2.1. Aroma Profile of Ilex
This is the first paper to present a comparison of the volatile compound profiles in Argentine
I. paraguariensis and European
I. aquifolium and
I. meserveae. The expected results of the study of the volatile fractions of European holly species were the appearance of rich monoterpene and monoterpenoid profiles, which have not yet undergone oxidation reactions during heat treatment. The analysis of volatile compounds in different varieties of
Ilex revealed a total of 81 compounds, of which the ten main ones are presented in
Table 1 (in addition,
Table S1 showing all compounds is available in the
Supplementary Data ). The results obtained show a definite difference in the volatile compound profile between Argentine
I. paraguariensis and European cultivars of
I. aquifolium and
I. meserveae. This difference was due to the fact that
I. paraguariensis was subjected to a prior drying process that contributed to the formation of more oxidation intermediates, characterized by a lower boiling point. The profiles presented by the different groups can represent the characteristic distribution of the chemotype within a given cultivar and act as specific marker indicators to assist in the identification of individual holly species. A divergence was also observed among the profiles of European cultivars. In
I. paraguariensis, the main volatile compounds were found to be 6-methyl-5-heptene-2-one (25.73 µg g
−1), (
E,
E)-3,5-octadien-2-one (22.82 µg g
−1), (
E,
E)-2,4-heptadienal (14.83 µg g
−1), hexanal (13.80 µg g
−1) and 2-methyl-2-pentenal (11.77 µg g
−1). The two main compounds found in
I. paraguariensis (6-methyl-5-heptene-2-one and (
E,
E)-3,5-octadien-2-one) were absent in the European varieties. In
I. aquifolium, (
E,
E)-2,4-heptadienal was also absent. and p-cymene (58.21–82.62 µg g
−1), α-phellandrene (3.68–5.35 µg g
−1) and α-pinene (1.98–3.94 µg g
−1) were the main compounds. In
I. meserveae, p-cymene (20.20–32.75 µg g
−1) was also among the main compounds, but the other compounds were (
E)-2-hexenal (12.28–20.97 µg g
−1) and 3-hexen-1-ol (6.34–11.85 µg g
−1).
Regarding the profile of volatile compounds in
I. paraguariensis, this has already been reported in several works. A study by Dallago et al. [
9] compared the profile of volatile compounds contained in
I. paraguariensis both before the drying process (fresh green leaves) and after the drying stage. The results presented differences in the number of Maillard reaction products formed and their percentage ratios. It was observed that the effect of a higher temperature and oxygen access favored the formation of more secondary products such as ketones, aldehydes, and carboxylic acids. Fresh leaf samples were found to be dominated by compounds from the alcohol group, while dry leaf samples were dominated by aldehydes and ketones. It was also found that long hydrocarbon chains are degraded first, followed by the oxidation of individual functional groups. One paper that shows values similar to those of our team in the profile of volatile compounds of
I. paraguariensis is the publication by Araujo et al. [
10]. They showed that the main aromatic compounds in mate are (
E,
E)-2,4-heptadienal (7.8%), (
E,
Z)-2,4-heptadienal (7.0%) and (
E,
Z)-3,5-octadien-2-one (5.2%). However, they obtained a much lower value for 6-methyl-5-hepten-2-one (1.4%), which in our study, proved to be the main component of the volatile profile. In a study by Marquez et al. [
11], they observed that geranyl acetate (9.38 µg g
−1), linalool (4.78 µg g
−1) and isomers (
E,
E)- and (
E,
Z)-2,4-heptadienal (3.60 and 1.21 µg g
−1) were the most abundant in Yerba mate leaves. Conversely, Martins et al. [
12] conducted a study on the volatile compounds released in relation to mechanical damage and herbivore exposure. In the control group, which was
I. paraguariensis, the main volatile compounds reported were decanal, nonanal, and limonene.
2.2. Terpenoid Profile
The analysis of terpenoids from two cultivars of European
Ilex, i.e.,
I. aquifolium and
I. meserveae, showed the presence of 12 compounds, of which the seven main ones are presented in
Table 2 (in addition,
Table S2 showing all compounds is available in the
Supplementary Data). The study showed similarities in the terpenoid profiles of the European cultivars. The dominant compounds in both
I. aquifolium and
I. meserveae were ursolic acid (7.29–16.11 mg g
−1), oleanolic acid (2.03–6.62 mg g
−1), α-amyrin (0.36–3.22 mg g
−1), lupeol (0.42–2.53 mg g
−1) and uvaol (0.37–2.48 mg g
−1). For comparison, the reference sample was
I. paraguariensis, whose terpenoid profile was similar to those of the European varieties, with the difference being that a significantly lower content of ursolic acid (1.23 mg g
−1) was observed in Yerba mate leaves.
Previous studies have also reported the presence of tritepenes in other
Ilex cultivars. Lupeol, betulonic acid, uvaol, ursolic acid, and α-amyrin have been identified in the leaves of
I. cornuta and
I. latifolia [
13]. Compounds from the terpenoid group were also documented in
I. centrochinensis and
I. macropoda. In the first species, the presence of lupeol and oleanolic acid was revealed, and in the second, betulin was isolated in addition to lupeol [
14]. Plants with a high content of pentacyclic tritepenes are often used in phytotherapy due to their valuable medicinal properties. Additionally, they are widely distributed in the plant world and are the subject of phytochemical and pharmacological research. One of them is
Ficus carica, a traditional plant in folk medicine used to combat pneumonia, diarrhea, inflammation, and indigestion. Ivanov et al. [
15] began to identify the constituents of the nonpolar fraction using GC-MS, and the results of their study show that pentacyclic triterpenoids α-Amyrin, β-amyrin and lupeol are present in ficus leaves. Wolbiś et al. [
16], in their study of the quats and leaves of
Prunus spinosa, which is known as a tea substitute, proved that more triterpene acids are present, and these are mainly ursolic acid (9.5 mg g
−1) and oleanolic acid (2.3 mg g
−1). Conversely, Kowalski [
17] obtained similar values for ursolic acid (14.98 mg g
−1) and oleanolic acid (approximately 5 mg g
−1) [
17] in the leaves of
Silphium integrifolium, whose infusion was used to treat urinary tract disorders. Cheun and Zhang [
18], in
Prunella vulgaris spikes, identified mainly betulinic acid, ursolic acid, and oleanolic acid, whose amounts were 4.7, 4.0 and 0.9 mg g
−1, respectively. A terpenoid fraction was also isolated from the herb
Hieracium pilosella [
19], used, in the treatment of skin diseases, among others, due to its astringent, antiseptic, and anti-inflammatory activities. This fraction consists of β-amyrin, lupeol, and α-amyrin. In conclusion, terpenoids are a widely distributed group among medicinal plants that show promise for the development of new, multifunctional bioactive agents.
2.3. Saponin Profile
Following our previous UHPLC-MS/MS analyses, the saponin profiles of
I. aquifolium and
I. paraguariensis were compared under the conditions described above [
8]. By comparing the saponin profiles of holly and commercial mate, we found that the saponin pattern in
I. aquifolium is less complicated than in
I. paraguariensis, as shown in
Table S3 (available in
Supplementary Materials). Therefore, we have tentatively designated 29 and 53 compound respectively, as triterpene glycosides or glycoside esters.
Qualitatively, the profiles were quite different: 16 compounds were found in both species, while 50 were unique to one or the second. In I. paraguariensis, one compound (911, 12.54 min; only in mate), tentatively assigned as matesaponin 1, was the dominant one, while another compound (911, 13.98 min; in both species) appeared to be the main saponin in I. aquifolium. However, numerous already-recognized saponins in the Ilex genus match the MS fragmentation of the last saponin, for example, ilexsaponin B2, latifoloside A, latifoloside B, or latifoloside D; thus, we cannot speculate on dereplication.
Considering the compounds classified as the same in both holly and mate, only six of sixteen had quite small differences below 10 RA% between species (911, 6.81 min; 825, 7.74 min; 927, 7.97 min; 911, 9.25 min; 1073, 9.53 min; 895, 14.68 min); these compounds, moreover, were also of generally small intensity. Four compounds differed between species in the range of 10–25 RA% (927, 8.30 min; 1219.61, 9.15 min probably matesaponin 4; 1057, 10.85 min; 1057, 12.15 min), while six others had a difference greater than 25RA%.
Taking into account the MS fragmentation of saponins, the following three types of triterpene aglycones were found based on MS/MS-resulting ions with m/z 455 for [C30H48O3−H]− (an equivalent of monohydroxylated triterpene acid, such as oleanolic or ursolic acid), 469 for [C30H46O4−H]− (an equivalent of didehydrogenated dihydroxylated triterpene acid) and 471 for [C30H48O4−H]− (an equivalent of dihydroxylated triterpene acid) in both species.
2.4. Fatty Acid Profile in Ilex Leaves
Analysis of fatty acid methyl esters (FAMEs) from European
Ilex cultivars revealed the presence of 17 compounds, and seven of the main FAMEs are summarized in
Table 3 (in addition,
Table S4 showing all compounds is available in
Supplementary Data). It was found that the fatty acid profiles of the Argentine and European varieties were similar to one another. The main compounds identified in the European varieties were palmitic acid (12.05–15.03 mg g
−1), linoleic acid (LA) (5.52–10.40 mg g
−1) and α-linolenic acid (ALA) (9.09–22.16 mg g
−1). Regarding the European cultivars, greater profile similarity was observed between
I. aquifolium Ferox Argentea and
I. meserveae Blue Angel due to similar amounts of LA (5.84 and 5.63 mg g
−1) and ALA (9.72 and 9.09 mg g
−1) acids. However, the other cultivars, i.e.,
I. aquifolium Alaska,
I. aquifolium Rubricaulis Aurea,
I. meserveae Golden Girl, and
I. meserveae Blue Boy, showed higher amounts of these acids, which were as follows: LA (8.40–10.08 mg g
−1) and ALA (19.28–22.16 mg g
−1).
I. paraguriensis as a reference sample showed the highest amounts of palmitic (18.08 mg g
−1) and oleic acid (9.03 mg g
−1). Stearic and α-linolenic acids were identified at similar levels, namely 6.39 and 5.92 mg g
−1 respectively. It was also observed that the level of linoleic acid was lower in the Argentinean cultivar compared to the European cultivars.
The fatty acid profile in
I. paraguriensis has also been reported by other researchers. In a paper published by Souza et al. [
6], the acid that was significantly prominent in terms of the amount in the leaves of
I. paraguariensis was α-linolenic acid, and the second acid that was most prominent, but in a much lower amount, was palmitic acid. Compared to the above analyses, the levels of palmitic and LA acids were lower. In addition, in the work of Reis et al. [
20] the fatty acid profile of
I. paraguariensis was demonstrated, where the main acids were also palmitic acid and α-linolenic acid, but their amounts were similar. As in the previous study, Reis et al. also reported lower contents of stearic acid, oleic acid, and linoleic acid. Chóez-Guaranda et al. [
21], in their study on the oxidative activity of fractions of
I. guayusa leaves, proved the presence of the following fatty acids in the hexane fraction: palmitic acid, oleic acid, and stearic acid.
Similar fatty acid relationships have been identified in medicinal plants in previous studies. Guil-Guerrero [
22], in a study on
Plantago major, determined the highest amount of α-linolenic acid (40.04%), followed by palmitic acid (16.59%) and linoleic acid (13.77%). The relationships among fatty acid profiles in medicinal plants were also recorded by Rutto [
23], who explored the dietary value of raw and processed
Urtica dioica. Stinging nettle showed the highest α-linolenic acid content (49.55%). The opposite situation to the previous case was also observed here, since the second highest acid content was attributed to linoleic acid (23.30%), followed by palmitic acid (17.06%). However, in the fatty acid profile of
Melissa officinalis leaves, linoleic acid was found to be the dominant acid (74.08%), while palmitic acid was next (15.77%) [
24]. In the leaves of
Salvia officinalis, Taarit [
25] identified those with 18 carbons in the molecule as the main fatty acids. The acid content decreases with the decrease in the number of double bonds in the acid (or with the decrease in the degree of unsaturation) and is as follows: α-linolenic acid (45.80%), linoleic acid (14.00%) and oleic acid (9.41%).
The polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio is used when estimating the effect of diet on the cardiovascular system. PUFAs are assumed to lower serum low-density cholesterol (LDL), as opposed to SFAs, which contribute to high serum cholesterol [
26]. It follows that the higher the PUFA/SFA ratio, the more positive the effect on the cardiovascular system. The most beneficial plants were
I. meserveae Golden Girl (1.90),
I. aquifolium Alaska (1.96) and
I. aquifolium Rubricaulis Aurea (1.70), showing the highest indices; thus, compared to
I. paraguariensis (0.57), they can be considered more beneficial to the circulatory system.
However, the PUFA/SFA ratio is too general and does not allow for an assessment of atherogenicity. Therefore, an atherogenicity index (IA) is used, which reveals the ratio of the sum of SFAs and the sum of unsaturated acids (UFAs). SFAs (C12:0, C14:0, C16:0) promote lipid adhesion to the cells of the circulatory and immune systems. However, UFAs inhibit the accumulation of atherosclerotic plaques and influence the reduction of phospholipids, cholesterol, and esterified fatty acids, which is why they are considered anti-atherosclerotic [
27]. According to this rule, products with lower IA may contribute to the reduction of total cholesterol and LDL cholesterol in plasma. The values of the IA index in different
Ilex cultivars range from 0.42 to 0.77. The lowest and most similar values are shown by
I. aquifolium Alaska (0.42),
I. aquifolium Rubricaulis Aurea (0.43),
I. meserveae Blue Boy (0.44), and
I. meserveae Golden Girl (0.50). In contrast, the IA indices of
I. aquifolium Ferox Argentea (0.69) and
I. meserveae Blue Angel (0.62) are similar to that of the reference sample of
I. paraguariensis (0.77). This suggests that European cultivars may have a more beneficial effect than
I. paraguariensis in lowering plasma cholesterol levels. An indicator that shows the tendency to form thrombi in blood vessels is the thrombogenicity index (IT). It characterizes the relationship between FAs that are considered prothrombogenic (C12:0, C14:0 and C16:0) and antithrombogenic (n-3, n-6 and MUFA). Therefore, a diet containing products with a lower IT ratio is more beneficial for the cardiovascular system [
28]. The index of thrombogenicity in
Ilex samples oscillates in the range of 0.20–0.69. The most favorable IT values, and thus indicating the most positive effect on the cardiovascular system, are observed in four European cultivars:
I. aquifolium Alaska (0.20),
I. aquifolium Rubricaulis Aurea (0.23),
I. meserveae Golden Girl (0.22), and
I. meserveae Blue Boy (0.22). However, the index of one of the European varieties of
I. aquifolium Ferox Argentea (0.62) is similar to that of
I. paraguariensis (0.69). In summary, both of these indices (IA and IT) can be used as a means to assess the effect of the composition of Fas on the cardiovascular system. Products with lower IA and IT compositions are associated with a reduced risk of coronary heart disease and better nutritional value.
Another indicator that can better illustrate the effect of the composition of Fas on cardiovascular disease than PUFA/SFA is the hypocholesterolemic/hypercholesterolemic index (HH). It describes the relationship between hypocholesterolemic fatty acids (cis-C18:1 and PUFA) and hypercholesterolemic Fas (C12:0, C14:0 and C16:0). As the value of the h/H index increases, the beneficial effect on the circulatory system also increases [
29]. Here, the highest values were also found in
I. aquifolium (2.68),
I. meserveae Golden Girl (2.63),
I. aquifolium Rubricaulis Aurea (2.41) and
I. meserveae Blue Boy (2.39). They were twice as high as those of the reference samples,
I. paraguariensis (1.24) and
I. aquifolium Ferox Argentea (1.17).
Unsaturated fatty acids show different weights in relation to the unsaturation index (UI). This reflects the complex proportions of FA, with different degrees of unsaturation compared to the total FA composition. This index can be used to evaluate the suitability of the products as alternative sources of high-quality PUFAs [
30,
31]. Regarding the Argentine
I. paraguariensis (110.07), all the European varieties tested obtained higher UI indices. The variety most similar to the reference sample was
I. meserveae Blue Angel (126.82). The highest UI values were obtained for
I. aquifolium (174.73) and
I. meserveae Golden Girl (175.87).
The results obtained indicate that European Ilex varieties show potentially positive effects on human health through lowering cholesterol levels, reducing thrombus formation, and reducing the risk of cardiovascular disease.