturated sodium carbonate solution and distilled water was added and placed in a thermostat (40 °C) for 30 min; the analysis was performed in triplicate. The results were calculated from the standard curve determined for gallic acid (GA)and expressed as mg of total polyphenols content per gram of extract performed per GA.

2.5. Determination of Antioxidant Activity Using the DPPH Method

The analysis was performed according to the method given by Yen and Chen [24]. The antioxidant activity was determined using the DPPH method, consisting of the colorimetric measurement of the degree of the reduction in DPPH free radicals (2,2-diphenyl-1-picrylhydrazyl, Merck, Poznań, Poland), for prepared extracts. Absorbance was measured using a Hitachi U-2900 UV-Vis model spectrophotometer (Hitachi High-Tech Corporation, Ibaraki, Japan); the absorbance of the solutions was measured at a wavelength of 517 nm using methanol as the reference solution. In this study, we used a 1% methanol solution of vitamin C as a control. To determine DPPH activity, 25 mL of methanol was added to about 0.5 g sample and left for 5 min in ultrasonic bath; then, the solution was filtered and used for testing and the analysis was performed in triplicate. The blank test consisted of mixing the reagents with water instead of the tested extracts. The results were expressed as the percentage of DPPH inhibition according to the formula given by Rossi et al. [21]:

X% = 100 − [At/Ar × 100]

At—absorbance of the test sample;

Ar—absorbance of the blank.

2.6. Determination of Antioxidant Activity Using the FRAP Method

The antioxidant activity was determined using the FRAP method for prepared infusions of dried plant material according to the method presented by Thaipong et al. [25] and Mulugeta et al. [26], with modifications. For the analyses, a FRAP (Merck, Poznań, Poland) reagent was prepared to consist of 0.3 M acetate buffer (pH = 3.6; Pol-Aura, Zabrze, Poland), 10 mM TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine; Merck, Poznań, Poland) in hydrochloric acid (Chempur, Piekary Śląskie, Poland), and 20 mM iron (III) chloride hexahydrate (Chempur, Piekary Śląskie, Poland). In order to analyze 0.5 g of the extracted samples and 25 mL of methanol, the solution was filtered and used for testing. After that, 100 μL of the extract was taken to determine antioxidant activity using the 3 mL the FRAP reagents. The solution was maintained at 37 °C for 10 min; analysis was performed in triplicate. After this time, the absorbance was measured at 593 nm using a blank containing a mixture of solvents and methanol. Absorbance was measured using a Hitachi U-2900 UV-Vis model spectrophotometer (Hitachi High-Tech Corporation, Ibaraki, Japan). The results were read from the formula curve made for the standard solution, Trolox, and the antioxidant capacity of the samples was expressed as the Trolox equivalent (mg Trolox/g extract).

2.7. HPLC Analysis of the Tested Extracts

Analyses were conducted according to the methodology developed by Łabuda and Paliński [27]. Analyses were conducted using a LaChrom liquid chromatograph equipped with a Merc Hitachi L-7455 DAD detector, L-7360 thermostat, L-7100 pump, L-7612 de-gasser, and a 20 μL dosing loop (Hitachi High-Tech Corporation, Ibaraki, Japan). All analyses were performed in the reversed-phase system using a LiChrospher 100 RP-18 250-4 column (Merck KGaA, Darmstadt, Germany). Analyses were performed using water and methanol (75 + 25 v/v), with 1% acetic acid as the mobile phase, at a flow rate of 0.8 mL/s. Compounds were identified based on a comparison of our the retention times and spectra of standards available in the literature with our obtained retention times and the spectra of the analyzed extracts.

2.8. Statistical Analysis

The obtained results are presented as the means and were statistically analyzed via ANOVA, and the averages were compared using Tukey’s HSD test at the probability level α = 0.05. Statistical analyses were calculated with Statistica 13.3 PL software (StatSof Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Antioxidant Activity and Phenolic Content

Medicinal and aromatic plants serve as prospective sources of natural bioactive compounds—including phenolic compounds—which hold significant potential for applications in the pharmaceutical, cosmetic, and food industries [5,11,28]. Particularly rich in these compounds are plants of the Lamiaceae family, which exhibit high biological activity. We have shown two Lamiaceae species—T. vulgaris and S. scardica—to have significant phytochemical potential, and found differences between the extraction efficiency, chemical compositions and the antioxidant activities of the herb extracts derived from them (

Table 1. Extraction and fractionation results of the tested plant material.

	Solvent	Extract Weight (g)		Efficiency (%)
Plant Material		Sideritis Aerial Parts	Thymi Aerial Parts	Sideritis Aerial Parts	Thymi Aerial Parts
	ACT	2.80	1.41	14.01	7.10
	MET	6.20	5.56	31.05	27.82
	EtOH-H2O	6.43	6.81	32.14	34.06
	EtOAc	1.81	1.33	9.07	6.66

,

Table 2. Polyphenol, flavonoid and phenolic acids content (per gram of extract) in the tested plant extracts.

Solvent	Sideritis	Thyme	Mean	Sideritis	Thyme	Mean	Sideritis	Thyme	Mean
	TPC (mg GA/g)			TFC (mg QE/g)			TPAC (mg CA/g)
ACT	189.39 a	144.87 dc	167.13 A	20.63 d	60.42 a	40.52 A	111.28 c	38.89 f	75.09 B
MET	164.11 cb	179.57 ba	171.84 A	12.34 f	15.7 e	14.02 B	92.72 d	141.09 a	116.9 A
EtOH-H2O	151.88 c	125.4 d	138.64 B	10.57 f	6.51 g	8.54 C	76.71 e	119.1 b	97.9 C
EtOAc	144.12 dc	121.39 d	132.76 B	23.49 c	55.85 b	39.67 A	76.69 e	15.94 g	46.31 D
Mean	162.38 A	142.81 B		16.76 B	34.62 A		89.35 A	78.75 B

TPC—total polyphenols content; TFC—total flavonoids content; TPAC—total phenolic acids content; ACT—acetone; MET—methanol; EtOH-H₂O—ethanol 70%; EtOAc—ethyl acetate. Data related to a given compound in rows/columns marked with the same upper- or lower-case letter do not differ significantly from each other.

and

Table 3. Antioxidant activity of the tested plant extracts.

Solvent	Sideritis	Thyme	Mean	Sideritis	Thyme	Mean
	FRAP mg/g Trolox			DPPH%
ACT	1.66 ba	1.49 b	1.57 A	86.07 b	55.67 e	70.87 C
MET	1.16 c	1.82 a	1.49 A	87.98 a	77.26 d	82.62 B
EtOH-H2O	1.09 dc	1.47 b	1.28 B	85.75 b	83.06 c	84.4 A
EtOAc	0.88 d	1.01 dc	0.94 C	85.75 b	55.16 e	70.46 C
Acidum ascorbicum						90.03
Mean	1.2 B	1.45 A		86.39 A	67.79 B

ACT—acetone; MET—methanol; EtOH-H₂O—ethanol 70%; EtOAc—ethyl acetate. Data related to a given activity in rows/columns marked with the same upper- or lower-case letter do not differ significantly from each other.

). Sideritis extracts were found to have a higher total polyphenols content and total phenolic acids content, as well as better antioxidant activity (DPPH), compared to thyme extracts. Conversely, thyme extract had higher total flavonoids content and showed more antioxidant activity (FRAP) than sideritis extract. A high total polyphenols content in extracts is not necessarily accompanied by a high total flavonoids content, as the proportions of specific flavonoids in the total polyphenols content can vary [10]. T. vulgaris is an established medicinal plant with notable antioxidative capacity [29]. Roby et al. [30] reported thyme extracts MeOH as having the highest antioxidative capacity, exceeding that of other plants, α-tocopherol and BHA. Antioxidant activity can also be estimated in MeOH extracts using ascorbic acid or total polyphenols content as a proxy measure, due to the strong correlation [25]. Köksal et al. [31] established that aqueous and EtOH thyme extracts have high antioxidant activity in terms of DPPH and ABTS radical scavenging activity, reducing potential, the inhibition of linoleic acid peroxidation, and iron/copper ion-reducing capacity. The differences in the chemical composition and antioxidant activity between the extracts under study were also attributed to the type of solvent (ACT, MeOH, EtOH-H₂O, and EtOAc) (Table 1 and Table 2). Whereas both MeOH extracts (sideritis/thyme) assayed had a similarly high total polyphenols content, the TMeOH proved superior to sideritis (SMeOH) in terms of total phenolic acids content (141.09 mg CA/g EX) and total flavonoids content (15.7 mg QE/g EX). Mokhtari et al. [32] compared the composition of two methanol extracts—one from salvia and one from thyme—and found that the salvia extract contained more total flavonoids (4.11 mg·QE/g·DW), but the thymi extract had more total polyphenols (8.89 mg·GAE/g·DW) and an improved extract yield (21.81%). In our study, the EtOH-H₂O performed the best in terms of extraction yield, at 34.06% (thyme) and 32.14% (sideritis), followed by MeOH (31.05% sideritis and 27.82% thyme) (Table 1), which exhibited the highest antioxidant activity when measuring DPPH activity (Table 2). However, the activity of the 1% vitamin C solution, used as a control, was 90.03%. Minarti et al. [33] obtained the highest yield, at 46%, for the n-hexane fraction—conversely, however, the fraction exhibited no antioxidant activity and contained few phenolic compounds and flavonoid compounds. The EtOAc fraction exhibited strong antioxidant activity, DPPH, and ABTS. The EtOAc, butanol, and H₂O fractions had more total polyphenols and total flavonoids, and higher antioxidant activity, compared to the n-hexane. This suggests that antioxidant activity, DPPH, ABTS, and FRAP are a function of secondary metabolites (phenolic compounds or flavonoid compounds).

S. scardica is one of the most bioactive and versatile medicinal plants. Its multi-tiered health benefits stem from the presence of phenol compounds—especially total phenolic acids content—in S. scardica extracts [34]. Sideritis extracts possess remarkable DPPH scavenging activity and ensure lipid peroxidation inhibition. Furthermore, Sideritis plants with high levels of polyphenolic compounds also exhibit the highest antioxidant activity [35]. Our findings indicate that SIEs are characterized by both a high total polyphenols content (162.38 mg GA/g) and high antioxidant activity (DPPH) (86.39%) (Table 2 and Table 3). This is corroborated by Koleva et al. [36], who showed that butyl-methyl-ether and MeOH extracts of S. scardica showed superior antioxidant activity to other extracts. Conversely, EtOAC, BuOH, and rosmarinic acid were determined to have moderate to weak antioxidant activity by a β-carotene bleaching assay. Our study similarly shows EtOAC sideritis extracts as having the poorest antioxidant activity, FRAP, compared to the others (0.88 mg/g Trolox).

The type of solvent is a major factor in extraction and a crucial determinant of the extract’s antioxidant activity [37]. We have shown that, on average, ACT and MeOH were the most effective solvents for extracting total polyphenols content and total phenolic acids content from the plant material under study, likely contributing to the superior AAS-FRAP that was observed (1.57 and 1.49 mg/g Trolox, respectively). TF yields, on the other hand, proved to be the highest when AC and EtOAc were used (Table 2). For thyme, MeOH performed the best in extracting total polyphenols content and total phenolic acids content (179.57 mg GA/g and 141.09 mg CA/g), whereas ACT was superior for total flavonoids content (60.42 mg QE/g). In the case of sideritis, ACT allowed for the extraction of the highest amounts of total polyphenols content (189.39 mg GA/g) and total phenolic acids content (111.28 mg CA/g), while for total flavonoids content, the best was EtOAc (23.49 mg QE/g). Roby et al. [30] determined the antioxidant activity and total polyphenols content of thyme, sage, and marjoram extracts across various extraction solvents using the DPPH and Folin–Ciocalteu methods. MeOH performed the best in extracting total polyphenols content (8.10, 5.95, and 5.20 mg GA/g DW for thyme, sage, and marjoram, respectively). Furthermore, the thyme MeOH extract was reported to have the highest antioxidant capacity, exceeding that of other plants, α-tocopherol, and BHA. This was corroborated by our study, which found that the thyme MeOH extract had the highest total phenolic acids content (141.09 mg CA/g EX) and antioxidant activity-FRAP (1.82 mg/g Trolox). Alcoholic extracts of thyme are rich in polyphenols and possess strong antimicrobial properties [38]. Mokhtari et al. [32] determined the total polyphenols content to be 8.89 mg GAE/g·DW and the total flavonoids content to be 3.87 mg·QE/g·DW for thyme MeOH extract, with thymol, apigenin, rosemary acid, and carvacrol being the most abundant phenolic compounds for thyme MeOH. EtOAc and hexane extract from thyme leaves were tested for their activity, with both (especially the former) showing antibacterial action [28].

3.2. The Extraction Conditions

Ethanol (EtOH) has been used for centuries and played a vital role in the production of medicines. Historically, galenic formulations were usually created by extracting plant materials with aqueous EtOH solutions. EtOH is currently considered to be more efficient when extracting phenol compounds and pigments [39]. Yeasmen and Islam [40] demonstrated that using an EtOH solvent on Tamarindus indica seeds provided better results than ACT in terms of extract yield, total polyphenols content, scavenging activity, reducing capacity, and antioxidant activity. Yanchev et al. [41] investigated how different solvents and extraction protocols affect yield, total polyphenols content, and total flavonoids content and antioxidant activity for S. scardica. H₂O, ACT, and EtOH-H₂O (20%, 50%, and 70% EtOH) were used as solvents, with preparation methods including infusion, brewing, and maceration. EtOH70 performed the best in terms of total polyphenols content, followed by ACT and water infusion. The highest extraction yields were achieved with EtOH50 and EtOH70 maceration, followed by water infusion, with EtOH70 also providing the highest antioxidant potential. Furthermore, the study showed a positive correlation between total polyphenols content and antioxidant activity. Mróz et al. [42] showed that EtOH70 was the most efficient extractant for various classes of Sideritis phytochemicals, including antioxidants. Tsibranska et al. [43] also found that H₂O-EtOH 20/80 performed the best in terms of polyphenol and flavonoid extraction. EtOH content in the solvents was inversely correlated with total extract yield, but positively correlated with levels of polyphenol compounds. Duque-Soto et al. [44] noted that, as assayed by TEAC (Trolox Equivalent Antioxidant Capacity), EtOH–H₂O in a ratio of 80:20 (v/v) was the best solvent for extracting antioxidants from all tested Lamiaceae MAPs. Wisam et al. [45] reported that the phenolic content of EtOH thyme leaf extract was markedly higher than that of an aqueous extract (20.31% and 13.44%, respectively). Köksal et al. [31] determined the phenolic profile of aqueous (AQ) and EtOH extracts of thyme. Total polyphenols content was 256.0 µg GA/mg dry extract and 158.0 µg GA/mg dry extract in AQ and EtOH extract of thyme, respectively. Total flavonoids content, however, was 44.2 µg and 36.6 µg QE/mg dry extract, respectively. Our study showed that EtOH-H₂O ranked second (after MeOH) in terms of total phenolic acids content extracted from thyme (141.09 mg CA·g EX), and second (again, after MeOH) as an SIEs solvent for antioxidant activity (DPPH) (85.75%). The EtOH-H₂O extracts also exhibited the strongest average DPPH radical reduction activity (84.4%) (Table 2). This finding suggests that polar solvents such as ACT, MeOH, and EtOH-H₂O are effective in extracting total polyphenols content, total flavonoids content and total phenolic acids content from thymi and sideritis herbs, which translates to high antioxidant activity in these extracts. EtOH and MeOH can extract polyphenols, flavonols, tannins, and terpenoids, while ACT is capable of extracting flavonoids, likely contributing to its high antioxidant activity [46]. Using polar solvents (MeOH) allows for more diverse secondary metabolites to be extracted from Ageratum leaves compared to a non-polar solvent. The polar fraction of the leaves contained alkaloids, flavonoids, tannins, saponins, and terpenoids [47]. In our study, ACT and EtOAc—solvents with differing polarities—achieved the highest average total flavonoids content extraction performance across all extracts (Table 2). Zazouli et al. [48] examined the chemical composition of Caralluma root extracts using solvents of increasing polarity (hexane, chloroform, dichloromethane, EtOAc, ACT, EtOH, and MeOH). There were substantial differences in total polyphenol yields from the various solvents. The total polyphenols content was significantly higher in the MeOH extract compared to the chloroform and EtOH fractions (p < 0.05). Conversely, more total flavonoids were found in the EtOH and EtOAc samples (834.7 and 844.2 CEQ/100 g DW, respectively). Polar solvents, including alcohol groups, are commonly used to extract organic compounds from natural products, including secondary plant metabolites. Due to their ability to increase cell permeability and penetrate cell interiors, these solvents tend to be more effective at extracting intracellular secondary metabolites (both polar and “less polar” metabolites) than non-polar solvents such as n-hexane. The non-polar solvents generally dissolve only non-polar compounds, lipophilic substances, and hydrophobic substances [47,48]. This may explain the differences in antioxidant activity across the extracts tested in our study, where the highest antioxidant activity (DPPH) was found for EtOH-H₂O extracts (84.4%), while antioxidant activity (FRAP) was the highest for ACT and MeOH extracts (1.57 and 1.49 mg/g Trolox, respectively).

3.3. Correlation Coefficients of the Antioxidant Activity and Phenolic Content of the Tested Plant Extracts

Spectrophotometric methods for determining antioxidative properties, such as the FRAP assay–ferric ion reducing antioxidant parameter and the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, are often used to study the antioxidant properties of plant-derived products [15,17,18]. The antioxidative property determinations made using these two methods are strongly correlated (r = 0.822) [49]. Our study found a moderate positive correlation between antioxidant activity (DPPH) and antioxidant activity (FRAP), but only for thyme (r = 0.591167) (

Table 4. Pearson’s correlation coefficients of antioxidant activities and phenolic compounds content.

Sideritis
Compound/AAC	TPC	TFC	TPAC	AAC–DPPH	AAC–FRAP
TPC	x	0.106035	0.895424 *	0.148167	0.83135 *
TFC	−0.26717	x	0.205463	−0.4218	0.066284
TPAC	0.57718 *	−0.93361 *	x	0.272149	0.917172 *
AAC–DPPH	0.274637	−0.99574 *	0.940963 *	x	0.048353
AAC–FRAP	0.825931 *	−0.55946	0.789627 *	0.591167 *	x
Thymus

AAC—antioxidant activity; TPC—total polyphenols content; TFC—total flavonoids content; TPAC—total phenolic acids content; * Significant at p < 0.05.

). Irawan et al. [50] compared the results regarding antioxidant activity (DPPH, FRAP and CUPRAC assays), noting that DPPH produced a different value (IC₅₀), showing poor antioxidant activity compared to CUPRAC and FRAP. Due to the differing sensitivities of DPPH, ABTS, CUPRAC, RP and FRAP, antioxidant activity measurements may vary significantly across the tests. Nevertheless, many reports have indicated that there is high correlation between these methods, especially between DPPH and FRAP [33,50,51]. The results of antioxidant activity assays are also affected by the type of material analyzed and the type of extract. For example, ABTS, DPPH, FRAP, and ORAC all provide similar antioxidant activity outputs for MeOH guava fruit extract, while FRAP features high reproducibility, and the highest correlation for ascorbic acid and total polyphenols content [25].

The biological activity of plant extracts is highly variable, influenced by the plant species and, especially, by the extraction technique. Temperature, time, and extraction rate are positively correlated with DPPH, FRAP, and levels of polyphenols compounds (p < 0.05) [52]. The study by Ulewicz-Magulska and Wesołowski [10] showed that Lamiaceae herbs are substantially richer in total polyphenols content and have higher antioxidant activity than the Apiaceae species. The strong correlations between antioxidant activity found across DPPH, ABTS, FRAP, and total polyphenols content assays confirm the major role played by phenolic compounds in determining the antioxidative properties of herbs. However, data on the correlation between phenolic compounds and antioxidant activity are far from consistent, varying with different plant materials, extraction methods, and antioxidant activity assays. Irawan et al. [50] report that total polyphenols content shows no correlation with flavonoid compounds but a strong correlation with antioxidant activity, whereas flavonoid compounds do not correlate with antioxidant activity. Aboukhalaf et al. [53] found a strong correlation between total polyphenols content, total flavonoids content, and antioxidant activity. Our own findings show that the antioxidant activity (FRAP) of the herbal extracts tested was significantly and very strongly correlated with total polyphenols content (r = +0.825931 and +0.831350), and affected by the type of raw material used (Table 4). In both SIEs and THEs, there was a very strong positive correlation between total phenolic acids content, total polyphenols content, and antioxidant activity (FRAP), with SIEs demonstrating the highest correlation in the total phenolic acids content/antioxidant activity (FRAP system; r = +0.917172). In contrast, when DPPH was used, the SIEs did not show significant correlations, but THEs showed a significant and very strong negative correlation between total flavonoids content and antioxidant activity (DPPH; r = −0.995743), as well as a very strong positive correlation between total phenolic acids content and antioxidant activity (DPPH; r = +0.940963). A correlation coefficient value higher than 0.61 and lower than 0.97 is indicative of a highly positive correlation [25]. Fernandes et al. [54] correlated total polyphenols content with the antioxidant capacity of marjoram, oregano, lemon balm, and rosemary extracts. The r-value between the antioxidant capacity and total polyphenols content was 0.8866 according to FRAP and 0.9706 according to DPPH. The high r suggests that DPPH radical scavenging activity can be reliably predicted using the Folin–Ciocalteau test to determine total polyphenols content, and confirms that the antioxidant activity is attributable to the phenolic compounds. In Djeridane et al.’s [8] study of extracts from several Algerian medicinal plants, the authors identified positive correlations between total polyphenols content and antioxidant activity (Trolox; R² = 0.7931), and between total flavonoids content and antioxidant activity (Trolox; R² = 0.7802), indicating that phenolics play a major role in antioxidant activity.

3.4. Coefficients of Antioxidant Activity Depending of the Phenolic Compounds and Type of Extract

Sasikumar et al. [9] found that the antioxidant activities of spices (Lippia adoensis, Nigella sativa, Piper capense, Thymus bschimperi, and Trachyspermum ammi) may vary according to the different solvents used for extraction (H₂O and MeOH). Most MeOH extracts demonstrated better DPPH radical scavenging capacity than aqueous extracts. High to moderate positive correlations were observed between total polyphenols content and in vitro antioxidant assays. Furthermore, there was a strong positive correlation between total polyphenols content and antioxidant activity when using DPPH for MeOH and H₂O herbal extracts, a pattern that held true for the extracts tested in the present study (Table 4). This was further confirmed by analyzing correlations among antioxidant activity, phenolic compounds, and four types of thyme/sideritis extract (

Table 5. Pearson’s correlation coefficients of antioxidant activities, phenolic compounds, and type of extract.

Compound/AAC		Extract
		ACT	MET	EtOH-H2O	EtOAc
TFC	TPAC	−0.998190 *	0.975796 *	−0.968122 *	−0.999104 *
	AAC–DPPH	−0.999250 *	−0.995786 *	0.982767 *	−0.999258 *
	AAC–FRAP	−0.686423	0.984452 *	−0.935777 *	0.961422 *
	TPC	−0.939186 *	0.890216 *	0.820933 *	−0.970955 *
TPAC	AAC–DPPH	0.999538 *	−0.991446 *	−0.993789 *	0.999815 *
	AAC–FRAP	0.658483	0.935648 *	0.988061 *	−0.961763 *
	TPC	0.956980 *	0.808368	−0.723206	0.970589 *
AAC–DPPH	AAC–FRAP	0.664808	−0.967907 *	−0.968210 *	−0.965447 *
	TPC	0.948541 *	−0.865067 *	0.729254	0.970166 *
AAC–FRAP	TPC	0.878578 *	−0.714550	−0.933343 *	0.503842

AAC—antioxidant activity; ACT—acetone; MET—methanol; EtOH-H₂O—ethanol 70%; EtOAc—ethyl acetate; TFC—total flavonoids content; TPAC—total phenolic acids content; TPC—total polyphenols content; * significant at p < 0.05.

).

The correlations between individual phenolic components and AAC depended on the type of solvent used in the extraction process. We found strong positive correlations between total polyphenols content and total phenolic acids content (ACT), total phenolic acids content and total flavonoids content (EtOAc, MeOH), total flavonoids content and total polyphenols content (MeOH, EtOH-H₂O), antioxidant activity (DPPH) and total polyphenols content (ACT, EtOAc), antioxidant activity (DPPH) and total phenolic acids content (ACT, EtOAc), antioxidant activity (DPPH) and total flavonoids content (EtOH-H₂O), antioxidant activity (FRAP) and total polyphenols content (ACT), antioxidant activity (FRAP) and total flavonoids content (EtOAc, MeOH), and antioxidant activity (FRAP) and total phenolic acids content (MeOH, EtOH-H₂O). The data suggest a relationship between the content of specific phenolic compounds and antioxidant activity, but the type of extract (solvent) is a major factor in this regard. The correlation between FRAP and total polyphenols content (Folin–Ciocalteu method) can be attributed to the same single electron transfer mechanism; however, FRAP cannot detect inert phenolic compounds and thiols [18]. On the other hand, the DPPH assay provides insight into various antioxidant properties due to the different reaction mechanism used, can detect the overall antioxidant capacity of a sample, and is not specific to a single antioxidant [15,18]. Minarti et al. [33] investigated the antioxidant capacity of MeOH extract from Macaranga hypoleuca leaves and its fractions using DPPH, ABTS, and FRAP, as well as using the total polyphenols content and total flavonoids content methods. The highest antioxidant activity (DPPH) and ABTS was found for the EtOAc fraction, whereas the highest antioxidant activity (FRAP) was found for the butanol fraction. The butanol fraction also exhibited the highest total polyphenols content and total flavonoids content (27.51 mg GAE/g and 88.42 mg QE/g., respectively). Our study showed a significant positive correlation between antioxidant activity (DPPH) and antioxidant activity (FRAP) for ACT extracts (r = 0.664808), and strong negative correlations for the other extracts (Table 5). Ahoua et al. [55] studied extracts from the dried and powdered leaves and fruits of several African plant species (using dichloromethane followed by methanol) and found a positive correlation between the DPPH and FRAP results (r = 0.599).

3.5. Phenolic Acid Profile of the Tested Plant Extracts

The results of phenolic acid identification using HPLC are presented in

Table 6. Phenolic acid profile of the tested plant extracts.

Phenolic Acids	ACT		MET		EtOH-H2O		EtOAc
	Thymi	Sideritis	Thymi	Sideritis	Thymi	Sideritis	Thymi	Sideritis
Chlorogenic acid		x		x		x		x
Cafeic acid	x		x	x			x
Protocatechuic acid		x
m-Cumaric acid	x
Ferulic acid	x	x	x	x	x

ACT—acetone; MET—methanol; EtOH-H₂O—ethanol 70%; EtOAc—ethyl acetate.

and Figures S2 and S3. In the extract of the sideritis aerial parts obtained using acetone as a solvent, three compounds were identified, including chlorogenic acid with a retention time of 3.15 min, protocatechuic acid (RT—6.99 min), and ferulic acid (RT—10.27 min) (Figure S2). In the MeOH extract of sideritis, three phenolic acids were also identified, including chlorogenic acid, caffeic acid, and ferulic acid, with retention times of 3.2 min, 5.04 min, and 11.2 min, respectively (Figure S2). In the EtOH-H₂O and EtOAc extracts, only chlorogenic acid was identified, with retention times of 3.33 min EtOH and 3.63 min EtOAc, respectively. The HPLC analysis of extracts obtained from the sideritis aerial parts allowed for the identification of four compounds belonging to the phenolic acid group, including chlorogenic, protocatechuic, ferulic, and caffeic acid. The ACT extract allowed for the identification of three of the four phenolic acids, i.e., chlorogenic, pro-tocatechuic, and ferulic acid, similarly to the MeOH extract, where, in this case, chlorogenic, caffeic, and ferulic acid were identified. Kaparakou et al. [19] determined only chlorogenic acid (RT-3.15) using LC-MS/MS-QTOF in sideritis hydroalcoholic extracts (70:30 MeOH/H₂O mixture), which was confirmed by our studies (in the ethanol extract, which was a mixture of alcohol and water, the presence of chlorogenic acid was also detected: RT-3.33). The present study showed that most of the detected phenolic acids were detected in the ACT extract obtained from S. scardica’s aerial parts, including chlorogenic, protocatechuic, and ferulic acids; the presence of these was also confirmed in the aqueous extract in the study conducted by Zhelev-Dimitrov et al. [20] using the UHPLC-HRMS method.

In the case of the analysis of extracts obtained from thyme aerial parts, three compounds were identified (Figure S3). In the ACT extract, three phenolic acids were identified: caffeic acid (RT—4.88 min), ferulic acid (RT—9.87 min), and m-coumaric acid (RT—15.65 min). In the MeOH thyme extract, the presence of caffeic acid and ferulic acid was detected, with retention times of RT—5.01 min and RT—10.21 min, respectively. In the EtOH-H₂O extract, ferulic acid was identified (RT—9.49 min), while in the EtOAc extract, caffeic acid (RT—5.04 min) was identified (Figure S3). Caffeic acid was identified in the thyme extract obtained using EtOAc solvent, which confirms the results obtained by Saleem et al. [56]. These researchers also detected the presence of other acids including chlorogenic and sinapic acid in the EtOH fraction, which was not confirmed in our study. In the study conducted by Sarfaraz et al. [57] using a gradient mobile phase using water, acetonitrile, and formic acid, gallic acid (RT—5.5 min), caffeic acid (RT—14.68 min), ferulic acid (RT—29.74 min), cinnamic acid (RT—37.96 min), and rosmarinic acid (RT—39.04 min) were identified in the MeOH extract obtained from the aerial parts T. vulgaris, while the presence of chlorogenic acid was not detected. Our studies also confirmed the presence of caffeic acid and ferulic acid in the MeOH extract of thyme, and we also detected the presence of chlorogenic acid. Similarly, Roby et al. [30] determined the presence of the following phenolic components in MeOH thyme extract: rosmarinic acid, methyl rosmarinate, caffeic acid, cinnamic acid, chlorogenic acid and quinic acid, ferulic acid, apigenin, luteolin, and quercetin. Differences in the identification of phenolic acids in thyme and sideritis extracts can be explained primarily by the variability of the chemical composition of the raw material (genetic, ontogenetic, and/or environmental).

4. Conclusions

T. vulgaris and S. scardica extracts exhibit high levels of phenolics compounds and significant antioxidant activity, pointing to their potential application as antioxidant sources in the food industry. They may also be used for their therapeutic benefits in the treatment of certain diseases. Sideritis extracts contained more polyphenols and phenolic acids, whereas thyme extracts had a higher flavonoid content, which led to difference sin their antioxidant activity, as measured using DPPH and FRAP methods.

We identified a significant correlation between antioxidant activity and total polyphenols content, pointing to phenolics as the main contributors to the antioxidative activity of the plants under study. Our study found also a strong positive correlation between AAS-FRAP SIEs and total polyphenols content/total phenolic acids content, whereas antioxidant activity (DPPH) does not show a correlation with the phenolic components of the extract. In contrast, THEs showed different correlations: antioxidant activity (DPPH) had a strong positive correlation with total phenolic acids content and a strong negative correlation with total flavonoids content, while antioxidant activity (FRAP) was strongly positively correlated with total polyphenols content and total phenolic acids content.

Choosing the right solvent seems essential to ensure the efficient extraction of phenolic compounds, which are strongly associated with antioxidant activity. We have shown that the nature of the solvent affects the total polyphenols content, total flavonoids content, and antioxidant activity. EtOH-H₂O (EtOH70%) extracts from S. scardica and T. vulgaris aerial parts are phenolics-rich, bioactive, safe, and environmentally friendly, making them valuable in the production of medicinal tinctures and other plant-derived pharmaceuticals. Other extracts (ACT, MeOH, EtOAc) with strong antioxidant activity can be used as effective preservatives. The identification of phenolic acids in the tested extracts allows us to conclude that the type of solvent used for a specific raw material (sideritis/thyme) determines the presence of such acids in the extract. In the extracts tested, more phenolic acids were identified when MeOH and ACT were used as solvents (2–3) compared to EtOH-H₂O and EtOAc (1). Chlorogenic acid was present in all sideritis extracts, while in most thyme extracts, caffeic acid was present (exception: EtOH-H₂O).