Infusions of Herbal Blends as Promising Sources of Phenolic Compounds and Bioactive Properties

Several plants have been used for medicinal applications and have been traditionally consumed as decoctions and infusions. Although some herbs are used alone as a beverage, they are often blended in mixtures to maximize their effects. Herein, the nutritional characterization of six infusions from herbal blends was evaluated using the official methods of analysis (AOAC international). A further characterization of the individual phenolic profile was also performed by HPLC-DAD/ESI-MSn, and finally bioactive potential was determined by evaluating the antioxidant, cytotoxic, anti-inflammatory, and antimicrobial activities of each blend. The wide variety of plants in each sample led to variability in the results for all analyzed parameters. However, blends containing 15% Laurus nobilis L. and 15% Juglan regia L. in their composition showed higher sugar content and energy contribution; higher concentration of phenolic compounds (phenolic acids and flavonoids); greater antioxidant, cytotoxic, and anti-inflammatory capacity; and also better antimicrobial effects against all the tested bacterial and fungal strains. Further studies will be necessary to evaluate the real synergistic effects that these two species show in the presence of other plants, and to evaluate their potential for application in various food, pharmaceutical, and nutraceutical products as infusion preparations.


Introduction
The search for new, safer, and sustainable high-added-value compounds relies to a certain degree on the continuous study of traditionally used medicinal plants [1]. These plants have been used since ancient times for their countless benefits, being associated with health-promoting properties [2,3]. Conventionally prepared as an infusion, decoction, or by maceration (also used for culinary purposes due to their fragrance and flavor), these herbs can be blended in different combinations of leaves, roots, barks, stems, and flowers, among other plant materials [4]. One of the botanical families containing an enormous range of species most commonly consumed as herbal tea is the Lamiaceae family, which brings together an enormous range of plants (e.g., Rosmarinus officinalis L. and Thymus mastichina L.) that have several recognized bioactive properties and beneficial health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. Table 1. Description and morphological characteristics of the six herbal blends provided by Ervital ® company.

Mix 1 Mix 2 Mix 3
Molecules 2020, 25, x 2 of 17 health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. The morphological characteristics of the six herbal blends were flowering aerial parts of Foeniculum vulgare Mill., Hypericum perforatum L., Mentha cervina L., Mentha pulegium L., Origanum vulgare subs. virens Hoffm. and Link, and Thymus mastichina L; flower heads of Calendula arvensis L., Chamaemelum nobile (L.) All., and Sambucus nigra L.; and leaves of Juglans regia L., Laurus nobilis L., Olea europaea L., Rosmarinus officinalis L., Rubus idaeus L., and Vitis vinifera L.

Nutritional Analysis and Sugar Composition
Molecules 2020, 25, x 2 of 17 health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. The morphological characteristics of the six herbal blends were flowering aerial parts of Foeniculum vulgare Mill., Hypericum perforatum L., Mentha cervina L., Mentha pulegium L., Origanum vulgare subs. virens Hoffm. and Link, and Thymus mastichina L; flower heads of Calendula arvensis L., Chamaemelum nobile (L.) All., and Sambucus nigra L.; and leaves of Juglans regia L., Laurus nobilis L., Olea europaea L., Rosmarinus officinalis L., Rubus idaeus L., and Vitis vinifera L.

Nutritional Analysis and Sugar Composition
Molecules 2020, 25, x 2 of 17 health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures.   health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. The morphological characteristics of the six herbal blends were flowering aerial parts of Foeniculum vulgare Mill., Hypericum perforatum L., Mentha cervina L., Mentha pulegium L., Origanum vulgare subs. virens Hoffm. and Link, and Thymus mastichina L; flower heads of Calendula arvensis L., Chamaemelum nobile (L.) All., and Sambucus nigra L.; and leaves of Juglans regia L., Laurus nobilis L., Olea europaea L., Rosmarinus officinalis L., Rubus idaeus L., and Vitis vinifera L.

Nutritional Analysis and Sugar Composition
Molecules 2020, 25, x 2 of 17 health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. The morphological characteristics of the six herbal blends were flowering aerial parts of Foeniculum vulgare Mill., Hypericum perforatum L., Mentha cervina L., Mentha pulegium L., Origanum vulgare subs. virens Hoffm. and Link, and Thymus mastichina L; flower heads of Calendula arvensis L., Chamaemelum nobile (L.) All., and Sambucus nigra L.; and leaves of Juglans regia L., Laurus nobilis L., Olea europaea L., Rosmarinus officinalis L., Rubus idaeus L., and Vitis vinifera L.

Nutritional Analysis and Sugar Composition
Molecules 2020, 25, x 2 of 17 health effects [5][6][7]. Another widely used botanical family is the Asteraceae family, comprising plants such as Calendula arvensis L. and Chamaemelum nobile L., which have been extensively studied [8][9][10] and also present a wide range of bioactive properties. The species I this family are of great commercial and economic value, with potential to be applied in the food and pharmaceutical industries. There are other plant families that are still underexplored, despite showing promising potential to be used as sources of high-added-value compounds, such as Lauraceae (e.g., Laurus nobilis L.), Oleaceae (e.g., Olea europaea L.), Vitaceae (e.g., Vitis vinifera L.), Apiaceae (e.g., Foeniculum vulgare Mill.), and Juglandaceae (e.g., Juglans regia L.) families, among others [11][12][13][14][15]. The demand of today's consumers for foods marketed as healthier and natural has led the food industry to seek new formulation products to meet their high expectations [16], such as blending herbs from different botanical families. These mixtures can present additive and synergistic effects and increase the content of compounds of interest, improving the nutritional value of these products, and also by acting as sources of functional ingredients to be incorporated into food products [16,17]. In this sense, the present work was performed to chemically characterize six herbal blends (complete description in Table 1), namely relating to their profiles in individual sugars and phenolic compounds. Moreover, it also aimed to evaluate their antioxidant, cytotoxic, anti-inflammatory, and antimicrobial properties, comparing these results with previously reported data on the individual plants, allowing a better understanding of the eventual synergistic effects existing in these innovative mixtures. The morphological characteristics of the six herbal blends were flowering aerial parts of Foeniculum vulgare Mill., Hypericum perforatum L., Mentha cervina L., Mentha pulegium L., Origanum vulgare subs. virens Hoffm. and Link, and Thymus mastichina L; flower heads of Calendula arvensis L., Chamaemelum nobile (L.) All., and Sambucus nigra L.; and leaves of Juglans regia L., Laurus nobilis L., Olea europaea L., Rosmarinus officinalis L., Rubus idaeus L., and Vitis vinifera L.

Nutritional Analysis and Sugar Composition
The total contents of fat, ash, and proteins in the infused blend were, as expected (and confirmed in previous studies by Caleja et al. [18]), below the limits of detection, and consequently below the limits of quantification of the equipment and protocols used to perform these analyses. Therefore, assuming total sugars as total carbohydrates, the calculation of energy was performed according to the following equation: energy (cal) = 4 × (mg carbohydrates). Data on the free sugar content and energy contribution of the six infusions are shown in Table 2. The qualitative and quantitative profiles of individual sugars in the six blends were quite different, with mix 1, mix 2, and mix 4 presenting fructose and glucose, and mix 3 and mix 6 only revealing the presence of fructose and sucrose, respectively. On the other hand, mix 5 was the only blend presenting these three sugars, also showing the highest concentration of total sugars (75 ± 2 mg/100 mL). Given the fact that this was the only blend containing O. vulgare and that it was its main constituent (60%), its high sugar content can be possibly ascribed to this species. In a previous study performed by Pereira et al. [19], a sample of O. vulgare, in which the subspecies was not identified, revealed a total sugar content of 19 ± 1 g/100 g of dry plant, also revealing the presence of trehalose; however, in refenced study the infusion was not assessed, so the results are not comparable to the ones presented herein. In mix 1, the most abundant species was R. officinalis (50%). For this herbal infusion, no sugars were detected in a previous study [19], which suggests that the sugar content of the mixture is due to the contributions of the other plants (C. nobile, L. nobilis, and J. regia). Indeed, Pereira et al. [19] reported a total sugar content of 15.0 ± 0.2 mg/100 mL for the C. nobile infusion and 10.46 ± 0.02 g/100 g of dried sample (not directly comparable) for L. nobilis. Regarding mix 2, in a previous study, F. vulgare infusion had a total sugar amount of 15.0 ± 0.9 mg/100 mL [19], lower than the one found for this mixture, which was composed of 40% of this plant. This suggests that the remaining plants in the blend had a higher contribution. Given the fact that S. nigra and H. perforatum were not present in the other herbal blends, this possibility was not discussed further.

Phenolic Compounds Characterization
The retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data, and tentative identification of the phenolic compounds in the six blends are presented in Table 3. The quantification results (mg/g extract) of the phenolic compounds present in the six blends are presented in Table 4. Fifty-two phenolic compounds were found in the six blends, among which twenty-eight were phenolic acids, twenty-three were flavonoids, and two were unknown compounds. All the plants in these blends were previously studied for their phenolic composition profiles. As such, the identification of all the compounds was performed using the bibliographic references described in the footnotes of Table 3. Twenty phenolic compounds were detected in mix 1, with peak 30 representing the main compound (luteolin-3'-O-glucuronide, 4.6 ± 0.1 mg/g extract). Additionally, this mixture had a high percentage of R. officinalis; its main phenolic compounds were not the ones described for the aqueous form of this plant (infusions and decoction) [20] or for hydroethanolic extracts [6], namely rosmarinic acid and its derivatives. In the case of the mixture, C. nobile [8] and L. nobilis [21] seemed to have a greater influence on the phenolic composition, with luteolin-O-glucuronide being the major peak detected. Tuberonic acid hexoside (peak 7, [M − H] − at m/z 387) showed fragments at m/z 207, which corresponds to the aglycone after loss of hexose [M−H−162] − , and has been previously described in other plants of the Lamiaceae family [22]. Nine compounds were tentatively identified in mix 2. This blend comprised F. vulgare, S. nigra, and H. perforatum, and previous studies on the individual phenolic profiles of these plants revealed quercetin glycosylated derivatives as the main compounds [14,15,23], which is in accordance with the results obtained herein, showing peak 20 (quercetin-3-O-rutinoside, [M − H] − at m/z 609) as the main compound (31.1 ± 1.3 mg/g extract). In mix 3, eight compounds were tentatively identified, with oleuropein (peak 44, [M − H] − at m/z 539) being the major compound found. This is probably due to the 25% content of M. pulegium, since these secoiridoid-type compounds are very common and abundant in the Oleaceae family [12,24]. Thirteen phenolic compounds were tentatively identified in mix 4, with rosmarinic acid being the major compound (13.03 ± 0.05 mg/g extract). Taking into account the high amount of M. cervina in the mixture, and considering a study describing rosmarinic acid as the major compound found in this plant [25], it is possible to state that M. cervina has a great influence on the phenolic composition of this blend. Regarding mix 5, twelve phenolic compounds were tentatively identified, and as in the previous mixture, rosmarinic acid was the major compound (33.9 ± 0.1 mg/g of extract). Likewise, the high percentage (60%) of O. vulgare should explain this result, since Origanum genus plants are characterized by containing rosmarinic acid and its derivatives as the main compounds [26]. Finally, ten compounds were tentatively identified in mix 6, with peak 31 ([M − H] − at m/z 447) being tentatively identified as kaempferol-O-hexoside, the major compound in this mix (35 ± 1 mg/g extract). This was an expected result, since the presence of this compound was described in the individual assessment of the phenolic composition of T. mastichina [6], L. nobilis [21], and J. regia [27]. Table 3. Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data, and identification of phenolic compounds in the six infused blends.     Regarding the total compositions of phenolic compounds, it was possible to verify that mix 6, mix 5, and mix 2 presented higher contents of total phenolic compounds (72 ± 1, 69.6 ± 0.2, and 65 ± 2 mg/g extract, respectively). In mix 5, the total phenolic compound quantity is mainly due to the presence of phenolic acids (67.1 ± 0.2 mg/g extract), which represents 96% of the total phenolic composition. In mix 6, the total phenolic compound quantity is mainly due to the total flavonoid content (47.1 ± 0.2 mg/g extract), which represents 65.4% of the total composition.

Bioactive Properties
The results for antioxidant, anti-inflammatory, and cytotoxic activities are shown in Table 5. For TBARS assays, mix 1 and mix 6 presented the lowest IC 50 values (4.5 ± 0.2 and 6.9 ± 0.3 µg/mL, respectively), which indicates high antioxidant activity. In fact, mix 1 revealed a higher lipid peroxidation inhibition capacity than the positive control, Trolox (5.8 ± 0.6 µg/mL). This activity could be explained by the presence of R. officinalis, reported in the literature as an excellent antioxidant plant, which is even applied in foodstuffs such as cottage cheese to increase shelf life [6]. Regarding the good results found for mix 6, the fact is that this blend presented the highest concentration of phenolic compounds, which are often reported as the main compounds responsible for the bioactive properties of plants. In a previous study, O. vulgare infusion presented an IC 50 value of 22.8 ± 0.5 µg/mL, which was a high concentration when compared to 9 ± 1 µg/mL, the IC 50 value obtained for mix 5 (with 60% of this plant), thus suggesting synergistic effects among the blended plants [26].
In terms of antihemolytic activity, the blend revealing the best results was mix 5, with an IC 50 value of 4.0 ± 0.6 µg/mL. In fact, this blend presented the highest concentration of phenolic acids, which could be related to its antioxidant properties. All blends, with the exception of mix 2, revealed lower IC 50 values than the positive control (85 ± 2 µg/mL). Studies developed by Caleja et al. [42] and Ribeiro et al. [43] proved the high antioxidant and antimicrobial potential of F. vulgare (present in mix 2) and R. officinalis (present in mix 1) and their capacity to increase the shelf life of functionalized foods. Moreover, in a previous study, S. nigra (present in mix 2) also revealed antioxidant properties (DPPH and FRAP assays) [44]. It is also reported in the literature that the combination of several plants in a drink beverage can provide a synergistic effect in terms of bioactivities, as their combination improves the antioxidant status and reduces oxidative stress [45]. Table 5. Antioxidant, anti-inflammatory, and cytotoxic activity of the six infused blends (mean ± SD). Regarding cytotoxic activity, except for mix2 and mix 4, all blends revealed the capacity to inhibit the growth of the studied tumor cell lines in concentrations ranging from 175 ± 18 (mix 6 in HepG2) to 320 ± 12 (mix 3 in NCI-H460) µg/mL. Another positive aspects is that none of the mixtures revealed toxicity for non-tumor cells up to 400 µg/mL. Similar observations could be made for anti-inflammatory activity, with mix 2 and mix 4 being the only blends not showing activity. Among the remaining infusions, mix 6 revealed the highest activity at a concentration of 262 ± 17 µg/mL. Considering the results reported by Dias et al. [11] and Vieira et al. [27], where L. nobilis and J. regia presented cytotoxic activity, in the present study it was expected that mix 2 and mix 4, containing these plants, would also present these properties, which was not observed. This was possibly due to the fact that such blends also included other species.

Mix
Finally, Table 6 shows the antimicrobial capacity of the six infused blends. It is possible to confirm the excellent antimicrobial potential of all mixtures against the analyzed microorganisms. S. aureus and P. funiculosum seemed to be the most sensitive microorganisms tested, with the infusions revealing lower minimal inhibitory concentration (MIC) and minimal bactericidal concentration/minimal fungal concentration (MBC/MFC) values than the positive controls. Among the tested blends, mix 1 revealed the highest antibacterial properties, with inhibitory and bactericidal concentrations ranging between 0.25 and 2 mg/mL. These results are in accordance with the ones obtained for R. officinalis (50% of the composition of mix 1) hydroethanolic extracts, which showed promising antimicrobial capacity [41]. On the other hand, mix 6 revealed the greatest antifungal activity, being able to inhibit fungal growth at 0.12 to 0.5 mg/mL and presenting fungicidal capacity in concentrations ranging from 0.25 to 5 mg/mL. This blend was more effective than the positive controls for all the tested fungi, except in terms of killing A. fumigatus. These results are in accordance with the ones obtained for Thymus sp. (70% of the composition of mix 6), which has been prescribed for the treatment of infectious diseases [33]. The antimicrobial capacity of Thymus sp. infusion has often been correlated with the presence of flavonoids and phenolic acids [6,21,46]. 95 • C, 5-8 min; mix 4: 4 g/L, 90 • C, 4-6 min; mix 5: 10 g/L, 90 • C, 5-7 min; and mix 6: 10 g/L, 90 • C, 5-7 min. All samples were filtered through Whatman # 4 paper, frozen at −20 • C, and then lyophilized. The extracts were protected from light and humidity until further analysis.

Nutritional Analysis and Sugar Content
Fat, carbohydrate, ash, and protein contents of the six dry infusion extracts were analyzed following the AOAC [48] procedures. Free sugars were analyzed following the method previously described by Barros et al. [28], using HPLC coupled to a refractive index detector (Knauer, Smartline 1000 and Smartline 2300 systems, respectively) and melezitose as internal standard. The results were expressed in mg/100 mL of infusion. Finally, the energetic value was calculated according the equation: energy (cal) = 4 × (mg proteins + mg carbohydrates) + 9 × (mg lipids).

Phenolic Compounds Composition
The dry infusion extracts were resuspended in water at a concentration of 10 mg/mL. The phenolic profile was determined by liquid chromatography (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) with a diode array detector (280, 330, and 370 nm wavelengths) equipped with an ESI source and working in negative mode (Linear Ion Trap LTQ XL, Thermo Scientific, San Jose, CA, USA) [49]. Chromatographic separation was achieved with a Waters Spherisorb S3 ODS-2C18 (3 m, 4.6 mm × 150 mm, Waters, Mil-ford, MA, USA) column thermostat at 35 • C. The solvents used were: (A) 0.1% formic acid in water, (B) acetonitrile. The established isocratic elution gradient was 15% B (5 min), 15% B to 20% B (5 min), 20-25% B (10 min), 25-35% B (10 min), 35-50% B (10 min), and re-equilibration of the column, using a flow rate of 0.5 mL/min [49]. The phenolic compounds were identified by comparing their retention times, UV, and mass spectra values with those obtained from standard compounds and with the literature. For quantitative analysis, 7-level calibration curves prepared with appropriate standards were used. The results were expressed in mg per g of dry extract (mg/g) as mean ± standard deviation of three independent analyses.

Antioxidant Activity
All samples were tested by two in vitro assays. The thiobarbituric acid reactive species (TBARS) assay uses a brain porcine homogenate measured by spectrophotometry at 532 nm [28]. This assay is used to determine the TBARS content in various samples, including those used to detect a sort of protective activity (usually antilipoperoxidant activity) using a fat or membrane model and an oxidant to induce the damage. The oxidative hemolysis inhibition (OxHLIA) assay is used to evaluate the antihemolytic activity of the extracts using sheep erythrocytes, measured by spectrophotometry at 690 nm [50]. Hemolysis was previously induced using 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH). The results were expressed as EC 50 values (sample concentration providing 50% of antioxidant activity, shown in µg/mL), and Trolox was used as a positive control for both assays.

Anti-inflammatory Activity
The anti-inflammatory activity was assessed following a procedure described by Svobodova et al. [51]. The dried extracts were re-dissolved in water at 8 mg/mL and evaluated in mouse lipopolysaccharide (LPS)-stimulated macrophage-like cell line RAW 264.7. The results were expressed as IC 50 values (sample concentration providing 50% of anti-inflammatory activity, µg/mL) and dexamethasone (50 µM) was used as a positive control.

Cytotoxic Activity in Tumor and Non-Tumor Cells
The cytotoxic potential was evaluated in four different human tumor cell lines (HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-small-cell lung cancer)) and a primary culture of non-tumor cells (PLP2 (porcine liver)). To monitor the growth of cell cultures, which were sub-cultured and plated in 96-well plates (density of 1.0 × 10 4 cells/well), a phase-contrast microscope was used, following the protocol defined by Guimarães et al. [8]. Ellipticin was used as a positive control, and the results were expressed as GI 50 values (sample concentration that inhibited 50% of cell growth, µg/mL).

Statistical Analysis
For each herbal blend, three individual samples were analyzed, and all assays were prepared in triplicate. The results were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's HSD test as p = 0.05, and are expressed as mean values with standard deviation (SD). When less than three results were present in each individual analysis, Student's t-test p-values were used to determine the significant difference, with p = 0.05. Both of these statistical treatments were carried out using the SPSS v.22.0 program.

Conclusions
The infusions of different blended species proved to be valuable sources of high-added-value compounds and also showed high capacity for bioactive activity. Given the wide variety of species in each sample, a great variability in results was also expected, rather than obtaining a mixture that stood out in terms of all the analyzed parameters. However, mix 5 had higher contents of soluble sugar, energy contribution, total phenolic acids, and a higher capacity to inhibit erythrocyte hemolysis and the proliferation of the HeLa tumor cell line. Mix 6 presented the highest content of total phenolic compounds (especially flavonoids), higher capacity to inhibit the growth of HepG2 tumor cell line, higher anti-inflammatory activity, and higher antifungal activity. Finally, mix 1 revealed a higher capacity to inhibit lipid peroxidation in the TBARS assay, higher capacity to inhibit the growth of the MCF-7 tumor cell line, and higher antibacterial potential, presenting the lowest MIC and MBC values for Gram-positive and Gram-negative bacteria (even lower than the positive controls). The fact that blends 1, 5, and 6 contain 15% L. nobilis and 15% J. regia in their composition could explain the better results obtained with these plant mixtures compared to the other ones that did not contain any of these species. As stated earlier, L. nobilis and J. regia have already been described as highly promising plants for obtaining high-added-value compounds with bioactive potential. Future studies will be necessary to evaluate the real synergistic effects presented by these two plants in the presence of others, and to evaluate their potential for further application in various food, pharmaceutical, and nutraceutical products. Funding: The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER (Fundo Europeu de Desenvolvimento Regional) under Programme PT2020 for financial support to CIMO (Centro de Investigação de Montanha) (UIDB/00690/2020); national funding by FCT, P.I., through the Institutional Scientific Employment Program as contracts for L. Barros