Comparative Analysis of Metabolic Variations, Antioxidant Profiles and Antimicrobial Activity of Salvia hispanica (Chia) Seed, Sprout, Leaf, Flower, Root and Herb Extracts

The purpose of this study was to evaluate the phytochemical profiles of the seeds, sprouts, leaves, flowers, roots and herb of Salvia hispanica and to demonstrate their significant contribution to antioxidant and antimicrobial activities. Applied methods were: HPLC-DAD coupled with post-column derivatization with ABTS reagent, untargeted metabolomics performed by LC-Q-Orbitrap HRMS, and two-fold micro-dilution broth method, which involved suspending a solution of tested compounds dissolved in DMSO in Mueller–Hinton broth for bacteria or Mueller–Hinton broth with 2% glucose for fungi. Metabolomic profiling using LC-Q-Orbitrap HRMS used in this study yielded the identification and preliminary characterization of one hundred fifteen compounds. The dominant class of compounds was terpenoids (31 compounds), followed by flavonoids (21 compounds), phenolic acids and derivatives (19 compounds), organic acids (16 compounds) and others (fatty acids, sugars and unidentified compounds). The organic and phenolic acids were the most abundant classes in terms of total peak area, with distribution depending on the plant raw materials obtained from S. hispanica. The main compound among this class for all types of extracts was rosmarinic acid which was proven to be the most abundant for antioxidant potential. All tested extracts exhibited considerable antibacterial and antifungal activity. The strongest bioactivity was found in leaf extracts, which presented bactericidal activity against Gram-positive bacteria (S. aureus, S. epidermidis, M. luteus and E. faecalis). The work represents the first compendium of knowledge comparing different S. hispanica plant raw materials in terms of the profile of biologically active metabolites and their contribution to antioxidant, antimicrobial and antifungal activity.


Introduction
Thanks to the development of advanced analytical technologies, it is now possible to better characterize the complex composition of many plant raw materials, with a focus on understanding their biologically active compounds. In this context, a very interesting plant species commonly used in phytotherapy as well as in health nutrition is chia-Salvia hispanica L. (Lamiaceae) [1,2]. Currently, S. hispanica can be field cultivated or cultivated in greenhouse conditions in many regions [3][4][5]. However, S. hispanica is a neglected and underutilized plant (NUCS) [6]. The plant is now known for its health-promoting properties [6][7][8]. Furthermore, chia seeds are well known in Traditional Chinese Medicine (TCM) and were used by the Mayan and Aztec tribes as food and an ingredient in many herbal mixtures. However, no specific healing properties were attributed to them at the time [1,9].
Currently, the main raw material obtained from S. hispanica is chia seed (Salviae hispanicae semen) [10]. The chemical composition and biological activities of chia seeds have been well described in the scientific literature, and therefore, commercial interest in this raw material continues to increase [11][12][13][14]. In 2009, the European Food Safety Authority (EFSA) issued a positive opinion on chia seeds, making them safe for use in the food industry [15]. In Canada, the seeds and chia seed oil are classified as "natural health product ingredient" [16]. However, few scientific studies deal with the analysis and commercial use of other raw materials derived from this plant, namely sprouts, leaves, flowers, and whole herbs.
On the other hand, interestingly, there are few papers dealing with phytochemical or biological studies of the other raw materials extracted from S. hispanica. Only some researchers focused on the sprouts and leaves of S. hispanica and proved that they could be an interesting plant resource [36][37][38][39][40][41][42]. It is known that chia leaves are a source of hydroxycinnamic acid and its derivatives, flavones (apigenin, luteolin, orientin, and vitexin), and flavonols (quercetin, kaempferol derivatives). In addition, studies on chia sprouts have shown that they are a source of proteins, minerals (especially calcium and magnesium), and vitamins (especially A, E, C, and the B group) [41,43]. In addition, sprouts contain a high concentration of unspecified plant polyphenols with strong antioxidant potential [44,45]. However, there are no scientific studies detailing and comparing the phytochemical profile and antioxidant and antimicrobial activity of chia flowers or herb.
The current scientific literature on the different morphological parts of S. hispanica is very limited. Studies have comprehensively described the composition and biological activity of chia seeds, but knowledge of the sprouts, flowers, leaves, or herb has not been exhaustively described. Current research on the analysis of the leaves is only related to the chemical analysis of the essential oil and the content of some biologically active compounds. Few studies describe the antioxidant capacity of chia leaf extracts.
The work aims to explore the scientific knowledge on the content of the main metabolites, focusing on the group of polyphenols present in different raw materials extracted from S. hispanica, as well as on their antioxidant, antimicrobial and antifungal activities. The work represents a comprehensive comparative analysis of S. hispanica plant material-seeds, sprouts, leaves, flowers, roots and herb. The study included phytochemical and qualitative analysis by the HPLC technique in conjunction with post-column derivatization and un-targeted metabolomic analysis. Quantitative analysis of the content of the predominant phenolic compound-rosmarinic acid-was performed using HPLC-DAD. The study of antioxidant activity with the indication of the main compounds responsible for this activity was performed by post-column derivatization with ABTS reagent. The study compared the antioxidant potential of the tested extracts from different morphological parts of S. hispanica. In addition, a comparative study of antibacterial and antifungal activity was also carried out using the microdilution broth method.

Metabolomic Profiling Using LC-Q-Orbitrap HRMS
The high-resolution, accurate mass via Orbitrap used in this study yielded the identification and preliminary characterization of one hundred fifteen compounds (Table 1). In Figure 1, the metabolite profiles as total ion chromatograms and heat maps with the signal intensity of individual analytes are reported. The largest class of compounds was terpenoids, with 31 compounds, followed by flavonoids (21 compounds), phenolic acids and derivatives (19 compounds), organic acids (16 compounds) and others such as fatty acids, sugars and unidentified compounds. The organic and phenolic acids were the most abundant classes in terms of total peak area with distribution depending on the type of part S. hispanica ( Figure 1A). This class was represented mainly by hydroxycinnamic acids and their derivatives. The main compound among this class for all types of extracts was rosmarinic acid (peak 49) with pseudo-molecular ions at m/z 359.0769 (C 21 H 17 O 11 − ) and fragmentation ions at m/z 197.0450 and m/z 161.0235 formed by cleavage of a caffeic acid and danshensu moieties. The deprotonated form of caffeic acid was also detected in compound 69, which was identified as salvianolic acid F ([M-H] m/z 313.07142). The extracts obtained from leaves, flowers and herbs were additionally characterized by a high content of ferulic acid (peak 54) and caffeic acid (peak 32) with [M-H] − ions at m/z 193.0496 and m/z 179.0336, respectively. In the case of ferulic acid, the fragmentation ion at m/z 134.0379 formed after the loss of carbon dioxide, and the methyl radical was observed. The caffeic acid precursor ion also generated characteristic major fragments at m/z 135.0441 due to the loss of carbon dioxide. Seed and sprout extracts, on the other hand, were characterized by a high content of salviaflaside (peak 39) with pseudo-molecular ions at m/z 521.1298 (C 24 H 25 O 13 − ). The parent ion of this rosmarinic acid glucoside produced fragment ions at m/z 359.0748 and m/z 161.0235, expected for rosmarinic acid and caffeic acid, respectively.
Another class of phytochemicals detected in S. hispanica extracts was flavonoids. Most of the identified compounds belonging to this class have been assigned to flavones. In almost all extracts, the content of flavone with peak number 72 was the highest. Another main group of phytochemicals present in S. hispanica extracts were terpenoids. The highest content of these compounds was found in flower extracts. The most abundant compound (peak 87) with quasimolecular ion at m/z 345.17046 (C 20 H 25 O 5 − ) has a unique fragmentation pattern with fragmentation ions at m/z 331.1508 and 315.1597 that have been previously observed for rosmadial and hydroxyrosmadial [46]. Therefore, this compound was assigned as a rosmadial derivative. In the extracts studied, especially those from sprouts, a high content of saccharides was also noted. Peaks 1 and 4 were tentatively identified as raffinose and sucrose, as they are often major transport sugars in salvia species [47]. In the case of extracts from leaves, flowers and herbs, the high content of compound 17 was also observed. Its precursor ion [M-H] − was found at m/z 271.08193, which indicates that its molecular formula is C 12

Antioxidant Profiling by Post-Column Derivatization with ABTS
Post-column derivatization of analytes with ABTS reagent was performed during HPLC analysis of extracts from different plant parts of S. hispanica. In the applied postcolumn derivatization, the principle action of the ABTS reagent is the same as in the case of spectrophotometric tests. The reduction reaction of the ABTS reagent leads to a significant shift in the visible UV spectrum, which results in a change in the absorption of the ABTS reagent (discoloration). Post-column introduction of the reagent into the on-line system and the presence of antioxidants in the eluate result in negative peaks in the chromatogram recorded at 734 nm ( Figure 2A). The profiles obtained after derivatization indicated that several compounds identified in S. hispanica extracts exhibit antioxidant activity. The greatest contribution to the overall antioxidant activity was made by rosmarinic acid (peak 49). Its activity covered 26 to 49% of the total antioxidant activity ( Figure 2B). In the case of seed extract, additionally, other derivatives of hydroxycinnamic acid, such as salviaflaside (peak 39) and dehydroxyl-rosmarinic acid-glucoside (45), showed antiradical potential visible as negative peaks on profile. In extracts from leaves, flowers and herbs, the presence of caffeic acid (peak 32) also caused the reduction of the ABTS radical. Other phytochemicals showing visible antioxidant activity were gluconic acid (peak 2), arbutin (peak 17), danshensu (peak 19), caftaric acid (peak 24) and chlorogenic acid (peak 27). The flower extract was characterized by a slightly different antioxidant profile compared to the rest of the extracts. In this case, additional antioxidants with a short retention time were noted (peaks 10, 12, 14). They were probably derivatives belonging to the group of pyrimidines and purines.  Table 1.
Recalculation of the area of negative peaks using the calibration curve of the standard antioxidant enabled the quantification of the total antioxidant activity of the extracts in an on-line system and its expression as Trolox equivalents ( Figure 2B). The highest antioxidant activity was found in flower extracts. Lower activity by about 40% and 50% was shown by extracts from leaves and herbs, as well as extracts from seeds and sprouts, respectively. Analysis of the average content of rosmarinic acid in extracts from seeds, sprouts, leaves, flowers and herb of S. hispanica showed that all analyzed morphological parts have a high content of rosmarinic acid. Among all the analyzed parts of S. hispanica, the average content of rosmarinic acid was the highest in the leaves (198.53 mg/100 g DW).
There was slightly less rosmarinic acid in the herb (185.12 mg/100 g DW). Rosmarinic acid was present in lower amounts in the flowers (149.45 mg/100 g DW), in the sprouts (134.27 mg/100 g DW) and in the least amount in the seeds (127.25 mg/100 g DW) ( Table 2).

Antibacterial and Antifungal Activities
The antibacterial and antifungal activities of the tested extracts are presented as the MICs, i.e., the lowest concentration of compound that inhibits visible growth of the microorganism and the MBCs, i.e., the lowest concentration that results in a ≥99.9% reduction of the microorganism inoculum upon subculture to a compound-free medium (Table 3). Vancomycin, ciprofloxacin and nystatin were used as the standard drugs. Tested extracts were more active against Gram-positive reference strains. Gram-negative bacteria tested showed 4-8 times higher MIC values in comparison to those for Gram-positive bacteria. The best bioactivity was indicated for leaves extract, which presented considerable bactericidal activity against Gram-positive bacteria (S. aureus, S. epidermidis, M. luteus and E. faecalis) counted by MBC/MIC index, which equals 1-4. A stronger inhibitory effect against Gram-negative reference strains was also presented by leaves extract. The leaves extract showed the best antimicrobial activity against two Gram-positive bacteria (S. epidermidis and S. aureus). More favorable antifungal activity of chia leaves was found for S. albicans (MIC = 5). The whole seeds extract exhibited greater activity against S. aureus, M. luteus and B. cereus compared to the ground seeds extract. However, ground seeds extract demonstrated greater activity against E. faecalis. The sprout extract showed the best effect and the lowest MIC against two Gram-positive bacteria (M. luteus and B. cereus). The results showed that the sprout extract exhibited the best antifungal activity against C. parapsilosis (MIC = 0.625.). The roots extract demonstrated the best antibacterial activity against M. luteus. Roots extract exhibited beneficial antifungal properties against C. parapsilosis and C. glabrata. The herb extract showed the least favorable antibacterial and antifungal activity. The antibacterial efficiency of tested extracts was in the order of leaves > sprouts > whole seeds > ground seeds > roots > herb. However, their activity was much lower compared to standard drugs routinely used in bacterial infection treatment. Not strong, but still better antifungal activity against Candida spp. reference strains were shown for sprout extract.

Discussion
The conducted research is the first comparative analysis providing phytochemical profiling and connected with its antioxidant potential as well as antimicrobial properties of different raw materials obtained from S. hispanica. Rosmarinic acid was identified as the major compound responsible for antioxidant activity. The comparison of the quantity of this compound in relation to the ABTS reagent was shown. Furthermore, except for the antioxidant potential, the antimicrobial and antifungal properties were profiled for the first time while studying different S. hispanica raw materials. The chemical characterization revealed the presence of various groups of compounds in the extract from seeds, sprouts, leaves, flowers, and herb, mainly terpenoids (31 compounds), flavonoids (21 compounds), phenolic acids and derivatives (19 compounds), organic acids (16 compounds) and others (fatty acids, sugars, unidentified compounds).
Chia seeds are the most recognized raw material obtained from S. hispanica, although they are not well phytochemically profiled. A few studies described the polyphenolic profile of chia seeds, but our results are more comprehensive. Rahman et al. [48] determined the polyphenolic profile and biological activity of chia methanolic seed extract. They identified only the total phenolic content using the out-of-date method of the Singleton and Rossi assay (1965). They indicated rosmarinic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, and quercetin as the major components using HPLC-DAD-MS/MS method. The results of Oliveira-Alves et al. [49] in identifying the main phenolic compounds in methanolic chia seed extracts by LC-DAD-ESI-MS/MS methods mostly correspond with compounds identified in our study, too. The researchers confirmed the presence of phenolic acids (protocatechuic acid, p-hydroxybenzoic acid, cis-p-coumaric, cis and trans-caffeic acids, hydroxycoumaric acid, cis-and trans-ferulic acids, ellagic acid, rosmarinic acid), flavonoids (quercetin, quercetin-hexoside, kaempferol-hexoside, myricetin, apigenin, daidzein, rutin, genistein), and procyanidins (procyanidin dimer B 1, 2 and 3, procyanidin dimer A). Our results were consistent with those of Martinez-Cruz et al. [23] on 70% methanol extracts with the UHPLC method in chia seeds. The researchers indicate the main compounds as rosmarinic acid, protocatechuic acid, caffeic acid, gallic acid and daidzein. The estimated amount of rosmarinic acid was 92.67 mg/100 g DW, 1.4 times lower than the amount obtained in our results for seeds. The antioxidant activity determined by the DPPH assay indicated the high antioxidant capacity (percentage of inhibition = 68.83%) of chia seeds, which was 2 times higher than estimated in our study. The presence of phenolic acids, especially rosmarinic acid, and other phenolic compounds from isoflavones and anthocyanins were supposed to be responsible for this activity. The results were also most similar to those of Abdel-Aty et al. [50], who determined polyphenols profile using HPLC analysis. They identified in chia seed extracts: phenolic acids (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, syringic acid, ferulic acid, synapic acid, rosmarinic acid, and cinammic acid) and flavonoids (quercetin, apigenin, chrysin). The researchers also proved that the most abundant phenolic acid identified in seeds extract was rosmarinic acid (0.320 mg/g DW). Dib et al. [51] quantified the groups of phenolic compounds in the hydromethanol extract of chia seeds. It was proved that chia seeds had a high content of total phenols (19.06 mg GAE/g DW), which were mainly represented by flavonoids (12.3 mg CE/g DW) and tannins (8.32 mg catechin equivalents (CE)/g DW). Moreover, the researchers assessed the antioxidant properties of the studied extract using DPPH and FRAP assay. The results indicated that the extract showed the highest DPPH scavenging potential with an IC50 of 0.27 mg/mL and FRAP assay with an EC50 of 0.06 mg/mL. In our study, qualitative and quantitative analysis of the main polyphenolic compounds present in chia seed extract demonstrated that the quantitatively dominant phenolic compound responsible for antioxidant activity was rosmarinic acid. The antioxidant activity of chia seeds has also been confirmed by other scientists, who have shown that chia seed extracts were able to scavenge DPPH radicals [52][53][54][55][56]. Tepe et al. [57] examined the antioxidant activity of an ethanolic extract of chia seeds and claimed that polyphenols present in chia seeds significantly inhibited oxygen free radicals. The same results were obtained by Craig et al. [58], who proved that the presence of polyphenols in chia seeds protects them from oxidative degradation. All performed studies confirm that rosmarinic acid was the main compound detected and quantified in chia seeds [23]. Similarly, the compound identified by our team-danshensu, is a simple polyphenol (3-(3,4-dihydroxyphenyl)lactic acid) corresponding to the hydrated form of caffeic acid [59]. This compound is also described in plants in the Lamiaceae family [60,61]. Our study compared the quantitative content of rosmarinic acid in S. hispanica seed extracts with other literature data. In addition to the present study, rosmarinic acid was also quantified in S. hispanica seed extracts at two other centers. In the present experiment, the content of rosmarinic acid in the seed extracts was 34.98 mg/100 g DW. which was almost 2 times less than the content determined by Pellegrini et al. [62] and 2.65 times less than the content estimated by Martinez-Cruz et al. [23]. Moreover, our study also examined the antioxidant properties of chia seed extracts and determined the percentage effect of rosmarinic acid content on this activity (which was indicated at 34.33%). We proved that other derivatives of hydroxycinnamic acids, such as salviaflaside and dehydroxyl-rosmarinic acid-glucoside, also exhibited antiradical visible potential.
Chia seeds are the main raw material obtained from S. hispanica, but according to current scientific studies, compared to seeds, chia sprouts could have better nutritional value and antioxidant capacity, making them a new promising plant raw material of potential medical and agri-food utilities [63,64]. Our studies showed the metabolite profile of chia sprout extracts for the first time. We identified new compounds that had never been identified by researchers before. In sprouts extract, we identified organic acids-gluconic acid, xylonic acid, threonic acid, tartaric acid, quinic acid, malic acid, citric acid, isocitric acid, homocitric acid, caftaric acid, salicylic acid, tuberonic acid hexoside, phenolic acids and their derivatives-dihydroxybenzoic acid hexoside, danshensu, neochlorogenic acid, caffeic acid, salviaflaside, rosmarinic acid, dehydroxyl-rosmarinic acid-glucoside, ferulic acid, salvianolic acid F, 4-hydroxybenzoic acid, methyl rosmarinate, feruoyl arabinose, rabdossin, caffeoyl glucose. Namely, sprout extracts were characterized by a high content of salviaflaside and jaceosidin. Moreover, compared to all extracts we analyzed, the extracts from sprouts contained high amounts of saccharides (mainly raffinose and sucrose). In our study, we proved that the main group of secondary metabolites of chia sprout extracts were derivatives of hydroxycinnamic acids. Organic acids and flavonoids occupy the next position. We have shown that the proportion (%) of rosmarinic acid in the antioxidant activity of sprout extracts determined using post-column derivatization of analytes with ABTS reagent was the highest compared to the other analyzed extracts from raw materials obtained from S. hispanica. The analysis showed that chia sprouts had the highest percentage content of rosmarinic acid, which contributed to antioxidant activity, at 49.5%. These results are innovative because there are no scientific studies analyzing the phytochemical profile and antioxidant properties of chia sprouts extract. Calvo-Lerma et al. [36] indicated that chia sprouts extract contains higher total polyphenol content than seeds (2.87 vs. 1.78 mg GA/g DW). Their determination of the total antioxidant activity using the DPPH assay showed higher results in sprouts in comparison to seed extracts (5.69 vs. 3.49 mg TX/g DW), which is consistent with the results obtained by our team. Abdel-Aty et al. [50] evaluated the effect of the germination process of S. hispanica seeds on total phenolic and flavonoid contents and antioxidant and antimicrobial properties. In the chia sprout methanolic (80%) extracts with the HPLC method, 12 phenolic acids (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, syringic acid, vanilic acid, ferulic acid, synapic acid, p-coumaric acid, rosmarinic acid, and cinammic acid) and 5 flavonoids (catechin, quercetin, apigenin, kaempferol, chrysin) were identified with concentrations ranging from 0.06 to 0.80 mg/g DW. Our results presented different data. We did not detect protocatechuic acid, vanilic acid, p-coumaric acid and cinammic acid in the sprout extracts. The researchers showed that the dominant phenolic compound found in chia sprout extracts was protocatechuic acid (0.50 mg/g DW), followed by rosmarinic acid (0.60 mg/g DW). In our study, we proved that rosmarinic acid was the abundant compound (134.27 mg/100 g DW) identified in sprout extracts.
Among phenolic acids, the most abundant in leaves extract was rosmarinic acid. These results are in line with those obtained by Amato et al. [40], who analyzed the methanolic extracts of chia leaf using the HPLC-ESI-MS method. In the study conducted by us in the chia plant material, we identified hydroxycinnamic acids and their derivatives, especially flavonoids, mainly flavones, such as apigenin, luteolin, orientin, vitexin, jaceosidin and phenolic acids with dominant amounts of rosmarinic, ferulic, isocitric, caffeic acid and their derivatives. These compounds have been commonly found in other members of the genus Salvia before [71,72]. Similar results were obtained by Zúñiga-López et al. [73], who identified the phenolic composition of chia leaves using the UHPLC-HRMS method. However, these researchers identified only 18 bioactive compounds in the chia leaf extracts: organic acids (dominant compounds: protocatechuic acid, p-coumaric acid, quinic acid, sinapic acid), phenolic acids derivatives (dominant compounds: chlorogenic acid, rosmarinic acid, caffeic acid, ferulic acid) flavonoids (dominant compounds: orientin, acetyl orientin, vitexin, coumaroyl, luteolin-O-glucuronide, kaempferol, genistein, naringenin, salvianolic acid F isomer, and dimethyl quercetin). Most of the detected compounds overlap with those identified in our study, but in our study, we identified 115 compounds (Table 1). There are very limited studies on the correlation between metabolomic analyses and the antioxidant properties of chia leaves. In our study, analysis of antioxidant activity determined by post-column derivatization with ABTS reagent of the chia leaf extracts showed that rosmarinic acid present in the leaf extracts was the most abundant compound responsible for this activity. Amato et al. [40], for potential antioxidant measurements of S. hispanica leaf extracts, used another three assays: oxygen radical absorbance capacity (ORAC), ORAC-Fluorescein (ORAC-FLORAC-FL index values) and 2,2 -diphenyl-1-picrylhydrazyl (DPPH). They proved that the methanolic extract of chia leaves exhibited higher antioxidant activity and indicated that rosmarinic acid was the most reactive compound, which is equivalent to the results obtained in our study. In our study, we broadened the results, and we pointed out that not only was rosmarinic acid responsible for antioxidant potential, but also other phytochemicals, such as caffeic acid, gluconic acid, arbutin, danshensu, caftaric acid and chlorogenic acid. Moreover, for the first time, we performed a recalculation of the area of negative peaks using the calibration curve of the antioxidant standard, which allowed us to quantify the total antioxidant activity of the leaf extracts in the on-line system. As a result, quantification of the total antioxidant activity of the extract expressed as Trolox equivalents and the percentage of the total antioxidant activity of the rosmarinic acids and other antioxidants present in the extracts showed that the antioxidant activity for the leaf extract was about 40-50%.
The study conducted by our team for the first time assessed the main metabolites and evaluated the antioxidant activity of S. hispanica flower extracts. The main group of secondary metabolites found in the flower extracts were organic acids (dominant compounds: gluconic acid, tartaric acid, citric acid, isocitric acid), flavonoids (dominant compounds: apigenin, jaceosidin), hydroxycinnamic acids and their derivatives (dominant compounds: caffeic acid, rosmarinic acid, ferulic acid, salvianolic acid F), and terpenoids (dominant compounds: hydroxyrosmadial, carnosic acid isomer, rosmadial derivative, rosmadial isomer, rosmanol, carnosol isomer). We showed that the main compound identified in flower extracts was rosmarinic acid. The maximum content of rosmarinic acid was determined in the flower extracts and was equal to 369.09 mg/100 g DW. Comparative analysis of the percentage contribution of rosmarinic acid to the antioxidant activity of all tested extracts from S. hispanica raw materials showed that the flower extracts contribute the least to antioxidant activity-26.3%. In our study, flower extracts demonstrated a slightly different antioxidant profile compared to the rest of the analyzed extracts. Probably the presence of derivatives belonging to the group of pyrimidines and purines was responsible for this activity.
Dziadek et al. [42] also determined the antioxidant power of S. hispanica herb extracts at 716.26 µmol TX/g DW. In our study, we demonstrated that rosmarinic acid contributes to this activity. The percentage contribution of rosmarinic acid to the antioxidant activity of herb extracts was 47.66% (185.12 mg/100 g DW). Abou Zeid et al. [74] studied the aerial parts of S. hispanica by the UPLC-ESI-MS/MS technique. They identified significantly fewer compounds than in our study (37 compounds) from phenolic acids, flavonoids, tannins, diterpenoids, lignans and triterpenoids. The individual compounds: caffeic acid, rosmarinic acid, ferulic acid, orientin, vitexin, danshensu, carnosol, jaceosidin, syringetin and luteolin, are similar to these confirmed in the present study. Furthermore, Abou Zeid et al. performed the analysis of antioxidant properties by the DDPH method and demonstrated the significant potential of the ethyl acetate extracts of S. hispanica aerial parts (herb).
There is limited research on the antibacterial and antifungal actives of S. hispanica seeds, sprouts, leaves, roots and herb extract. Our study showed that chia leaf extracts exhibited the highest antibacterial and antifungal activity compared to the other tested extracts. Chia leaf extracts showed stronger antibacterial activity against two Gram-positive bacteria (S. epidermidis and S. aureus). More favorable antifungal activity of chia leaves was found for S. albicans. The sprout extracts demonstrated the best effect and lowest MIC against two Gram-positive bacteria (M. luteus and B. cereus). The results proved that the sprout extracts showed the best antifungal activity against C. parapsilosis. The herb extracts showed the least favorable antibacterial and antifungal activity. The antibacterial efficacy of the tested extracts was in the order of leaves > sprouts > herb. However, their activity was significantly lower compared to that of standard drugs routinely used to treat bacterial infections. Abdel-Aty et al. [50] examined the antimicrobial activity of chia sprouts raw chia seed extracts against Gram-negative bacteria (E. coli O157-H7 ATCC 51,659, Salmonella typhi ATCC 15,566 and Pseudomonas aeruginosa NRRL B-272) and one Gram-positive bacterium (Staphylococcus aureus ATCC 13,565). The range of MIC for the chia sprout extracts was lower (0.40-0.65 mg/mL) in comparison to the dry chia seed extracts. In our study, the activity of the chia sprout extracts was a bit less (the MIC range value 0.625-10 mg/mL). The antimicrobial activity of chia protein hydrolysates obtained from seeds was studied by Coelho et al. [75]. The protein hydrolysates exhibited favorable inhibitory activity against S. aureus to a greater extent compared to E. coli, which is in line with our results confirming the more favorable antimicrobial activity of all analyzed extracts against S. aureus compared to E. coli. Güzel et al. [76] investigated the antibacterial and antifungal activity of ethanol extract of chia seeds against reference strains of S. aureus, B. subtilis, E. coli, A. baumannii, A. hydrophila, C. albicans, C. tropicalis and C. glabrata showing their higher activity compared to those presented in this study. According to the recent literature, the composition and content of key bioactive compounds in chia seeds can vary depending on external factors such as geographic origin, climatic conditions, agricultural practices, extraction procedures and antimicrobial activity procedures [9,77]. These factors may affect the efficacy of the extract under study and may result in different outcomes compared to other studies. In addition, Güzel et al. demonstrated that chia seed extract exhibited the highest antifungal activity against C. glabrata, but the result was not as high as with fluconazole (MIC values: 31.25 µg/mL and 3.90 µg/mL, respectively). In our study, we found significantly higher antifungal activity of the whole seed extract compared to the ground seed extract. The antimicrobial effect of chia seeds is likely due to their rich chemical composition. Chia seeds are a source of fatty acids, accounting for about 30%, which include linoleic acid (17-26%) and linolenic acid (50-57%). Chia seeds are also a source of vitamins, macronutrients and micronutrients [9,78]. The presence of numerous antioxidants in chia seeds, such as omega-3 fatty acids, may determine their antimicrobial properties. Chia seeds contain kaempferol and quercetin, which have scientifically proven antibacterial properties. It can be inferred that kaempferol binds to an enzyme in bacterial cells and blocks a process essential for bacterial function. Chia seeds also contain caffeic acid and p-coumaric acid, which have proven antimicrobial activity [79,80]. Moreover, adding chia seeds to food products can increase their microbiological stability and prevent contamination without additional preservatives. Numerous human pathogens have been scientifically proven to be experimentally sensitive to the inhibitory effects of phenolic acids, flavonoids, tannins and anthocyanins, especially against several specific strains of Gram-positive bacteria (S. aureus, L. monocytogenes, M. luteus, E. faecalis, C. botulinum and B. subtilis) and Gram-negative bacteria (E. coli, S. typhimurium, S. enterica, P. mirabilis, Y. enterocolityca, S. dysenteriae, S. flexneri, P. fluorescens, P. aeruginosa and V. cholerae) and the fungal pathogen C. albicans [69,76,79,[81][82][83][84][85][86].
In our study, we examined for the first time the antibacterial and antifungal properties of the root extract of the S. hispanica plant. The roots extract demonstrated beneficial antifungal properties against C. parapsilosis and C. glabrata.

Plant Material
The seeds from which the plant material for analysis was obtained were from Guatemala,

Preparation of Extracts
For chromatographic analysis, the powdered lyophilizates (50 mg) were extracted with a methanol solution (70%, 0.5 mL). The extraction was assisted by ultrasound (15 min). The extracts were centrifuged (13,000 rpm, 15 min), and the supernatants were collected. The extraction step was repeated for the solid residue with another portion of methanol (70%, 0.5 mL). The combined supernatants (~1 mL) were subjected to chromatographic analysis.
To prepare the extracts used for the analysis of antibacterial and antifungal properties, samples of dry powdered plant tissue were weighed at 4 g DW each. The material was extracted with methanol in a volume of 100 mL. Extraction was carried out in an ultrasonic bath model 3 times for 20 min each. The extracts were filtered through tissue paper strainers. The material was extracted using blotting paper, which was poured into crystallizers after draining. The material was left to evaporate for 3 days. After 3 days, the material was eluted with methanol, and the weighed extract was placed in 7 mL (16 × 66 mm) polypropylene tubes from Rymed Company (Dabrowa Gornicza, Poland).

Untargeted Metabolomics by LC-Q-Orbitrap HRMS
The Salvia hispanica hydromethanolic extracts were investigated using a Dionex Ultimate 3000 UHPLC system (Thermo ScientificTM, Dionex, San Jose, CA, USA). Chromatography separations were performed using SynergiTM Hydro-RP A (150 × 4.5 mm, 4 µm, Phenomenex) column. Mobile phases A (water) and B (acetonitrile), both acidified with formic acid (0.1% v/v), were pumped at a flow rate of 0.8 mL/min 1 , according to the following gradient pattern: 0 min, 5% B; 20 min, 50% B; 25 min, 100% B; 27 min, 100% B and finally, the initial conditions were held for 8 min as a re-equilibration step. The injection volume was 4 µL. The chromatographic unit was coupled to a Q ExactiveTM Focus quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI II). The HESI parameters in negative polarity included: sheath gas flow rate, 35 arb; auxiliary gas flow rate, 15 arb; sweep gas flow rate, 3 arb; spray voltage, 2.5 kV; capillary temperature, 350 • C; S-lens RF level, 50; heater temperature, 300 • C. Full scan data in the negative mode was acquired at a resolving power of 70,000 FWHM; AGC target, 1e6; max IT, auto. A scan range of m/z 100-1200 was chosen for the compounds of interest. The parameters of data-dependent MS2 were as follows: resolution, 17,500; isolation window, 3.0 m/z; normalized collision energy, 30; AGC target, 1e6; max IT, auto. Mass calibration was performed once a week, in both positive and negative modes, using mixture containing n-butylamine, caffeine, Met-Arg-Phe-Ala (MRFA) and Ultramark 1621.
Raw data from high-resolution mass spectrometry were elaborated with Compound Discoverer (v. 2.1, Thermo, Waltham, MA, USA). Major metabolite identification was based on accurate mass and mass fragmentation pattern spectra against MS-MS spectra of compounds available on customized database of different classes of phytochemicals created on the basis of literature data on the Salvia species and implemented in the software. Raw data from three experimental replicates and a blank sample were processed using a workflow presented in Kusznierewicz et al. [87].

Antioxidant Profiling by Post-Colum Derivatization with ABTS
Antioxidant profiles were obtained for S. hispanica hydromethanolic extracts using an HPLC-DAD system (Agilent Technologies, 1200 series, Waldbronn, Germany) coupled with a Pinnacle PCX Derivatization Instrument (Pickering Laboratories Inc.) and a UV-Vis detector (Agilent Technologies). The chromatographic column and conditions of chromatographic separation were the same as in the case of LC-HRMS analysis. The post-column derivatization with ABTS reagent was carried out according to Kusznierewicz et al. [88,89] with slight modification. Stream of methanolic ABTS solution (1 mM) was introduced to the eluate stream at a rate of 0.1 mL/min and then directed to the reaction loop (1 mL, 130 • C). Reduction of ABTS radical by extract components was monitored at 734 nm. The antioxidant activity of the major reducing analytes was quantified with the use of Trolox calibration curve and expressed as Trolox equivalents. The percentage contribution of the rosmarinic acid to the antioxidant activity of extracts was estimated on the assumption that 100% is the sum of the negative peak areas integrated into chromatograms obtained after derivatization with ABTS.

Rosmarinic Acid Determination
For quantitative determination of rosmarinic acid, the calibration curve was generated by integrating the areas of absorption peaks (330 nm) determined during HPLC-DAD analysis of serial dilutions of authentic standard. The chromatographic system, column and conditions of separation were the same as in the case of antioxidant profiling (Section 4.5).

Antibacterial and Antifungal Activities
All extracts were screened for antibacterial and antifungal activities by two-fold microdilution broth method. Minimal inhibitory concentration (MIC) of tested compounds were evaluated for the panel of reference Gram-positive bacteria: Staphylococcus aureus ATCC 25923, S. aureus ATCC BAA-1707, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240, Bacillus cereus ATCC 10876 and Gram-negative bacteria: Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027 and yeasts: Candida albicans ATCC 102231, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030. The procedure for conducting antimicrobial activity testing has been described in detail before [90]. Briefly, the solutions of tested compounds dissolved in dimethylosulfoxide (DMSO) were suspended in Mueller-Hinton broth for bacteria or Mueller-Hinton broth with 2% glucose for fungi. Then the series of two-fold dilutions were carried out in the sterile 96-well polystyrene microtitrate plates (Nunc, Denmark), obtaining concentration range from 10 to 0.078 mg/mL in the appropriate medium. Simultaneously, the inocula of 24 h cultures of microorganisms in sterile physiological saline (0.5 McFarland standard density) were prepared and added to each well, obtaining final density of 5 × 10 5 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts; CFU-colony forming units. Proper positive (inoculum without tested compound) and negative (compound without inoculum) controls were added to each microplate. Vancomycin, ciprofloxacin and nystatin were used as the standard reference reagents. After incubation (35 • C, 24 h), the growth of microorganisms was measured spectrophotometrically at 600 nm (BioTEK ELx808, BioTek Instruments, Inc, Winooski, VT, USA). MICs were marked at the lowest dilution of extract without the growth of bacteria or fungi. Then, 5 µL of the suspension from each well, including controls, was subcultured on the agar plates in order to determine the minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC). The plates were incubated at 35 • C for 24 h. The MBC/MFC was determined at the lowest concentration of extracts inhibiting the growth of microbes. MBC/MIC index was also calculated to show bacteriostatic or bactericidal effect of tested extracts.

Conclusions
In the current literature, there are no scientific studies on the comparative analysis of different plant raw materials obtained from S. hispanica. In addition, studies on the antioxidant, antimicrobial and antifungal activities of extracts from different chia raw materials are severely limited. Our work is innovative because it conducts an in-depth characterization and analysis comparing the antioxidant, antimicrobial and antifungal properties of all morphological parts of S. hispanica. In this study, for the first time, the phytochemical profiling and comparative analysis of various morphological parts/organs of S. hispanica extracts was conducted. We showed that in S. hispanica raw materials, the largest class of compounds were terpenoids, followed by flavonoids, phenolic acids and derivatives, organic acids, and other compounds, such as fatty acids and sugars. Conducted analyses proved that organic and phenolic acids were the most abundant class of phytochemicals identified in studied extracts. Rosmarinic acid, belonging to the hydroxycinnamic acids group, was the quantitatively dominant compound found in all tested extracts.
The greatest contribution to overall antioxidant activity was made by this compound. Rosmarinic acid contribution to total antioxidant activity was the highest for sprout, herb and leaf extracts (49.5, 47.7 and 47.1%, respectively) and the lowest for seed and flower extracts (34.3 and 26.3%, respectively). The contribution to the antioxidant activity of sprout, herb and leaf extracts was 144, 139 and 137 times stronger compared to seeds extract.
The results of the antibacterial and antifungal activities of the tested extracts proved their higher activity against Gram-positive than Gram-negative reference strains. In Gramnegative bacteria, the tested extracts showed 4-8 times higher MIC values compared to those for Gram-positive bacteria. The antibacterial efficiency of tested extracts was in the order of leaves > sprouts > whole seeds > ground seeds > roots > herb. However, compared to the leaf and herb extracts, the sprout extracts showed better antifungal activity against Candida spp. reference strains.
In conclusion, the results obtained in our study indicate that not only the seeds but also other morphological parts of S. hispanica may be a potential source of novel raw materials containing compounds with strong antioxidant, antimicrobial and antifungal potential.