Green Extraction Combined with Chemometric Approach: Profiling Phytochemicals and Antioxidant Properties of Ten Species of the Lamiaceae Family

Abstract

The pharmacological potential of Lamiaceae plants is primarily linked to their high content of phenolic acids and flavonoids, known for strong antioxidant properties. This study investigated the antioxidant activity of ten widely used Lamiaceae herbs—oregano, lavender, basil, savory, garden thyme, wild thyme, sage, rosemary, lemon balm, and mint—prepared as traditional infusions and microwave-assisted extracts. The antioxidant capacity was evaluated using spectrophotometric assays, and total phenolics and flavonoids were quantified via spectrophotometry and HPLC. Chemometric analysis (PCA) was applied to explore correlations among antioxidant parameters. The results demonstrated excellent antioxidant activity across all samples. The IC50 for DPPH radicals was in the range from 3.73(0.13) to 8.03(0.17) μg/mL and that for ABTS radicals was from 2.89(0.12) to 8.55(0.34). The CUPRAC antioxidant assay delivered values in the range from 351.93(11.85) to 1129.68(44.46) μg TE/mg DE. The FRAP method produced values from 1.27(0.03) to 6.60(0.26) μmol Fe/mg DE. The presence of gallic acid was detected in all examined samples, with lemon balm and lavender exhibiting the highest concentrations across both applied extraction methods. Notably, lavender showed especially high levels of p-hydroxybenzoic acid and chlorogenic acid. Microwave-assisted extraction generally yielded higher levels of bioactive compounds compared to infusion. These findings highlight the potential of Lamiaceae herbal extracts, particularly those obtained through microwave-assisted extraction, as valuable sources of dietary antioxidants for everyday use.

1. Introduction

Normal metabolic processes are followed by the production of reactive oxygen species (ROS) and free radicals daily [1,2]. In aerobic cells, oxygen is the ultimate electron acceptor during oxidation in the production of energy in the form of ATP [2]. Disruption of oxygen reduction leads to the production of highly reactive molecules known as free radicals, such as hydrogen peroxide (H₂O₂), superoxide anions (•O₂⁻), hydroxyl radicals (OH⁻), and singlet oxygen (¹O₂⁻) [1,2]. An imbalance between these compounds and the antioxidant defense mechanism results in the promotion of oxidative stress [3,4].

Exposure to chronic oxidative stress is associated with numerous diseases: neurodegenerative and neuropsychiatric, metabolic (diabetes mellitus), cardiovascular diseases, and cancer, as well as inflammatory conditions, genotoxic effects, and the aging process [3,5,6]. Studies have established that consequential infertility can occur in women [7]. Negative lifestyle behaviors involving alcohol consumption, smoking, chronic psychological stress, radiation, use of different drugs and xenobiotics, and environmental pollution are factors that also can accelerate the oxidative stress [2,5]. Oxidative damage can be prevented, controlled, or delayed by antioxidants [4].

Antioxidants are stable molecules with the ability to neutralize free radicals in biological cells [3,4]. The human body is capable of synthesizing molecules with antioxidant activity [8]. In addition, exogenous sources, particularly from dietary supplementation, can further enhance the body’s antioxidant defense mechanisms [3]. The benefits of these sources lie in their suitability for enhancing the antioxidant status in a household environment, one of which is the consumption of plants rich in secondary metabolites with antioxidant properties [3].

Attractive targets of secondary metabolites are polyphenols, which are proven to be effective antioxidant agents and can be found only in the plant world [1,5,6]. In their chemical composition, they have at least one aromatic ring with one or more hydroxyl groups that dictate their antioxidant potential. The most important representatives and one of the bearers of antioxidant activity are phenolic acids and flavonoids [1].

The Lamiaceae family, commonly referred to as the mint family, consists of approximately 249 genera and nearly 7886 species, positioning it as the sixth most diverse plant family [9,10]. Its long history of human utilization is primarily attributed to its ease of cultivation [10,11]. Today, numerous aromatic species (Mentha spp., Lavandula angustifolia, Rosmarinus officinalis) have found applications in the culinary arts, cosmetics, and most notably in traditional and modern medicine, with significant scientific interest focused on secondary metabolites such as phenolic acids and flavonoids [6,11,12]. They are partially hydrophilic, allowing them to be administered as aqueous infusions, which represents a convenient dosage form for the patient [12].

Extraction methods are the first step in the isolation and purification of bioactive compounds [13]. Traditional infusion methods and more advanced techniques, such as microwave-assisted extraction (MAE), have been shown to be effective for extracting these active compounds in household settings, making Lamiaceae species accessible as natural sources of antioxidants for domestic use [14,15,16]. These “green” extraction methods are not only cost-effective and environmentally friendly but also align with the growing consumer demand for natural remedies over synthetic pharmaceuticals [1,14,15]. The aim of this study was to compare the chemical composition and antioxidant activity of ten herbal teas from the Lamiaceae family, traditionally used in households, using two sustainable extraction methods—conventional infusion and microwave-assisted extraction. The analyses included the quantification of total phenolics and flavonoids, identification and quantification of individual phenolic compounds by HPLC, and assessment of antioxidant capacity through four complementary assays (ABTS, DPPH, FRAP, and CUPRAC). Additionally, chemometric analysis was employed to explore correlations between the chemical composition and antioxidant potential of the examined extracts.

2. Materials and Methods

2.1. Materials

Ethanol was obtained from J.T. Baker (Netherland), methanol and sodium carbonate from POCH (Gliwice, Poland), quercetin from Extrasynthese (Genay Cedex, France), gallic acid (GA), p-hydroxybenzoic acid (pHBA), caffeic acid (CafA), p-coumaric acid (pComA), rosmarinic acid (RA), ferulic acid (FA), chlorogenic acid (ChlA), epicatechin (Epi), naringenin (Nar), acetonitrile, and aluminum chloride from Sigma Aldrich (St. Louis, MO, USA), 2,2-diphenyl-1-picrilhydrazil (DPPH) reagent and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS reagent) from Alfa Aesar (Karlsruhe, Germany), Trolox and 2,4,6-tris-(2-pyridyl)-1,3,5-triazine (TPTZ reagent) from Acros Organic (Geel, Belgium), neocuprine, copper (II) chloride, ammonium acetate, acetic acid, ferric (III) chloride, and hydrochloric acid from Lachner (Brno, Czech Republic), and Folin–Ciocalteu reagent from Merck (Darmstadt, Germany). Ultra-pure water was utilized for preparing all solutions. Unless otherwise specified, all solvents and reagents were of analytical-grade purity.

2.2. Plant Preparation

The samples of herbal drugs were single-component teas, of the family Lamiaceae, from the Institute for Medicinal Plant Research “Dr Josif Pančić”, Belgrade, Serbia: oregano (Origani herba, Origanum vulgare L., No. 13460423), lavender (Lavandulae flos, Lavandula angustifolia Mill., No. 09050323), mint (Menthae piperitae folium, Mentha piperita L., No. 00131023), lemon balm (Melissae folium, Melissa officinalis L., No. 05740223), rosemary (Rosmarini folium, Rosmarinus officinalis L., No. 02290324), sage (Salviae folium, Salvia officinalis L., No. 03020224), garden thyme (Thymi herba, Thymus vulgaris L., No. 35691122), wild thyme (Serpylli herba, Thymus serpyllum L., No. 32391022), savory (Saturejae montanae herba, Satureja montana L., No. 05830223), and basil (Basilici herba, Ocimim basilicum L., No. 27540922). Plant material was pulverized in a blender, and the mean particle size was determined to be 2 mm using a sieve set (CISA Cedaceria Industrial, Lliçà de Vall, Spain). Traditional (infusion) and modern extraction (MAE) were performed to identify the most suitable solvent and extraction technique for everyday use. To prepare the infusion, 1 g of the herbal drug was poured into 200 mL of boiling distilled water, and the extraction was carried out for 10 min (according to the manufacturer’s instructions). Modern extraction is performed with cold distilled water and a microwave oven (Midea) for 1 min. The given samples were exposed to 180 W microwave radiation for three cycles of 20 s each (total extraction time 60 s), in order to avoid overheating of the samples. Additionally, pauses were made between the cycles; the samples were taken out of the microwave oven and cooled to room temperature, since excessive heating could lead to the degradation of active compounds. The short extraction time and intermittent cooling between cycles were critical in minimizing solvent evaporation. Moreover, the samples were covered during microwave treatment, and no visible change in solvent volume was observed, indicating that the volume of distilled water remained effectively constant throughout the process. As modern extraction provides faster isolation of active components, the length of extraction was shorter compared to the traditional method. The extracts were filtered through filter paper with a diameter of 125 mm, manufactured by Munktel (Germany), and then evaporated to dryness. The yield was calculated based on the crude drug (CD), while all other measurements were expressed relative to the dry extract (DE). All data are presented as the mean values (standard deviations) from triplicate measurements.

2.3. Analysis of Total Phenolic Content

The colorimetrical method with the Folin–Ciocalteu (FC) reagent was used to determine the amount of total phenolic compounds in the extracts [17]. The reaction was performed in a dark place at room temperature, where the reaction mixture containing 0.1 mL of extract dissolved in water (1 mg/mL), 0.5 mL of freshly prepared 0.2 M FC reagent, and 0.4 mL of sodium carbonate solution (7%, m/v) was kept for 30 min. The absorbance of the resulting solution was assessed at 760 nm using a UV/Vis spectrophotometer (model 8453 Hewlett Packard, Agilent Technologies, Santa Clara, CA, USA). The total phenolic compound concentration was determined in mg of gallic acid equivalents (GAE) per gram of dried extract. Measurements were conducted in triplicate.

2.4. Total Flavonoid Content Estimation

The total flavonoid content in the examined extracts was measured spectrophometrically, applying a method based on the formation of a flavonoid–aluminum complex [17]. We mixed 2.5 mL of extract with 1 mL of 2% AlCl₃ solution, and incubation was carried out at room temperature for 15 min, after which the absorbance of the mixtures was estimated at 430 nm. The flavonoid content was quantified in mg of quercetin equivalents (QE) per gram of dried extract.

2.5. DPPH Activity

The DPPH method was used to examine the antioxidant activity [17]. This method involved herbal extracts being dissolved in methanol, as well as mixing different volumes (10–50 μL) of herbal extracts in test tubes with 1 mL of DPPH solution (0.1 mM in methanol) and diluting them to final volume of 4 mL with methanol. The control sample was prepared with the same volume, without test compounds. Methanol was used as a blank, and absorbance (A) was estimated at 515 nm in triplicate. In order to calculate the Radical Scavenger Capacity (%RSC), the following equation was applied:

%RSC = 100 × (Acontrol − Asample)/Acontrol

(1)

The IC50 value, representing the concentration of the test sample leading the free radical concentration to reduce by 50%, was calculated graphically and presented as μg of the extract per ml of the final solution in the measuring cell.

2.6. Cupric Reducing Antioxidant Capacity (CUPRAC) Assay

This assay is based on the reduction of Cu²⁺ ion to Cu¹⁺ in the form of the neocuproine complex [18]. Briefly, 0.2 mL of extract was placed in a test tube along with 1 mL of neocuprine, 1 mL of copper (II) chloride, and 1 mL of ammonium acetate buffer (pH 7), made up to total volume of 4.1 with distilled water. After leaving in the dark for 30 min at room temperature, the absorbance was measured at 450 nm with Trolox used as a standard antioxidant, and the activity of the given extracts was expressed as µg of Trolox equivalent per mg of dry weight (µg TE/mg DW).

2.7. Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was performed by following the procedure in [18], based on the reduction of the ferric–tripyridyltriazine complex to its ferrous form and producing a blue-colored compound, with its absorbance maximum at 595 nm. To the test tube, 3 mL of previously prepared FRAP reagent, prepared by mixing 0.3 M acetic acid buffer (pH 3.6) with 2,4,6-tris-(2-pyridyl)-1,3,5-triazine (TPTZ 0.01 M in ethanol), and FeCl₃·6H₂O (0.02 M ferric chloride, in 0.02 M aqueous HCl) in a volume ratio of 10:1:1, was added along with 0.1 mL of the extract and diluted with distilled water to the volume of 3.4 mL. Following the incubation at 37 °C, absorbance was recorded at 595 nm, and the activity was expressed as mmol of Fe (II) ion equivalents per g of dry weight from a previously constructed standard curve using FeSO₄.

2.8. ABTS Assay

For the ABTS radical scavenging activity assay, we followed the procedure of [18] by producing the ABTS radical, achieved by mixing 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt stock solution in distilled water with potassium persulfate and allowing it to stand in the dark for 12–16 h. After this period, the solution was further diluted with methanol in order to obtain the working solution, with the absorbance of 0.7(0.02) units at 734 nm. The sample was formed by mixing 1.8 mL of ABTS working solution with 0.2 mL of extract of different concentrations and made up to the total volume of 4 mL with methanol. After 6 min of incubation at room temperature, the reduction of absorbance was measured at 734 nm with methanol + ABTS mixture as a reference for 100% activity. The activity was expressed as the IC50 value in µg/mL.

2.9. High-Performance Liquid Chromatography (HPLC)

For the qualitative and quantitative analysis of flavonoids and phenolic acids in plant extracts, an appropriate HPLC method was applied [19]. Chromatographic analysis was performed using a Dionex HPLC system (Thermo Fisher Scientific, Cheshire, UK). The mobile phase consisted of 0.1% acetic acid in deionized water and 0.1% acetic acid in acetonitrile, with a flow rate of 1 mL/min. A 5 µL sample was injected. The elution followed a gradient program: 3.25 min-10% B; 8 min-12% B; 15 min-25% B; 15.8 min-30% B; 25 min-90% B; 25.4 min-100% B. A Zorbax Eclipse C18 column (4.6 mm × 150 mm, i.d., 5 µm particle size, Agilent Technologies, USA) was used. The column temperature was set to 25 °C, and the pressure was maintained at 134 bar. Detection was carried out at 280 nm. The total analysis time for each sample was 30 min.

2.10. Chemometric Analysis

Principal component analysis (PCA) was performed with Statistica v. 12 software (Stat Soft Inc., Tulsa, OK, USA). Herbal extracts obtained by MAE and infusion represented the cases, while the antioxidant activity parameters of these extracts determined by different assays, as well as the amounts of total phenolic and flavonoid compounds, were the variables. All data were standardized prior to calculation.

3. Results

3.1. Extraction Yield, Total Phenolic Content, and Flavonoid Content

During this research, environmentally friendly water extracts were examined, prepared in two ways that can be used every day in the household—a traditional technique (infusion) and preparation of tea in a microwave oven. These two techniques represent green approaches to the daily preparation of teas, and their activity was compared. The values for the extraction yield, total phenolic content, and total flavonoid content are shown in

Table 1. Extraction yield (mg/g CD), values of total phenolic content (mg eq GAE/g DE), and values of total flavonoid content (mg eq QE/g DE), (mean (SD)).

	Yield (mg/g CD)		TPC (mg eq GA/g DE)		FLV (mg QE/g DE)
	Infusion	Microwave	Infusion	Microwave	Infusion	Microwave
basil	90.73(2.32)	130.22(4.17)	39.58(0.15)	40.26(1.51)	4.01(0.06)	4.46(0.09)
wild thyme	92.81(3.5)	97.61(2.8)	25.30(0.61)	37.85(2.10)	5.13(0.07)	7.46(0.12)
savory	82.75(1.47)	106.47(6.45)	59.36(0.09)	69.85(0.34)	9.90(0.14)	11.23(0.08)
oregano	116.88(5.18)	163.35(5.19)	36.08(1.17)	48.64(0.27)	7.37(0.04)	7.71(0.07)
sage	115.00(4.57)	150.85(6.13)	53.97(0.36)	64.46(0.05)	5.00(0.02)	6.72(0.09)
garden thyme	111.89(7.24)	124.63(4.89)	37.51(0.85)	60.42(0.29)	6.56(0.12)	8.80(0.06)
mint	196.83(5.28)	250.75(8.16)	40.01(0.56)	48.24(1.69)	8.18(0.11)	8.97(0.12)
lavender	121.12(3.25)	162.03(4.16)	33.05(0.55)	47.63(0.11)	3.14(0.04)	5.27(0.14)
rosemary	30.94(1.02)	33.70(1.08)	43.80(0.73)	46.54(0.14)	7.06(0.06)	10.45(0.05)
lemon balm	217.43(8.65)	226.55(6.94)	51.24(0.61)	53.02(0.50)	2.62(0.02)	4.21(0.07)

. The content of total phenols in traditional infusions was in the range of 25.30(0.61)–59.36(0.09) mg GAE/g DE, and in microwave extracts, of 37.85(2.10)–69.85(0.34) mg GAE/g DE, which confirms that there is a difference in yields in relation to the applied method, but it is not statistically significant. The flavonoid content in traditional extracts ranged from 2.62(0.02) to 9.90(0.14) mg QE/g of dry extract, while in extracts obtained by microwave-assisted extraction, it was higher and ranged from 4.21(0.07) to 11.23(0.08) mg QE/g of dry extract.

3.2. DPPH and ABTS Test

The IC₅₀ values representing the radical scavenging activity of the extracts against DPPH radicals obtained by the traditional infusion method ranged from 3.73(0.13) to 8.03(0.17) μg/mL, while values for microwave-assisted extraction ranged from 2.63 (0.11) to 7.70 (0.26) μg/mL. A similar trend was observed with the ABTS assay, which is based on a comparable mechanism of radical scavenging. The IC₅₀ values obtained by the ABTS method ranged from 2.89 (0.18) to 8.55 (0.41) μg/mL for traditional infusions and from 1.34 (0.11) to 8.39 (0.61) μg/mL for microwave-assisted extracts (

Table 2. IC50 values for DPPH and ABTS (μg/mL), (mean (SD)).

	DPPH (µg/mL)		ABTS (µg/mL)
	Infusion	Microwave	Infusion	Microwave
basil	7.71(0.24)	7.23(0.18)	8.55(0.41)	8.39(0.61)
wild thyme	4.68(0.11)	3.96(0.18)	4.44(0.23)	4.20(0.32)
savory	3.73(0.13)	2.63(0.11)	6.12(0.38)	4.84(0.34)
oregano	7.73(0.49)	4.88(0.25)	2.89(0.18)	1.89(0.09)
sage	5.07(0.14)	4.53(0.21)	5.14(0.41)	3.35(0.22)
garden thyme	8.03(0.17)	7.67(0.26)	5.33(0.47)	4.16(0.32)
mint	4.35(0.23)	3.81(0.21)	6.64(0.52)	4.88(0.34)
lavender	7.22(0.13)	5.88(0.37)	6.89(0.53)	5.00(0.43)
rosemary	7.10(0.15)	6.84(0.32)	5.84(0.39)	4.34(0.38)
lemon balm	6.01(0.24)	5.70(0.28)	3.32(0.27)	1.34(0.11)

).

3.3. FRAP and CUPRAC Assay

The ability to neutralize free radicals by reducing metal ions using the FRAP method showed a range for traditional infusions from 1.27 (0.03) to 6.60 (0.26) mmolFe/g DE, while for microwave extraction samples, the range was from 1.88 (0.06) to 14.60 (0.12) mmolFe/g DE. The highest reduction values were determined in the lemon balm sample—for infusion, 6.60 (0.26) mmolFe/g DE, and 14.60 (0.12) mmolFe/g DE for the microwave-extracted sample. The results for the CUPPRAC assay for infusions were in the range from 351.93 (11.85) to 1129.68 (44.46) µg TE/mg DE, while for microwave extracts, they were from 333.37 (5.90) to 1284.69 (38.37) µg TE/mg DE (

Table 3. Values of FRAP (mmolFe/g DE) and CUPRAC (µg TE/mg DE) assay (mean (SD)).

	FRAP mmolFe/g DE		CUPRAC µg TE/mg DE
	Infusion	Microwave	Infusion	Microwave
basil	1.27(0.03)	1.88(0.06)	351.93(11.85)	333.37(5.90)
wild thyme	3.50(0.01)	4.11(0.04)	550.05(43.82)	780.92(47.07)
savory	2.64(0.03)	3.08(0.11)	554.42(15.62)	559.33(41.13)
oregano	5.46(0.03)	11.51(0.09)	956.12(28.84)	1110.58(43.52)
sage	4.08(0.13)	3.84(0.13)	901.54(54.87)	615.00(42.95)
garden thyme	4.50(0.09)	3.18(0.09)	779.83(47.47)	587.71(43.98)
mint	2.03(0.01)	3.32(0.07)	521.12(15.9)	679.95(21.37)
lavender	2.18(0.07)	3.46(0.20)	554.42(29.61)	597.53(7.38)
rosemary	2.87(0.07)	4.34(0.08)	608.45(48.38)	786.92(7.14)
lemon balm	6.60(0.26)	14.60(0.12)	1129.68(44.46)	1284.69(38.37)

).

3.4. HPLC

The quantitative analysis demonstrated considerable differences in the phenolic profiles between infusions and microwave-assisted extracts of herbal teas from the Lamiaceae family (

Table 4. Phytochemical profile of infusion and microwave-assisted extracts (mg/100 g crude drug) (mean (SD)).

Infusion (mg/100 g CD)

GA

ChlA

pHBA

CafA

CimA

VA

ComA

FA

RA

Qu

Epi

Nar

basil

23.4(0.3) a

7.5(0.5) a

n.d.

n.d.

1.1(0.1) a

17.9(0.5) e

n.d.

25.6(1.8) a

1.1(0.1) a

45.0(2.1) a

n.d.

30.3(2.1) a

wild thyme

25.0(0.5) b

n.d.

n.d.

n.d.

n.d.

n.d.

2.9(0.1) a

n.d.

n.d.

n.d.

n.d.

n.d.

savory

30.3(1.9) d

11.6(0.3) c

5.0(0.1) a

n.d.

n.d.

n.d.

7.1(0.7) b

25.2(1.6) a

n.d.

12.5(0.3) b

n.d.

29.6(1.1) b,c

oregano

29.4(1.5) c

8.2(0.5) a

1.4(0.1) b

n.d.

n.d.

n.d.

2.8(0.2) a

24.8(1.3) a

n.d.

15.8(0.3) c

n.d.

29.2(1.0) c

sage

26.2(1.2) b

14.5(0.4) d

6.9(0.4) d

39.8(1.5) a

0.14(0.0) b

4.5(0.2) b

36.3(0.6) f

32.3(2.5) c

19.8(0.6) c

31.1(1.6) d

30.5(1.5) a

30.6(1.3) a

garden thyme

25.8(1.2) b

n.d.

n.d.

n.d.

n.d.

n.d.

2.6(0.2) a

n.d.

n.d.

11.7(0.4) e

n.d.

n.d.

mint

23.8(0.9) a

10.0(0.8) b

1.0(0.1) b

n.d.

n.d.

1.6(0.1) a

34.3(0.7) e

59.4(2.5) e

29.3(1.4) d

18.5(0.9) f

25.6(1.8) b

29.3(0.8) a,b,c

lavender

32.2(2.2) e

28.9(0.4) f

64.8(0.3) e

50.9(0.3) c

n.d.

12.4(0.5) c

22.3(0.5) d

30.2(2.6) b

9.5(0.4) b

13.8(0.8) g

48.3(1.4) c

n.d.

rosemary

27.7(1.5) c

n.d.

n.d.

39.4(0.3) a

n.d.

n.d.

13.5(0.8) c

n.d.

n.d.

n.d.

n.d.

n.d.

lemon balm

38.0(2.3) f

18.4(0.4) e

4.8(0.3) c

42.9(0.2) b

n.d.

14.1(0.6) d

125.3(6.1) g

51.1(3.6) d

38.8(0.3) e

33.9(2.1) h

26.4(1.2) d

29.9(0.9) a,b

Microwave (mg/100 g CD)

GA

ChlA

pHBA

CafA

CimA

VA

ComA

FA

RA

Qu

Epi

Nar

basil

24.6(1.1) a

11.0(0.8) a

1.8(0.1) a,b

39.4(2.7) a,b

n.d.

0.1(0.0) a

21.9(0.9) a

28.7(0.7) a

0.5(0.0) a

12.2(0.4) a

n.d.

n.d.

wild thyme

27.3(1.2) c,d

15.5(0.7) b

0.5(0.0) a

38.7(2.4) a,b

2.3(0.1) a

8.9(0.2) b

75.9(0.4) b

28.6(1.8) a

18.6(0.4) e

23.0(1.8) d

27.7(1.1) a

30.1(1.7) b

savory

26.8(1.3) b,c

40.1(2.1) c

4.5(0.3) a,b,c

42.5(3.1) c

n.d.

2.4(0.1) c

40.2(2.3) c

33.5(1.7) b

10.4(0.8) b

18.7(1.2) c

26.0(1.3) b

29.6(1.8) a

oregano

30.1(2.0) e

8.8(0.2) d

n.d.

n.d.

0.3(0.0) b

n.d.

8.3(0.4) d

25.6(0.9) c

n.d.

22.7(1.7) d

n.d.

30.1(2.2) b

sage

25.2(1.6) a,b

13.9(0.8) e

7.9(0.3) c

38.9(2.6) b

n.d.

10.2(0.7) d

86.8(2.6) e

35.7(1.6) d

15.6(0.7) c

26.7(1.5) e

37.0(2.1) c

30.8(2.3) b

garden thyme

28.5(0.9) d

10.6(0.5) a

n.d.

37.8(2.4) a

n.d.

22.1(1.1) e

80.4(3.1) f

33.9(1.1) b

16.8(0.8) c,d

31.3(1.8) g

29.0(1.4) d

32.8(2.1) c,d

mint

25.4(0.8) a,b

24.5(1.4) f

6.5(0.4) b,c

41.4(2.1) c

n.d.

19.1(0.9) f

94.8(1.8) g

129.8(5.8) f

83.5(2.4) f

29.4(1.7) f

33.2(1.6) e

31.6(1.7) b,c

lavender

42.5(1.9) f

19.2(1.2) g

105.6(5.4) d

38.6(2.8) a,b

1.9(0.1) c

17.2(1.0) f

56.6(1.2) h

39.8(2.1) e

17.2(0.7) d,e

39.7(2.1) h

70.9(3.4) f

33.7(1.8) d

rosemary

28.4(1.0) d

n.d.

n.d.

n.d.

n.d.

n.d.

9.2(0.2) d

n.d.

n.d.

15.6(0.9) b

n.d.

29.5(1.3) a

lemon balm

30.7(2.1) e

26.6(1.7) h

7.3(0.5) c

39.2(2.2) a,b

n.d.

4.8(0.3) g

69.0(1.4) i

28.6(0.7) a

18.5(0.9) e

13.5(1.1) a

25.1(1.4) g

n.d.

Different superscript letters within the same column indicate significant differences between means at the 0.05 level.

). MAE produced significantly higher concentrations of phenolic acids and flavonoids compared to the traditional infusion method, confirming its enhanced efficiency in releasing bioactive compounds (Figure A1, Figure A2, Figure A3, Figure A4, Figure A5, Figure A6, Figure A7, Figure A8, Figure A9, Figure A10, Figure A11 and Figure A12). For example, the content of rosmarinic acid, a major antioxidant compound, significantly increased in mint and lavender samples after microwave-assisted extraction. The concentrations reached 83.5 and 17.2 mg/100 g CD, respectively, compared to 29.3 and 9.5 mg/100 g CD obtained from infusions. The presence of gallic acid was detected in all examined samples, with lemon balm and lavender exhibiting the highest concentrations across both applied extraction methods. Notably, lavender showed especially high levels of p-hydroxybenzoic acid and chlorogenic acid following MAE (105.6 and 19.2 mg/100 g CD, respectively), highlighting its rich and diverse polyphenolic composition.

The Pearson correlation revealed several statistically significant relationships between the analyzed phenolic compounds and flavonoids (

Table 5. Pearson’s correlation coefficients between individual phenolic compounds in Lamiaceae herbal extracts.

	GA	ChlA	pHBA	CafA	CinA	VA	ComA	FA	RA	Qu	Epi	Nar
GA	1	0.289	0.710 **	0.290	0.280	0.296	0.326	0.034	0.073	0.281	0.529 *	0.161
ChlA	0.289	1	0.259	0.411	0.004	0.189	0.315	0.423	0.294	0.266	0.431	0.223
pHBA	0.710 **	0.259	1	0.363	0.414	0.392	0.104	0.108	0.067	0.284	0.759 **	0.006
CafA	0.290	0.411	0.363	1	0.130	0.469 *	0.671 **	0.352	0.430	0.193	0.667 **	−0.086
Epi	0.529 *	0.431	0.759 **	0.667 **	0.376	0.630 **	0.620 **	0.471 *	0.500 *	0.491 *	1	0.270
VA	0.296	0.189	0.392	0.469 *	0.365	1	0.630 **	0.547 *	0.557 *	0.763 **	0.630 **	0.388
ComA	0.326	0.315	0.104	0.671 **	0.151	0.630 **	1	0.611 **	0.738 **	0.469 *	0.620 **	0.340
FA	0.034	0.423	0.108	0.352	0.004	0.547 *	0.611 **	1	0.930 **	0.469 *	0.471 *	0.424
RA	0.073	0.294	0.067	0.430	0.014	0.557 *	0.738 **	0.930 **	1	0.402	0.500 *	0.332
Qu	0.281	0.266	0.284	0.193	0.449 *	0.763 **	0.469 *	0.469 *	0.402	1	0.491 *	0.719 **
CinA	0.280	0.004	0.414	0.130	1	0.365	0.151	0.004	0.014	0.449 *	0.376	0.296
Nar	0.161	0.223	0.006	−0.086	0.296	0.388	0.340	0.424	0.332	0.719 **	0.270	1

** Correlation is significant at the 0.01 level (2-tailed). * Correlation is significant at the 0.05 level (2-tailed).

). A strong positive correlation between p-hydroxybenzoic acid and epicatechin (r = 0.759, p < 0.01) indicates a possibility of co-occurrence or a similar biosynthetic pathway. Moreover, caffeic acid showed a significant correlation with a group of compounds, including epicatechin (r = 0.667, p < 0.01), vanillic acid (r = 0.469, p < 0.05), and para-coumaric acid (r = 0.671, p < 0.01), which indicates its crucial role in the phenolic profile of the tested samples. Ferulic acid and rosmarinic acid displayed a very strong correlation (r = 0.930, p < 0.01), indicating the possibility of their co-extraction or similar chemical behavior during the preparation of samples. In addition, quercetin showed a significant positive correlation with vanillic acid (r = 0.763, p < 0.01) and naringenin (r = 0.719, p < 0.01), implying a potential synergistic antioxidant effect among the flavonoids. Certain compounds, including gallic acid and para hydroxy benzoic acid, also demonstrated a positive correlation (r = 0.710, p < 0.01), indicating their simultaneous presence in specific extracts.

4. Discussion

4.1. Extraction Yield

In all analyzed plants, a higher yield was obtained after microwave extraction compared to infusion (Table 1). Modern methods take less time, reduce the risk of thermal degradation, and give higher yields. MAE is based on the heating of a dipole and/or ionic solvent by absorption of microwave energy, where the solvent is in direct contact with the plant material [20]. The highest yield of MAE was recorded for mint and was 250.75 mg/g of crude drug (CD), while when using the traditional method, a high yield value of 196.83 mg/g CD was also recorded. Mint tea, as one of the most commonly used teas, gave extremely good yields when using both extraction techniques. The obtained values agree with other works, where a significant yield was also recorded using the mentioned methods on the mint plant itself. It is also important to emphasize that the application of MAE made it possible to reduce the process time, while increasing the extraction yield, which was also recorded in our research [21]. The lowest yields were recorded for rosemary (infusion 30.94 mg/g CD and MAE 33.70 mg/g CD), which indicates that in certain plants, the choice of the method itself does not significantly affect the final yield result.

4.2. Total Phenolic Content

In all samples obtained with microwave extraction, the total phenolic content was higher (Table 1). The highest contents were obtained by infusion in samples of savory, sage, and lemon balm, and they were over 50 mg of gallic acid per 1 g of dry extract. Similar results were shown in the study by Kozlowska et al., while lower values were obtained in the study of pulverized drugs [21]. In both studies, ethanolic and/or methanolic extracts were used as samples, and the difference in results may be due to the extraction solvent as well as the way the samples were stored [6]. A lower content of total phenols was obtained in the samples of thyme, rosemary, and lavender compared to the samples from the study of Gallego et al., where the content of the prepared emulsions was quantified [22]. This difference can be explained by different sample preparations. The total phenolic content obtained through traditional extraction in the oregano sample was 36.08 (1.17) mg GAE/g of dry extract. This value is lower compared to the total phenol content reported in the studies by Ali et al. (140.59 mg GAE/g dry extract) and Skendi et al. (55.0 mg GAE/g DE) [6,23]. The differences in these results can be attributed to variations in extraction solvents, sample storage methods, and sample quality. The lowest yield was determined in the wild thyme samples (25.30 (0.61) mg GAE/g dry extract for infusion and 37.85 (2.10) mg GAE/g dry extract for MAE). The yield in the basil extract (39.58 (0.15) mg GAE/g DE) is slightly higher than the value reported by Albayrak et al. (23.15 mg GAE/g DE) and comparable to the value found by Ali et al. (39.91 mg GAE/g DE) [6,24].

4.3. Flavonoid Content

The flavonoid content varies depending on the choice of extraction method (Table 1). The lowest flavonoid content obtained by traditional extraction was found in the lemon balm sample (2.62 (0.02) mg QE/g of dry extract), while the highest was in the savory sample (9.90 (0.14) mg QE/g of dry extract). One of the most commonly used spices is oregano, and the values for its flavonoid content were 7.37 (0.04) and 7.71 (0.07) mg QE/g of dry extract, which is in line with an Australian study quantifying flavonoids from plants of different qualities, where the yields for oregano were 5.15 mg QE/g and 5.21 mg QE/g of dry extract [6,25]. The microwave-assisted extract with the highest yield was also found in savory (11.23 (0.08) mg QE/g of dry extract), while the lowest was in lemon balm (4.21 (0.07) mg QE/g of dry extract). Additionally, in our research, the flavonoid content in thyme (6.56 (0.12) and 8.80 (0.06) mg QE/g of dry extract) was almost identical with a study investigating flavonoids as key antioxidant agents in the same plant sample (6.59 mg QE/g of dry extract) [5]. In the case of mint, the flavonoid content closely matched a similar study where the extraction duration was 3–5 min [26]. For most samples, the phenolic and flavonoid contents were higher in microwave-assisted extracts compared to traditional infusions. This finding is consistent with the Bulgarian study by Petkova et al., where the extraction time for traditional infusions was shorter (3–5 min, depending on the sample) [26].

4.4. DPPH and ABTS Test

During this research, the highest DPPH radical scavenging activity was observed in savory, with IC₅₀ values of 3.73 (0.13) μg/mL for the infusion extract and 2.63 (0.11) μg/mL for the microwave extraction method (Table 2). These results were compared with previous studies that also reported a strong in vitro antioxidant activity. It is believed that the pronounced antioxidant effect of this plant is due to its high contents of secondary biomolecules, primarily flavonoids, and phenolic compounds, which are known to exert protective and inhibitory effects on certain tumor cells in the human body [27]. The strongest radical scavenging activity against ABTS was recorded in lemon balm samples obtained by microwave extraction (IC₅₀ = 1.34 μg/mL; Table 2). This value was found to be approximately ten times lower (i.e., more potent) compared to a study in which the antioxidant activity of pulverized herbal drugs was evaluated using the DPPH assay (IC₅₀ = 10.16 μg/mL), which can be attributed to a higher content of phenolic compounds [24]. Differences in results can partly be explained by the distinct methodologies used for assessing antioxidant activity, as the ABTS assay—being more sensitive to phenolic compounds—offers greater precision in detecting the antioxidant potential of plant extracts [28]. This distinction is also evident when comparing the DPPH and ABTS results for lemon balm, which is related to the fundamental difference in their chemical principles: the DPPH assay involves a stable free radical, while the ABTS assay measures the neutralization of a radical cation [29]. Our findings are consistent with those of another study in which infusion and methanolic extracts of lemon balm (IC₅₀ = 0.45 (0.04) μg/mL and 0.57 (0.21) μg/mL, respectively) were frozen until further analysis. Oregano also exhibited strong antioxidant activity. IC₅₀ values against ABTS were 2.89 (0.18) μg/mL for the infusion and 1.89 (0.09) μg/mL for the microwave-assisted extract. In the case of DPPH, the corresponding IC₅₀ values were 7.72 (0.09) μg/mL (infusion) and 4.88 (0.15) μg/mL (microwave extract). Oregano extracts showed notable differences in radical scavenging activity when comparing DPPH and ABTS assays, similar to the trend observed in lemon balm. These differences may also be attributed to the varying sensitivity of each method, with the ABTS assay complementing and enhancing the interpretability of DPPH results [29]. These findings are also consistent with previously published data [1]. Mint also showed considerable radical scavenging activity, with IC₅₀ values against DPPH of 4.35 (0.23) μg/mL (infusion) and 3.81 (0.21) μg/mL (microwave extract). These results align with earlier studies, which attributed the antioxidant potential of mint to the presence of compounds such as chlorogenic acid, caffeic acid, and rutin [30]. High antioxidant activity was also recorded for mint in the ABTS assay, with IC₅₀ values of 6.64 μg/mL for the infusion and 4.88 μg/mL for the microwave extract. Aqueous extracts demonstrated significantly greater antioxidant potential than essential oils (IC₅₀ = 59.19 (0.65) μg/mL) [31]. However, the aqueous extracts in our study showed lower activity compared to alcoholic extracts of the same plant reported by Farnad et al., indicating that alcohol is a more effective solvent for extracting phenolic compounds [30,32].

4.5. FRAP and CUPRAC Assay

Similar studies have confirmed the excellent antioxidant potential of this plant, particularly reflected in its high FRAP and CUPRAC activity [33,34]. A fourfold higher activity was recorded in the infusion and an eightfold higher activity in the extract obtained by microwave extraction compared to the study by Ulewicz-Magulska et al., in which fresh plant material was examined [1]. The difference in results may be due to the quality and different preparation methods of the samples. High values were also recorded in the oregano sample (5.46 (0.03) mmol Fe/g DE and 11.51 (0.09) mmolFe/g DE), rosemary (2.87 (0.07) mmolFe/g DE and 4.34 (0.08) mmolFe/g DE), and garden thyme (3.18 (0.09) mmolFe/g DE and 4.50 (0.09) mmolFe/g DE) (Table 3). Studies on the antioxidant activity of spices and medicinal herbs, due to different storage conditions, showed slightly lower values [1]. Lavender has been confirmed as a significant antioxidative agent in oxidative stress conditions, due to its substantial polyphenolic content, as shown in the study by Hmidini et al. [35]. The significance of lavender as an antioxidant agent in oxidative stress conditions was also confirmed by the aforementioned study, due to the significant content of polyphenolic compounds [35]. Basil showed the lowest antioxidant activity in both types of extracts, with values of 1.27 (0.03) mmolFe/g DE for the infusion and 1.88 (0.06 mmolFe/g DE for the microwave extracts. In comparison with a similar study, although basil showed lower antioxidant activity, it was satisfactory due to its polyphenol content, making it a significant food ingredient [36]. The CUPRAC method, which functions under pH conditions close to physiological levels and uses a similar metal ion reduction mechanism to FRAP, provides a more reliable assessment of antioxidant activity [18]. The best antioxidant activity of lemon balm was confirmed by the CUPRAC method (1129.68 (44.46) µg TE/mg DE and 1284.69 (38.37) µg TE/mg DE), which is in line with the literature [33]. Good antioxidant activity was also shown in the garden thyme sample, with values of 587.71 (43.98) µg TE/mg DE and 779.83 (47.47) µg TE/mg DE for the infusion and the microwave extract. Compared to a similar study, the essential oil showed a higher antioxidant activity (1578.09 (59.67) mg TE/g), highlighting the significance of different forms of plant material use [37]. The lowest antioxidant activity was determined in the basil sample: 333.37(5.90) µg TE/mg DE and 351.93 (11.85) µg TE/mg DE. Also, the results for basil, though lower, indicate a satisfactory level of antioxidant activity, which may be significant for its use in nutrition and therapy. The obtained results provide a broader picture of antioxidant activity, considering that the FRAP method is performed under acidic conditions, limiting it to certain polyphenols that are stable under these conditions, while the CUPRAC method can detect those that are not stable in acidic environments [18].

Although the microwave extracts generally contained higher concentrations of phenolic compounds, their antiradical activity values (DPPH and ABTS) were sometimes lower than those of infusions. This discrepancy could be attributed to several factors. First, microwave-assisted extraction may lead to partial degradation or structural modification of certain phenolics, reducing their radical scavenging efficiency. Second, the composition of phenolic compounds—not just their total amount—plays a crucial role, as not all phenolics contribute equally to antioxidant activity. Furthermore, interactions between compounds or matrix effects in the extract could also modulate the overall activity. Similar trends observed in FRAP and CUPRAC assays suggest that antioxidant behavior depends not only on the concentration but also on the chemical nature and redox potential of individual constituents.

4.6. PCA

Principal component analysis (PCA) was applied in order to differentiate herbal extracts based on their antioxidant activity, total phenolics, and flavonoid content. Within the initial data matrix, different herbal extracts represent the objects of PCA, while antioxidant activity, total phenolics, and the flavonoid content represent the variables. Through PCA, by decomposing the original data into loading and score vectors, new variables—principal components (PC)—were obtained. The first two PCs (PC1 and PC2), accounting for most of the variation in the dataset, comprise 77.82% of the total variance of the initial data matrix (Figure 1).

The results of the PCA are presented in Figure 2. The variability described by PC1 mostly correlated with ABTS free radical scavenging and the cupric and ferric reducing capacities of extracts. CUPRAC and FRAP are strongly positively correlated, while ABTS is found in opposition to CUPRAC and FRAP. PC2 was correlated with total flavonoids and the phenolic content, as well as with the DPPH radical scavenging activity (Figure 2a).

PC1 allowed the separation of herbal extracts with high antioxidant potential (located on the negative side of PC1) from those with low antioxidant activity (located on the positive side of PC1). As shown in Figure 2b, oregano and lemon balm extracts were clustered together in the lower left quadrant of the loading plot, based on having the highest antioxidant activity among the analyzed species in the CUPRAC, FRAP, and ABTS assays. It is also evident that the lemon balm and oregano extracts obtained by MAE are well separated from the infusions of these species, which can be explained by the superior antioxidant properties of MAE extracts.

Furthermore, PC1 allowed the distinction of two groups of herbal extracts based on the extraction technique applied for extract preparation (MAE and infusion) in the case of savory, mint, sage, rosemary, and garden and wild thyme. PCA analysis showed that microwave extracts of savory, mint, sage, rosemary, and garden and wild thyme were clustered together in the upper left quadrant of the loading plot, being associated with high total flavonoid and phenolic contents and, similarly, a modest ABTS radical scavenging activity. Moreover, it can be observed on the loading plot (Figure 2b) that the microwave extract of savory is well separated from the other samples as a result of accumulating the highest amounts of total flavonoid and phenolic compounds in this extract and having the highest DPPH radical scavenging capacity. PC2 allowed further separation of the savory infusion from the other herbal infusions, which can be attributed to it having the highest total phenolic and flavonoid content, as well as DPPH radical scavenging in comparison to the other infusions. Herbal extracts, clustered in the lower right quadrant, are characterized by low DPPH and ABTS radical scavenging ability, as well as low CUPRAC and FRAP.

4.7. HPLC

Quantitative analysis revealed a significantly higher content of phenolic acids and flavonoids in microwave-assisted extracts compared to traditional infusions (Table 4). The dominant compounds among phenolic acids were gallic, caffeic, ferulic, and coumaric acids, while quercetin and naringenin were the most prominent flavonoids (Figure A1, Figure A2, Figure A3, Figure A4, Figure A5, Figure A6, Figure A7, Figure A8, Figure A9, Figure A10, Figure A11 and Figure A12). In the infusions, flavonoid content decreased in the following order: sage > lemon balm > basil > mint > lavender > oregano > savory. For microwave-assisted extracts, the order was lavender > sage > mint > garden thyme > wild thyme > savory > oregano > rosemary > lemon balm > basil. These findings are similar to the study by Skendi et al., where the flavonoid content followed the order of lemon balm > savory > thyme > oregano > rosemary [23]. It is important to emphasize that the variability of polyphenols in nature and their contents in different plant materials can be influenced by numerous factors, including physiological, genetic, and environmental conditions, as well as cultivation and storage practices. Additionally, plant processing and the analytical methods employed may also significantly affect the results. A positive correlation between advanced extraction techniques and the yield of phenolic compounds in herbal matrices was also confirmed in a similar study [28]. Furthermore, the presence of caffeic acid in sage, rosemary, and lemon balm additionally establishes their chemotaxonomic relevance. The observed differences in phenolic profiles not only demonstrate the rich botanical diversity within the Lamiaceae family but also clearly emphasize that the choice of extraction method significantly influences the efficiency of phytochemical compound recovery. In this context, MAE emerges as a more effective technique for achieving the maximal antioxidant potential.

5. Conclusions

This study highlights the substantial antioxidant capacity of Lamiaceae herbal teas, which are often consumed as part of daily nutrition. The combined application of detailed HPLC profiling with several antioxidant evaluation methods (DPPH, ABTS, FRAP, CUPRAC) confirmed the presence and activity of essential phenolic acids and flavonoids. Principal component analysis revealed the patterns among the antioxidant data, while correlation analysis further uncovered the roles of specific compounds. This study primarily showed that microwave-assisted extraction consistently yielded higher levels of bioactive compounds as well as better antioxidant activity in comparison with standard preparation methods. These results confirm the importance of herbal teas from the Lamiaceae family as powerful natural antioxidants and suggest that an improvement of preparation methods could additionally enhance the beneficial properties of the aforementioned herbal teas for overall health.