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Article

The Identification of Polyphenolic Compounds and the Determination of Antioxidant Activity in Extracts and Infusions of Peppermint, Lemon Balm and Lavender

by
Urszula Sadowska
1,
Ruth Armenta Villavicencio
2,
Kinga Dziadek
2,
Joanna Skoczylas
2,
Szymon Kamil Sadowski
3 and
Aneta Kopeć
2,*
1
Faculty of Mechanisation and Energy Technologies in Agriculture, University of Agriculture in Krakow, ul. Majora Łupaszki 6, 30-198 Krakow, Poland
2
Department of Human Nutrition and Dietetics, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland
3
Doctoral School in the Social Sciences, Faculty of Philosophy, Institute of Psychology, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(2), 699; https://doi.org/10.3390/app14020699
Submission received: 28 November 2023 / Revised: 28 December 2023 / Accepted: 12 January 2024 / Published: 14 January 2024
(This article belongs to the Special Issue New Insights into Natural Antioxidants in Foods: 2nd Edition)
Versions Notes

Abstract

:
Bioactive compounds are molecules that may have therapeutic potential with influence on oxidative stress, pro-inflammatory state and metabolic disorders. Herbs are recognized as a significant source of natural antioxidants. The aim of this study was to determine the antioxidant properties of peppermint (Mentha piperita L.), lemon balm (Melissa officinalis L.) and lavender (Lavandula angustifolia Mill.). Fresh plant materials were harvested and obtained from the Experimental Station of Agriculture University in Kraków, Poland. Proximate analysis (protein, fat, ash and dry matter) was performed on herbs. Total polyphenol content and polyphenol profile antioxidant activity (ABTS and FRAP methods) were measured in an extract prepared with deionized distilled hot water (infusion), 70% methanol or 70% methanol acidified with formic acid. It was found that the water extract of peppermint had the highest polyphenol content and antioxidant activity measured by the FRAP method. The highest antioxidant activity measured with the ABTS method was in methanolic lemon balm extract. The highest amount of total phenolic compound was determined in the water extract of peppermint. The water and acidified methanol extract of peppermint contained a higher amount of naringin, rutin, hesperidin and rosmarinic acid. Evaluated plants can be used as natural antioxidants instead of synthetic antioxidants in the food and pharmaceutical industries.

1. Introduction

In the European Union, the area under cultivation of herbal plants is about 70,000 sq. h., and the main producers are France, Spain, Austria, Germany and Poland [1]. Peppermint, lemon balm and lavender are popular species of herbs grown in Poland. These three species of herbs belong to the same botanical family, Lamiaceae, formerly called labiaceae [2]. The Lamiaceae plant family is known as an abundant source of bioactive substances, which include, among others, phenolic compounds [3,4], and has many medicinal properties [5,6]. The antioxidant effect of herbs is particularly desirable in the diet called the “Western diet” due to its richness in easily oxidizable fatty acids. The oxidation of unsaturated fatty acids causes the formation of free radicals, which are harmful in larger amounts for human organisms [7]. In the group of phenolic compounds, phenolic acids deserve special attention as they can prevent the development of cardiovascular and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s [8], inflammation, diabetes and cancer [5,8].
Due to the high oxygen demand, the brain is the most susceptible organ to oxidative damage [9,10,11,12]. The brain’s defense system can be modulated by antioxidants like polyphenols. Natural antioxidants from plants are widely recognized as having protective effects from free radicals in the human organism by reducing or reversing cellular damage and slowing the rate of neuronal cell loss [8,10,11]. The studies provided herein provide evidence for the neuroprotective influence of the herbs examined in this research, specifically lemon balm, lavender and peppermint [8,12].
The growing threat of lifestyle diseases stimulates the search for better methods for determining antioxidant activity [13]. The extraction method is of fundamental importance for obtaining the desired bioactive chemical components from plant material and their subsequent characterization [14]. The efficiency of active compounds depends on the extraction method, grinding of the raw material and the properties of the extraction solvent [15]. Each plant material has its own characteristic properties in terms of phenolic extraction [16]. The need to choose the most appropriate extraction methodology results from the fact that when different methods are used for the same plant material using the same solvent, the extraction efficiency may vary significantly [17]. According to Tajner-Czopek et al. [18], the cheapest way to obtain plant extracts is water extracts, although better results are observed when using ethanol or methanol, especially when acidified [19,20]. There are also few studies comparing the nutritional value of herbs and the polyphenolic composition of plants produced in the same soil, climatic and agrotechnical conditions. Comparison of the antioxidant capacity of various extracts of the studied herb species produced under the same conditions, especially in the context of their subsequent use as functional food ingredients, is interesting from both a technological and nutritional point of view [19,20]. Therefore, the aim of this study was to determine and compare the antioxidant properties of various extracts obtained from peppermint, lemon balm and lavender cultivated in the same agrotechnical, climatic and soil conditions against the background of their basic composition.

2. Materials and Methods

2.1. Plant Material

This research used the peppermint herb (Mentha piperita L.), lemon balm (Melissa officinalis L.), local ecotype Schoko variety, and inflorescences of lavender (Lavandula angustifolia Mill.), Hidcote Blue Strain variety, collected in full flowering. Peppermint and lemon balm herbs were cut with pruners at a height of about 5 cm above the soil surface. Plants of these species were cut in the herbaceous vegetative phase before flowering. The harvest took place in sunny weather. All species were grown in the same climatic and soil conditions. The experiment was located on the experimental station of the University of Agriculture in Krakow (50°04′ N, 19°51′ E, 211 m MSL, slope 2°). The crops were grown on soil with a granulometric composition of clay sand. The research material came from older, several-year-old plantations where the originally planted plants were already at a significant density. No fertilization was applied to the crops. During the vegetation period, only mechanical weed control was carried out (if necessary). Pesticides were not used during the cultivation of herbs.

2.2. Proximate Analysis

Peppermint herb, lemon balm and lavender were cleaned and dried after the harvesting. Proximate analysis of herbs was carried out based on AOAC methods [21], as was previously reported [22]. Dry mass (DM) in fresh samples was measured based on the AOAC methods [22] in the laboratory oven (Memmert GmbK, Schwabach, Germany). Part of herbs were dried by freeze-drying in a lyophilizer (Christ Alpha 1–4, Gefriertrocknungsanlangen, Germany), and dried samples were used for the proximate analysis. The level of protein was measured with the Kjeldahl method (AOAC no. 978.04) with the units designed for the digestion and distillation of samples (FOSS Digester and Autodistillation Unit KjeltecTM 8200; (Tecator Foss, Hillerød, Sweden). Shortly after, about 0.500 g of each herb was mineralized with 14 cm3 of sulphuric acid in presence of catalysators (CuSO4 and K2SO4 (POCh, Gliwice, Poland). Next, the samples were distilled and titrated. The protein content was calculated with formula reported previously with the conversion factor of 6.25 [22]. The crude fat content was measured in plant material (about 1 g of sample) in accordance with the Soxhlet method (AOAC no. 935.38). The equipment Soxtec Avanti’s 2050 Auto Extraction Unit was used (Tecator Foss, Hillerød, Sweden). For the extraction of the crude fat, the petroleum ether was used in amount of 80 mL (POCH, Gliwice, Poland) per sample [21]. The ash level was determined in the electric muffle furnace (SNOL 82/110, Utena, Lithuania). The total carbohydrate content of dry mass of exanimated herbs was calculated based on the following formula: total carbohydrates = 100 − (protein + raw fat + ash) [22,23,24].

2.3. Extract Preparation

Dried samples of peppermint herb (Mentha piperita L.), lemon balm (Melissa officinalis L.) and lavender (Lavandula angustifolia Mill.) were crushed into a powder. About 0.5 g of dry herbs were mixed with different solvents, e.g., 80 mL 100 °C dd water (infusion), 40 mL methanol (POCH, Gliwice, Poland) or 80 mL methanol acidified with formic acid (0.1% of formic acid; Chempur, Piekary Śląskie, Poland). Samples were shaken in laboratory shaker (Elpin Plus, type 357, Lubawa, Poland) with protection from light. After 2 h, extraction samples were centrifuged and filtered through filter paper. Next, extracts were stored in the freezer at a temperature of −20 °C until the time of analysis [22,25].

2.4. Determination of Antioxidant Activity

The antioxidant activity of herbs was measured with ABTS•+ radical ((2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) [26] and the FRAP methods (ferric-reducing antioxidant power) [27], as was previously described [25]. Shortly after, the working solution for ABTS analysis was obtained by diluting the ABTS•+ stock solution with 70% methanol to receive the absorbance between 0.740 and 0.750 at 734 nm. A total of 5–50 μL of the extract of each herb was transferred into a test tube with 1 mL solvent used for the infusion or extract preparation. Afterward, a diluted extract with 2 mL of working solution of the ABTS•+ was mixed. The mixture was protected from the light at 30 °C for 6 min. The absorbance of sample was measured at a wavelength of 737 nm using spectrophotometer (UV-1800, RayLeigh Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China).
The total reducing capability was determined with FRAP method based on the method described by Benzie and Strain [27]. The extracts of tested herbs (10–100 μL) were put into a test tube and made up to 1 mL with 70% methanol. Next, 3 mL of working solution of the FRAP reagent was added to the diluted extract. The mixture was kept protected from light at room temperature for 10 min. The absorbance of the samples was measured at 593 nm.
The results obtained for ABTS and FRAP methods were compared to the standard curve of concentration of Trolox standard dilution and expressed in μM Trolox/1 g DM of assessed herbs.

2.5. Total Polyphenol Content and Polyphenolic Profile

The total polyphenol concentration in infusions, methanol and acidified methanolic extract of examined herbs was measured with Folin–Ciocalteu reagent [28] as was reported by Dziadek et al. [25]. The results were calculated as the chlorogenic acid equivalent (CGA) in mg per 100 g of DM of sample as was previously reported [25].
For the HPLC polyphenolic compounds analysis, all types of extracts of herbs were used to determine polyphenolic compounds via the HPLC method using the Prominence-i LC 2030C D3 Plus system (Shimandzu, Kyoto, Japan) along with a DAD detector and a Luna Omega 5 µm Polar C18, 100A, 250 × 10 mm Phenomenex column (Torrance, CA, USA). The separation of phenolic compounds was carried out at 40 °C. The mobile phase was a mixture of two eluents: A, 0.1% formic acid in water (v/v), and B, 0.1% formic acid in methanol (v/v). The mobile phase flow rate was 1.2 mL/min. The analysis was carried out with the following gradient conditions: from 20% to 40% B for 10 min, 40% B for 10 min, from 40% to 50% B for 10 min, from 50% to 60% B for 5 min, 60% B for 5 min, from 60 to 70% B for 5 min, from 70% to 90% B for 5 min, 90% B for 5 min, from 90% to 20% B (the initial condition) for 1 min, and 20% B for 4 min. This resulted in a total run time of 60 min. The injection volume of tested samples was 20 μL. A stock standard solution (100 mg/L) of each polyphenolic compound was prepared in 70% acidified methanol (0.1% formic acid (POCH, Gliwice, Poland). The calibration curves of the polyphenol standards were made by the dilution of stock standard solutions in 0.1% formic acid in 70% methanol (v/v). All the solutions were filtered through a 0.22 µm filter, as was previously reported [22,23].
The following polyphenolic compounds were determined based on the following standards: gallic acid, catechin, chlorogenic acid, 4-hydroxybenzoic acid, epicatechin, caffeic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, sinapinic acid, naringin, rutin, hesperidin, rosmarinic acid, myricetin, quercetin, luteolin, kaempferol, apigenin, isorhamnetin, hispidulin, acacetin, carnosol and carnosic acid (Sigma, Saint Louis, MO, USA) (based on LabSolution ver. 5.93 Shimandzu Corporation (Kyoto, Japan)). The levels of quercetin, luteolin, apigenin, and myricetin were determined only in their free aglycones form.

2.6. Statistical Analysis

Proximate and polyphenolic profile analyses were performed in duplicate. For antioxidant activity and total phenolic content, three replications of samples were performed. The arithmetic mean and standard deviations (SD) were calculated. All calculations were made with Statistica 13 package (Stat Soft, Inc., Tulsa, Oklahoma). One-way analysis of variance was used. The significance of differences was evaluated with Duncan test at the significance level α = 0.05, and results were significant below the level p ≤ 0.05.

3. Results

The dry mass content and approximate composition of basic nutrients of the studied herb species are presented in Table 1.
The dry matter content varied among the herb species tested. Peppermint of the Schoko variety had the lowest content, with an average of 22.49 g/100 g FM. However, lemon balm and lavender did not differ significantly in this parameter (Table 1). It was determined that peppermint had a higher protein content compared to other herbs. No differences were found between the tested herb species in terms of crude fat content. The tested herbs also differed in their total carbohydrate content. Lavender and lemon balm were richer in total carbohydrates (88.76 and 88.18 g/100 g DM, respectively) compared to peppermint. The tested herb species contained a similar amount of ash, ranging from 0.22 to 0.48 g/100 g of DM.
Based on the results obtained, different contents of total polyphenolic compounds and different degrees of antioxidant activity were found depending on the plant species and the solvent used (Table 2). The highest ABTS values were found in methanolic lemon balm extract (0.57 µmol Trolox/1 g DM). However, methanolic peppermint extract also had a similar level of antioxidant properties. In turn, the lowest ABTS values were obtained in solutions with acidified methanol. Regardless of the solvent used, lemon balm was the leading species and showed the highest antioxidant potential using the ABTS radical, while lavender had the lowest. The high antioxidant potential of mint and lemon balm has also been confirmed by the FRAP method. In this method, the greatest antioxidant potential was observed in aqueous solutions. The highest values were observed for peppermint and then lemon balm, 6117.76 and 2770.60 µmol Trolox/1 g DM, respectively. Methanolic extracts were less effective in terms of activity, and in this case, mint and lemon balm obtained similar values that were not statistically different at p = 0.007. Similarly to the use of water extracts, lavender was characterized by the lowest antioxidant capacity. When acidified methanol was used, no significant differences were found between the tested herb species (p = 0.721).
The total content of polyphenols in the tested herb species was also assessed using various solvents. The highest polyphenol content was found in the water extract. In this case, the highest values were obtained for peppermint, followed by lemon balm, and the lowest were obtained for lavender (Table 2). In the extract using acidified methanol, mint and lemon balm were at the forefront, and no significant difference was found between them. The total content of polyphenols present in both extracts decreased significantly when lavender was used in this study.
The content of selected phenolic compounds was affected by the type of herbs and extraction. The higher content of naringin, rutin, hesperidin and rosmarinic acid was determined in peppermint water and acidified methanol extract (Table 3). In lemon balm and lavender, the higher level of rosmarinic acid was measured in higher amounts in both types of extract.

4. Discussion

Among the analyzed herbs, peppermint had the lowest dry matter content and was an average of 22.49 g/100 g FM. The difference in dry matter content between peppermint and lemon balm was as much as 13.18 g/100 g. Similar differences between mint and lemon balm are presented by the results of Kaur et al. [29]; in this case, the difference was 14.5 g. Similar results of dry matter content for peppermint were achieved in the studies of Uribe et al. [30] (27.5%). In turn, Chrysargyris et al. [31], on the first date of harvesting lemon balm, provided the DM content in the range of 21.06–24.48% and emphasized that it depended on the method of cultivation and irrigation of the crop. The dry matter content results presented in this study are much higher, which may be related to the plant development phase [24], as well as the use of other varieties and climatic conditions. In this study, however, these species were collected in the same developmental phase, so the differences achieved should be attributed to separate genotypes. The obtained test results indicate that lavender was characterized by an average dry matter content of 35.59%. Comparing the two-year results of fresh and dried inflorescence yields of lavender presented in the work of Seidler-Lozykowska et al. [32], in Polish conditions, it can be estimated that the cited authors obtained values similar to those presented in this work.
Data from the literature regarding the content of basic nutrients in the analyzed species indicate large discrepancies. They may result from different genotypes, plant development stages, soil and climatic conditions. For example, in the work of Uribe et al. [30], much lower values of the protein content in peppermint were obtained (3.44 g/100 g DM). In this case, the peppermint came from a local market, and little is known about it. Turcu et al. [33] measured the content of protein at the level of 17.09% in peppermint purchased from a local producer in Romania. Also, in Romanian conditions, Tomescu et al. [34], when examining the protein content for various species of the Lamiaceae family, obtained the highest value of 8.25% and the lowest of 4.81%. In our own research results, only lavender was within the given range, obtaining 7.68 g/100 g of DM.
In turn, higher results than the protein content presented in this study were obtained by Biel et al. [35] for lemon balm (17.25 g/100 g) DM and mint (14 g/100 g DM).
The research conducted by Belščak-Cvitanović et al. [36] shows that the protein content decreased in peppermint (3.12 g/kg), lavender (3.07 g/kg) and lemon balm (2.53 g/kg). It should be emphasized, however, that different research methods were used in these studies. Protein content was measured using the Bradford method, which makes it difficult to compare with the data obtained in our own research. However, as in our own research, among the species analyzed, peppermint had the highest protein content.
In turn, Flores Calderón et al. [37], analyzing the same plant species, obtained the following values: lemon balm: 17.77 g/100 g; peppermint: 12.88 g/100 g; and narrow-leaved lavender: 10.49 g/100 g. The result obtained in these tests for peppermint is most similar to that obtained in this study (12.60 g/100 g DM). These authors also analyzed the fat content and obtained 6.55 g/100 g for lavender, 6.12 g/100 g for lemon balm and 5.10 g/100 g for mint, although these are much higher results compared to those presented in our study. It should be noted that lavender turned out to be the species richest in this ingredient (3.37 g/100 g DM), although in this case, it was not statistically confirmed. Tomescu et al. [34] obtained fat content ranging from 8.96 to 5.64% in different species of the same family. In these studies, Melissa officinalis L. had a fat content of 5.85%. In turn, in the work of Uribe et al. [30], significantly lower fat values were obtained for peppermint, ranging from 0.24 g/100 gsm for fresh mint to 3.18 g/100 g for mint vacuum-dried at 50 °C. In a study by Mainasara et al. [38], fresh peppermint had 0.50% fat, while dried peppermint had 5%.
In this study, the estimated total carbohydrate content ranged from 84.31 g/100 g DM for peppermint to 88.18 g/100 g DM for lemon balm and 88.76 g/100 g DM for lavender. The work of Flores Calderón et al. [37] indicates lower total carbohydrate contents for the same species in the same order: 59.02 g/100 g DM, 57.27 g/100 g DM and 58.38 g/100 g DM, respectively. In turn, Belščak-Cvitanović et al. [36] examined the basic composition of the dried herbs mint, lemon balm and lavender from Croatia, obtaining a carbohydrate content of 79.30 g/kg DM for lemon balm, 82.83 g/kg DM for narrow-leaved lavender and 113.7 g/kg for peppermint.
The ash content in the herbs analyzed in this study was low and ranged from 0.22 to 0.48 g/100 g dm. No statistical significance was found between the analyzed species. Similarly, in the study by Biel et al. [35], no significant differences were found in the ash content in mint and lemon balm; however, higher values were obtained: 11.41 g/100 g and 11.67 g/100 g. Flores-Calderón et al. [37] showed ash contents of 8.30 g/100 g in mint, 11.02 g/100 g in lemon balm and 11.71 g/100 g in lavender. In turn, Uribe et al. [30] found 3.3 g/100 g DM in fresh peppermint. Research conducted by Khali et al. [39] indicated large differences in ash content for different species of herbs grown in Jordan. In their research, the authors used air-dried plants for 7 days and analyzed the composition of 10 species of herbs. In these tests, they obtained ash content from 2.02% for Thymus capitatus L. to 17.3% for Corianderum sativum and 17.5% for Petroselinum crispum. The explanation for this variability is probably due to environmental factors and botanical variability.
The obtained test results showed a different degree of antioxidant activity and a different content of total polyphenolic compounds depending on the plant species and the extract used. Many authors draw attention to the differences between the antioxidant properties of different species of herbs belonging to the family Lamiacea and the varied content of polyphenolic compounds [40,41,42,43].
However, it should be noted that comparison of the obtained results with other scientific literature is often not possible due to the use of different research methods, different solvents and a different way of expressing the results. Many authors pay attention to the problem of comparing results presented in different works due to different research methods [44,45].
Testing the antioxidant properties using the ABTS method clearly confirmed the greatest potential of lemon balm, regardless of the extract used in the tests. Jiménez-Zamora et al. [44] reached similar conclusions when examining water infusions of these species. The ABTS values obtained from lemon balm were significantly different from the others (except oregano). These authors reported that the ABTS value for lemon balm was 10.97 mmol Trolox/L, while it was 3.49 mmol Trolox/L for peppermint and 3.27 mmol Trolox/L for lavender. After three months of storage at various temperatures, about 50% of antioxidant activity was lost. Chrysargyris et al. [31] confirmed the high oxidative capacity of lemon balm, which is in the range of 27.95–43.08 mg Trolox/g FM for the first harvest and 32.40–45.57 mg Trolox/g FM for the second. In these studies, extracts were prepared using 50% methanol. Different results were obtained by Nikolic et al. [46] by comparing the antioxidant capacity of the same species with unknown growing conditions in methanol: water (80:20, v/v) extracts. The highest values were obtained for peppermint (2.59 mg Trolox/g FW), followed by lavender (2.54 mg Trolox/g FW) and lemon balm (1.39 mg Trolox/g FW).
The high antioxidant capacity of lemon balm has also been confirmed in the FRAP method, especially in methanol solution. However, in this case, no statistical difference was found between lemon balm and mint. In the literature on the subject, it is difficult to find a simultaneous comparison of the antioxidant properties of the same species in different solvents. Jiménez-Zamora et al. [44], testing water infusions of these species, obtained the highest values for lemon balm (14.20 mmol Trolox/L) compared to peppermint (6.17 mmol Trolox/L) and lavender (3.72 mmol Trolox/L). The results obtained by Benković et al. [47] using aqueous extracts clearly indicate lemon balm as the dominant species in terms of antioxidant properties. Similarly, Chrysargyris et al. [31] confirmed the high antioxidant capacity of lemon balm, which varies depending on the production and harvesting method in the range of 83.82–150.70 mg Trolox/g FW for the first harvest and 55.96–105.08 mg Trolox/g FW for the second harvest. In these studies, extracts were prepared using 50% methanol. In turn, Nikolic et al. [46] compared the antioxidant capacity of the same species using the FRAP test and obtained different results. Peppermint had the highest antioxidant potential (1.88 mg Trolox/g FW), followed by lavender (1.19 mg Trolox/g FW) and lemon balm (0.40 mg Fe/g FW). In this case, the tests used methanol:water solutions (80:20, v/v).
In this study, we have found that the highest level of polyphenolic compounds was measured in the infusion of peppermint as compared to other extracts (Table 2). Similarly, the same order for these herb species was observed by Butorová et al. [45]. These authors have used 50% ethanol–water extracts for polyphenol determination. The total content of polyphenols in peppermint was also higher compared to lemon balm in the study by Luță et al. [48] and was in the range of 91.80–101.43 for mint and 40.90–65.84 mg/g eq for lemon balm expressed as tannic acid. Moreover, they showed that the total content of polyphenols in both species increased after the application of mineral and biological fertilization. Similar results were found in the study of Aktsoglou et al. [49], who extracted total polyphenols using 10 mL of 80% methanol and obtained 0.467, 0.340 and 0.560 mg GAE/kg FW. These authors also found that the total phenolic compound depended on the application of the protein solution in various concentrations (0.0%, 0.25% and 0.50%) during the growth in a hydroponic system. It should also be noted that regardless of the solvent used, lavender had a much lower polyphenol content compared to other species. However, as research by Dobros et al. [50] indicates, the content of polyphenolic compounds may be related to the genotype used in the research.
However, from a general point of view, frozen samples of medicinal plants are characterized by a lower content of phenolic compounds [47].
Uribe et al. [33] assessed the total polyphenol content in a 50% ethanol solution for beer mint. They obtained the lowest value for fresh mint (12.43 mg) and the highest (27.12 mg) gallic acid equivalents (GAEs) (g D.M.) for mint vacuum dried at 70 °C.
The phenolic compound profiles of peppermint in our study are different from data published by Dorman et al. [19] and Lv et al. [51]. Dorman et al. [19] reported that leaves of peppermint crops in Finland contained mainly cinnamic and caffeic acid. Lv et al. [51] reported that in organic and conventionally grown peppermint obtained from Norway, IA, USA, the dominant phenolic compound was catechin, followed by epigallocatechin gallate and syringic acid. These differences can be explained by the various conditions of climate and maybe by using different solvents for the extraction of phenolic compounds. In our study, we have used water or acidified methanol, while Dormen et al. [19] used various extracts, including acetonitrile, methanol and acidified ethanol. In lemon balm, the rosmarinic acid was found in higher amounts. Our results are similar to data published by Barros et al. [52], who reported that commercial species and plants grown in in vitro cultures had a lower content of phenolic compounds. Also, Arceusz et al. [53] reported that in lemon balm originating from Poland, the dominant phenolic compound was rosmarinic acid in methanolic extracts and infusions. It was also reported that the concentration of phenolic compounds, as well as the profile, depends on the cultivar and origin of the seeds [20]. We have also found that rosmarinic acid was found in a higher amount in lavender (Lavandula angustifolia Mill) in water and acidified methanolic extract. Also, Adaszyńska-Skwirzyńska and Dzięcioł [54] reported that the major phenolic acid was rosmarinic acid. In the case of flavonoids, these authors reported that apigenin luteolin and quercetin were the most dominant flavonoids.
The type of solvent used can affect the amount and type of compounds extracted into the solution. The results obtained in this paper indicate that the infusions are characterized by a higher total polyphenol content compared to the alcoholic extracts. This may be due to the use of hot water. The high temperature may have released some of the polyphenols bound to other components. A similar observation was reported by Theuma and Attard [55]. They suggested that polyphenols have a higher solubility in hot water than in cold methanol—they are more accessible during extraction because of the breakdown of the cell wall and cellular constituents releasing polyphenolic compounds. Similar results were also obtained by Adel and Prakash [56]. The results showed that the antioxidant activity did not correlate with polyphenol content. This is because the antioxidant properties of plants depend on enzymatic and non-enzymatic antioxidant defense systems, including other components such as carotenoids, chlorophyll and antioxidant vitamins [57].

5. Conclusions

In our research, the highest antioxidant properties using the ABTS method were found in the methanol extract of lemon balm. In turn, in the FRAP method, the greatest antioxidant potential was observed in aqueous solutions for peppermint and then for lemon balm.
The extractability of polyphenols depended on the solvent used in the extraction and the plant species. The highest amounts were found in water extract and peppermint. This was confirmed in the HPLC analysis. The water and acidified methanol extract of peppermint contained a higher amount of naringin, rutin, hesperidin and rosmarinic acid.
The results obtained here are important due to their usefulness for further research based on the analyzed plant species and potential use in food (food industry). Based on the results of this research, it can be concluded that the method for extraction of the bioactive compounds should be selected for the type of herb.

Author Contributions

Conceptualization, U.S. and A.K.; methodology, U.S., A.K., K.D. and J.S.; formal analysis, U.S. and A.K.; investigation, R.A.V., K.D., J.S. and U.S.; resources, U.S. and A.K.; data curation, R.A.V., K.D., J.S. and U.S., writing—original draft preparation, R.A.V., U.S. and S.K.S.; writing—review and editing, U.S., S.K.S. and A.K.; funding acquisition, U.S. and A.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the Ministry of Science and Higher Education of the Republic of Poland for the University of Agriculture in Kraków, Poland.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data and samples are available from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Table 1. Dry mass (g/100 g FM *) and proximate composition of different varieties of tested herbs (g/100 g DM **).
Table 1. Dry mass (g/100 g FM *) and proximate composition of different varieties of tested herbs (g/100 g DM **).
ComponentHerbsp-Value
PeppermintLemon BalmLavender
Dry mass22.49 ± 0.35 a35.67 ± 0.43 b35.59 ± 0.76 b0.000
Protein12.60 ± 0.06 b8.92 ± 0.52 a7.65 ± 0.51 a0.003
Fat2.61 ± 0.32 a2.47 ± 0.37 a3.37 ± 0.04 a0.091
Total carbohydrates84.31 ± 0.40 a88.18 ± 0.97 b88.76 ± 0.45 b0.012
Ash0.48 ± 0.02 a0.43 ± 0.08 a0.22 ± 0.11 a0.089
* FM—fresh mass; ** DM—dry mass. Results are expressed as mean ± SD; mean values with different letters (a,b) within the individual rows (other than the last column) are statistically different (p ≤ 0.05).
Table 2. Antioxidant activity (µmol Trolox/1 g DM) and total polyphenols (mg/100 g DM) content in the tested herbs.
Table 2. Antioxidant activity (µmol Trolox/1 g DM) and total polyphenols (mg/100 g DM) content in the tested herbs.
MethodsExtractsHerbsp-Value
PeppermintLemon BalmLavender
ABTSInfusion0.35 ± 0.03 b0.45 ± 0.04 c0.25 ± 0.02 a0.000
Metanol0.33 ± 0.07 ab0.57 ± 0.21 b0.25 ± 0.02 a0.049
Acidified Methanol0.18 ± 0.00 b0.38 ± 0.06 c0.03 ± 0.01 a0.000
FRAPInfusion6117.76 ± 94.13 c2770.60 ± 48.96 b1795.47 ± 88.39 a0.000
Methanol1541.20 ± 409.88 b1279.88 ± 214.69 b490.15 ± 17.50 a0.007
Acidified Metanol1506.27 ± 112.24 a1588.32 ± 84.60 a2096.26 ± 1625.30 a0.721
Total
Polyphenols		Infusion23,470 ± 29 g18,102 ± 83 f4530 ± 39 bc0.000
Methanol5282 ± 196 c6648 ± 36 bd3538 ± 883 b0.063
Acidified Methanol11,815 ± 196 e11,629 ± 657 e2146 ± 269 a0.000
Results are expressed as mean ± SD; mean values with different letters (a–g) within the individual rows (other than the last column) are statistically different (p ≤ 0.05).
Table 3. Polyphenolic profile of peppermint, lemon balm and lavender (mg/100 g DM).
Table 3. Polyphenolic profile of peppermint, lemon balm and lavender (mg/100 g DM).
CompoundPeppermintLemon BalmLavender
InfusionAcidified MethanolInfusionAcidified MethanolInfusionAcidified Methanol
Gallic acid2.90 ± 0.03 b3.05 ± 0.01 b2.60 ± 0.12 c0.00 ± 0.00 a3.4 ± 0.14 d0.00 ± 0.00 a
Catechin38.59 ± 1.97 e18.33 ± 0.04 b11.37 ± 0.08 a23.57 ± 0.27 c35.56 ± 0.03 d43.59 ± 1.06 f
Chlorogenic acid36.18 ± 0.0864.87 ± 0.144.38 ± 0.0025.57 ± 1.021.76 ± 0.01 a3.09 ± 0.01 b
4-Hydroxybenzoic acid7.36 ± 0.04 e10.83 ± 0.09 f3.87 ± 0.04 b2.69 ± 0.01 c2.81 ± 0.061.61 ± 0.00
Epicatechin53.89 ± 3.18 d39.70 ± 1.17 c25.65 ± 1.20 a72.18 ± 1.00 e33.28 ± 0.08 b26.88 ± 1.32 a
Caffeic acid9.32 ± 0.03 a11.44 ± 0.06 d8.65 ± 0.87 a4.97 ± 0.02 c3.93 ± 0.00 b9.18 ± 0.02 a
Vanillic acid3.42 ± 0.03 a3.52 ± 0.16 a7.67 ± 0.17 d4.01 ± 0.05 b3.81 ± 0.10 b5.26 ± 0.01 c
Syringic acid3.93 ± 0.04 c13.04 ± 0.22 a22.55 ± 0.68 b32.83 ± 0.06 c13.18 ± 0.04 a22.92 ± 0.69 b
p-Coumaric acid12.33 ± 0.0 d17.32 ± 0.02 e1.60 ± 0.00 a2.11 ± 0.01 b3.62 ± 0.01 c18.37 ± 0.06 f
Ferulic acid17.72 ± 0.26 a21.78 ± 0.56 b17.53 ± 1.19 a10.3 ± 0.13 c18.52 ± 0.01 a21.59 ± 0.05 b
Sinapinic acid32.46 ± 9.6 a21.06 ± 0.30 b36.82 ± 2.38 b53.31 ± 1.23 c38.93 ± 0.03 a39.83 ± 0.01 a
Naringin1479 ± 0.39 e2578 ± 0.24 f106.34 ± 1.8 c202.93 ± 2.02 d68.31 ± 0.22 a84.98 ± 0.36 b
Rutin436 ± 1.22 e562 ± 0.50 f110.62 ± 0.32 c126.47 ± 0.46 d21.12 ± 0.54 b16.99 ± 0.52 a
Hesperidin604 ± 0.78 e892 ± 0.98 f41.06 ± 1.49 c43.95 ± 0.01 d22.97 ± 0.01 b17.76 ± 0.61 a
Rosmarinic acid1158 ± 14.95 c2077 ± 2.0 d2520 ± 2.05 e4406 ± 92 f155 ± 0.27 a230.67 ± 0.04 b
Myricetin279 ± 0.01 e137 ± 1.29 f13.09 ± 0.09 a87.84 ± 0.22 c27.31 ± 0.06 b65.22 ± 0.07 d
Quercetin111 ± 0.23 e136 ± 0.14 f9.39 ± 0.02 c3.53 ± 0.04 a7.94 ± 0.13 b12.93 ± 0.04 d
Luteolin170 ± 1.79 d570 ± 0.01 e2.49 ± 0.20 b3.28 ± 0.00 b0.00 ± 0.00 a8.94 ± 0.08 c
Kaempferol10.21 ± 0.21 f2.12 ± 0.06 b4.29 ± 0.12 c4.63 ± 0.01 d0.87 ± 0.01 a6.08 ± 0.00 e
Apigenin6.64 ± 0.01 b15.27 ± 0.01 b55.36 ± 0.99 e137.55 ± 0.07 f0.72 ± 0.00 a26.48 ± 0.05 d
Isorhamnetin16.88 ± 0.36 c15.81 ± 0.67 c5.95 ± 0.38 b136.25 ± 0.06 f0.48 ± 0.01 a19.21 ± 0.08 d
Hispidulin9.01 ± 0.00 e18.48 ± 0.04 f5.31 ± 0.22 d2.00 ± 0.04 b0.00 ± 0.00 a4.24 ± 0.01 c
Acacetin20.15 ± 0.05 c71.67 ± 0.03 d5.15 ± 0.26 b3.64 ± 0.01 b0.00 ± 0.00 a0.00 ± 0.00 a
Carnosol10.17 ± 0.02 a33.11 ± 0.07 c44.51 ± 0.016 d26.08 ± 1.14 b10.8 ± 0.00 a25.61 ± 0.32 b
Carnosic acid5.61 ± 0.057 c34.53 ± 0.15 f1.28 ± 0.09 a14.81 ± 0.04 d3.68 ± 0.13 b31.84 ± 1.55 e
Results are expressed as mean ± SD; mean values with different letters (a–f) within the individual rows are statistically different (p ≤ 0.05).
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Sadowska, U.; Armenta Villavicencio, R.; Dziadek, K.; Skoczylas, J.; Sadowski, S.K.; Kopeć, A. The Identification of Polyphenolic Compounds and the Determination of Antioxidant Activity in Extracts and Infusions of Peppermint, Lemon Balm and Lavender. Appl. Sci. 2024, 14, 699. https://doi.org/10.3390/app14020699

AMA Style

Sadowska U, Armenta Villavicencio R, Dziadek K, Skoczylas J, Sadowski SK, Kopeć A. The Identification of Polyphenolic Compounds and the Determination of Antioxidant Activity in Extracts and Infusions of Peppermint, Lemon Balm and Lavender. Applied Sciences. 2024; 14(2):699. https://doi.org/10.3390/app14020699

Chicago/Turabian Style

Sadowska, Urszula, Ruth Armenta Villavicencio, Kinga Dziadek, Joanna Skoczylas, Szymon Kamil Sadowski, and Aneta Kopeć. 2024. "The Identification of Polyphenolic Compounds and the Determination of Antioxidant Activity in Extracts and Infusions of Peppermint, Lemon Balm and Lavender" Applied Sciences 14, no. 2: 699. https://doi.org/10.3390/app14020699

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