The Changes in Bioactive Compounds and Antioxidant Activity of Chia (Salvia hispanica L.) Herb under Storage and Different Drying Conditions: A Comparison with Other Species of Sage

Studies on herb chia (Salvia hispanica L.) are very limited. Therefore, the aim of this study was to assess how different drying methods and periods of storage affect the bioactive properties of the herb Salvia hispanica and to compare it with other species of sage (Salvia officinalis L. and Salvia sclarea L.). In fresh herbs, directly after drying (freeze-drying, natural drying, and drying at 30, 40, and 50 °C), and after storage (3, 6, and 12 months), the following analyses were performed: content of total carotenoids and total polyphenols, polyphenol profile (including 25 compounds), and antioxidant activity. Additionally, the basic chemical compositions of the herbs were analyzed. To the best of our knowledge, the content of total carotenoids and the quantitative polyphenol profile in Salvia hispanica and Salvia sclarea were evaluated for the first time. The obtained results showed that the barely investigated herb Salvia hispanica is rich in polyphenolic compounds and shows high antioxidant activity. In all the tested species, rosmarinic acid was the most abundant polyphenolic compound. The use of different drying methods allowed us to determine that freeze-drying was the most effective for preserving polyphenols and carotenoids. Long-term storage up to 12 months resulted in a gradual reduction in antioxidant activity and in the content of polyphenols and carotenoids.


Introduction
Herbs have been used for centuries as natural remedies to fight some diseases. With the development of synthetic drugs in the early 20th century, the use of herbs in medicine declined significantly. However, in recent years, there has been growing interest in folk medicine involving medical plants and herbs [1][2][3].
One group of beneficial medicinal plants is the genus Salvia, which belongs to the Lamiaceae family and includes approximately 900 species [4]. It is cultivated throughout the world for use in food industries as spices (to flavor meats such as pork, sausage, and poultry) as well as in cosmetics [5,6], newly formulated biomaterials [7][8][9], and active food packaging [10]. Studies have indicated that several species of sage (including Salvia officinalis L. and Salvia sclarea L.) show biological activities such as antioxidant, antibacterial, antifungal, anticancer, antimutagenic, anti-inflammatory, and antidepressant 930.05) were determined according to the AOAC [30] methods. The content of digestible carbohydrates was calculated using the following equation [31]: digestible carbohydrates = 100 − (protein + crude f at + ash + dietary f iber)

Determination of Total Carotenoids
The content of total carotenoids was determined by extracting carotenoids from the samples using an acetone-hexane mixture (4:6 v/v) (Chempur, PiekaryŚląskie, Poland), according to the Polish standard with some modifications [32]. Samples containing 1 g of fresh or 0.2 g of dried sage herbs were weighed into a porcelain mortar. Thereafter, approximately 0.3 g of sand was used to extract the dye using an acetone-hexane mixture. The extracts were poured into a test cylinder. Then, the extract volume was read. The extracts were shaken and left in a dark place for 30 min. The absorbance was measured at 450 nm using a spectrophotometer (UV-1800, Rayleigh, Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China). Results were calculated based on the β-carotene (Sigma-Aldrich, St. Louis, MO, USA) calibration curve.

Preparation of the Extracts
The content of total polyphenols, the polyphenol profile, and the antioxidant activity were determined in methanolic extracts. To prepare the extracts, 0.3 g of fresh or 0.2 g of dried herbs, were used, with 60 mL of 0.1% formic acid in 70% methanol (v/v) (POCH, Gliwice, Poland). The extracts were prepared by shaking for 2 h at room temperature (water bath shaker type 357, Elpan, Lubawa, Poland). Then the samples were filtered using filter paper. The filtrates were stored at a temperature of −20 • C for further analysis.

Determination of Total Polyphenols
The level of total polyphenols was estimated using Folin-Ciocalteu reagent (Sigma-Aldrich, Saint-Louis, MO, USA), as previously described [33]. Results were expressed as mg of gallic acid (GA) per 100 g DW (dry weight) of the sample.

Determination of Polyphenol Profile
High-performance liquid chromatography (HPLC) was used to evaluate the polyphenol profile. The analysis was conducted using a Prominence-i LC-2030C 3D Plus system (Shimadzu, Kyoto, Japan) equipped with a diode array detector (DAD). The separation was performed on a Luna Omega 5 µm Polar C18, 100 A, 250 × 10 mm column (Phenomenex, CA, USA) at 40 • C. The mobile phase was a mixture of two eluents: A-0.1% formic acid in water (v/v) and B-0.1% formic acid in methanol (v/v). The flow rate of the mobile phase was 1.2 mL min −1 . The analysis was carried out with the following gradient conditions: from 20% to 40% B in 10 min, 40% B for 10 min, from 40% to 50% B in 10 min, from 50% to 60% B in 5 min, 60% B for 5 min, from 60 to 70% B in 5 min, from 70% to 90% B in 5 min, 90% B for 5 min, from 90% to 20% B (the initial condition) in 1 min, and 20% B for 4 min, resulting in a total run time of 60 min. The injection volume was 20 µL.
The quantification of individual polyphenols in methanolic extracts was performed by establishing calibration curves using the standards. The calibration curves for the standards were linear with R 2 > 0.995.

Determination of Antioxidant Activity
The antioxidant activity was determined using the ABTS method (2,2 -azino-bis(3ethylbenzthiazoline-6-sulfonic acid)), as previously reported [34]. The obtained results were expressed as micromoles of Trolox equivalent per gram (DW) of the sample.

Statistical Analysis
Statistical analysis of the obtained data was performed using the program Statistica version 13.1, Dell Inc., Tulsa, OK, USA, 2016. The results were analyzed using one-way analysis of variance (ANOVA). Significant differences between mean values (n = 3) were compared using Duncan's test.

Basic Chemical Composition
Salvia sclarea was characterized by significantly the highest level of ash (18.39 g·100 g −1 DW) and protein (14.49 g·100 g −1 DW) ( Table 1). The highest amount of crude fat was determined in Salvia officinalis (4.06 g·100 g −1 DW), while the lowest was found in Salvia hispanica (1.67 g·100 g −1 DW) herb. The highest content of dietary fiber (52.98 g·100 g −1 DW) and, at the same time, the lowest level of digestible carbohydrates (24.82 g·100 g −1 DW) were measured in Salvia officinalis, in comparison to the other species.

The Content of Total Carotenoids
Among the fresh herbs, significantly the highest content of total carotenoids was found in Salvia sclarea (109.06 mg·100 g −1 DW) ( Table 2). The drying methods and storage significantly affected the content of these compounds ( Table 3). The freeze-dried herbs showed the highest content of carotenoids (71.04 mg·100 g −1 DW in Salvia sclarea, 64.87 mg·100 g −1 DW in Salvia hispanica, and 57.98 mg·100 g −1 DW in Salvia officinalis), indicating that this method caused the smallest loss in comparison to other drying methods. On the other hand, samples dried at 30 • C were characterized by the lowest level of these compounds (42.24 mg·100 g −1 DW in Salvia sclarea, 27.93 mg·100 g −1 DW in Salvia hispanica, and 23.60 mg·100 g −1 DW in Salvia officinalis), resulting from the highest losses. Storage for 3, 6, and 12 months caused an increasing loss in carotenoid content in all tested species of sage. The highest loss was observed after 12 months of storage. The loss of carotenoids in the studied samples during storage seemed not to depend on the drying method.

The Content of Total Polyphenols
In the fresh herbs, Salvia hispanica and Salvia officinalis were characterized by a significantly higher content of total polyphenols (9.76 g·100 g −1 DW and 9.47 g·100 g −1 DW, respectively) in comparison to Salvia sclarea (4.95 g·100 g −1 DW) ( Table 2). The drying method affected the level of these compounds significantly (Table 4). In the herbs Salvia hispanica and Salvia officinalis , an increase in the content of polyphenols was found in the samples after freeze-drying and natural drying. However, the highest content was observed after freeze-drying (12.89 g·100 g −1 DW in Salvia officinalis and 11.84 g·100 g −1 DW in Salvia hispanica). In these herbs, a loss in polyphenolic compounds was found in samples after drying at 30 • C, 40 • C, and 50 • C. The lowest level of these compounds was determined in these herbs after drying at 50 • C (6.11 g·100 g −1 DW in Salvia officinalis and 5.49 g·100 g −1 DW in Salvia hispanica). However, for the Salvia sclarea samples, all drying methods caused an increase in the content of polyphenols. The greatest changes were found after natural drying (8.52 g·100 g −1 DW). Storage resulted in a decrease in levels of polyphenolic compounds in all the tested samples. The lowest levels of these compounds, caused by the highest loss, were found after 12 months of storage.
In the fresh herb of Salvia hispanica, 4-hydroxybenzoic acid, ferulic acid, syringic acid, acacetin, catechin, epicatechin, hispidulin, kaempferol, and quercetin were not identified. Rosmarinic acid (1358.80 mg·100 g −1 DW), sinapinic acid (196.71 mg·100 g −1 DW), naringin (199.07 mg·100 g −1 DW), and rutin (197.59 mg·100 g −1 DW) were the dominant polyphenols in this species. In the fresh herb of Salvia officinalis, apigenin, catechin, and hispidulin were not detected. However, the highest contents of rosmarinic acid (1488.28 mg·100 g −1 DW), hesperidin (346.43 mg·100 g −1 DW), naringin (523.85 mg·100 g −1 DW), and rutin (317.77 mg·100 g −1 DW) were measured. In the fresh herb of Salvia sclarea, p-coumaric acid, acacetin, catechin, hispidulin, kaempferol, myricetin, and quercetin were not found. The following polyphenolic compounds were dominant in this species of sage: rosmarinic acid (346.32 mg·100 g −1 DW), rutin (127.41 mg·100 g −1 DW), and carnosol (296.72 mg·100 g −1 DW). The results indicated that the method of drying significantly affected the level of individual polyphenolic compounds. Furthermore, differences in the polyphenol profiles before and after drying were found. In the herb of Salvia hispanica after drying, 4-hydroxybenzoic acid, ferulic acid, syringic acid, epicatechin, hispidulin, kaempferol, and quercetin were additionally determined. On the other hand, gallic acid was detected only in the fresh sample, after freeze-drying, and after natural drying. In samples of Salvia officinalis after drying, apigenin (with the exception of the sample after natural drying), catechin, and hispidulin were additionally identified. However, in the dried herb of Salvia sclarea, pcoumaric acid, acacetin, kaempferol, myricetin, and quercetin were detected (the exceptions were p-coumaric acid, kaempferol, and quercetin in the sample after natural drying). In all samples, storage resulted in a decrease in the content of particular polyphenolic compounds. However, the rates of loss during storage were different for individual compounds. Results are expressed as mean ± SD (n = 3). DW-dry weight; nd-not determined. Mean values with different letters (a-k) within the individual rows are statistically different (p < 0.05).

Antioxidant Activity
The results indicated that the fresh herbs Salvia hispanica and Salvia officinalis were characterized by higher antioxidant activity (713.26 µmol Trolox·g −1 DW and 651.48 µmol Trolox·g −1 DW, respectively) than Salvia sclarea (568.49 µmol Trolox·g −1 DW) ( Table 2). In the herbs Salvia hispanica and Salvia officinalis after freeze-drying, natural drying, and drying at 30 • C and 40 • C, an increase in the ability to scavenge free radicals (highest in the samples after freeze-drying and natural drying) was observed (Table 8). In these groups of samples, drying at 50 • C caused a decrease in the antioxidant activity. In the herb Salvia sclarea the highest increase in antioxidant capacity was found in the sample after natural drying, and the lowest was measured after drying at 50 • C. After 3, 6, and 12 months of storage, a decrease in the ability to scavenge free radicals was observed.

Discussion
As there are only very limited studies on the herb Salvia hispanica, the basic chemical composition, bioactive compounds, and antioxidant activity were analyzed and compared to other known species of sage (Salvia officinalis and Salvia sclarea). As drying is one of the most commonly used methods for herb preservation and shelf-life extension, the effects of different drying methods and periods of storage on the bioactive properties of the analyzed species of sage were evaluated.
In the tested samples, dietary fiber was the most abundant ingredient and fat was the least abundant. In the available literature, there are limited data regarding the basic chemical composition of sage. Peiretti and Gai [29], who studied Salvia hispanica, reported a lower content of ash (7.7 g·100 g −1 DW) and protein (5.7 g·100 g −1 DW) compared to our results. Other results indicated that different herbs from Lamiaceae family were also characterized by a lower content of ash, protein, carbohydrates, and dietary fiber [35][36][37].
The content of total carotenoids in fresh Salvia officinalis was 63.48 mg·100 g −1 DW. Daly et al. [38], Cvitković et al. [39], and Murkovic et al. [40], who studied commonly consumed herbs, reported a lower level of total carotenoids in sage. However, Martins et al. [41] showed higher values for Salvia officinalis leaves, compared to our results ob-tained for the whole herb (leaves and stems). This suggests that carotenoids are mainly accumulated in the leaves of the herb. This was confirmed also on the basis of research on different herbs [42]. To the best of our knowledge, the content of total carotenoids in Salvia hispanica and Salvia sclarea herb has not previously been determined.
Carotenoids are thermally and oxidatively labile compounds. They contain a chromophore group, which consists of seven or more conjugated double bonds. This structure allows the compounds to absorb visible light, giving them the appropriate color. This system is also responsible for the high thermal and oxidative sensitivity [43]. The obtained results showed that all the drying methods used caused a decrease in the carotenoids content in sage. The lowest loss was found after freeze-drying. During freeze-drying, a low temperature is used, and contact with oxygen is limited, which results in reduced degradation of these compounds [44]. On the other hand, the greatest losses of carotenoids were measured in samples after drying at 30 • C. This could be due to easier oxidation of these components at higher temperatures in comparison to freeze-drying, as well as longer contact with oxygen compared to drying at higher temperatures (40 • C and 50 • C) [44]. Furthermore, thermal processes can damage some cellular structures, thereby releasing oxidative enzymes that decompose some antioxidant compounds such as carotenoids [45]. Generally, consumers store herbs at room temperature, and therefore the studied samples were stored under these conditions. A low temperature preserves quality, including the content of bioactive components [46]. On the other hand, storage at room temperature may cause loss of these compounds, as observed in our study. It is difficult to indicate the drying method that allows for the preservation of carotenoids during storage to the highest extent.
The content of total polyphenols in Salvia hispanica was 9.76 g·100 g −1 DW. To the best of our knowledge, there are no previous data on content of total polyphenolic compounds in the herb Salvia hispanica. Our results indicated that the herb Salvia officinalis contained 9.47 g·100 g −1 DW of total polyphenols. However, Jeshvaghani et al. [47] showed that Salvia officinalis leaves had 8.64 mg·100 g −1 DW of polyphenolic compounds, while Roby et al. [48] reported a value of 0.595 g·100 g −1 DW. Dent et al. [49], who studied ethanol and water extracts of sage from Croatia, also obtained lower values. The herb Salvia officinalis from Bulgaria, studied by Atanassova et al. [50], had 27.94 mg·100 g −1 DW of polyphenolic compounds. The level of total polyphenols in Salvia sclarea was 4.95 g·100 g −1 DW. Jeshvaghani et al. [47] obtained a higher value of 10.33 g·100 g −1 DW, while Taârit et al. [16] reported a lower level of polyphenols of 2.44 g·100 g −1 DW. Our results showed that the herb Salvia hispanica contained polyphenols at the same level as the herb Salvia officinalis. This suggests that the unknown Salvia hispanica herb, like the well-known Salvia officinalis, may have similar beneficial properties.
In all the tested species of sage, rosmarinic acid was the most abundant polyphenolic compound. The results of other studies also indicated that rosmarinic acid was the dominant polyphenolic compound in Salvia officinalis. Dent et al. [51] studied Salvia officinalis leaves from four wild field sites in the Mediterranean region of Croatia. Farhat et al. [52] analyzed Salvia officinalis from two different regions in north Tunisia at the vegetative, flowering, and fruiting stages. Hamrouni-Sellami et al. [53] evaluated Salvia officinalis from southern Tunisia, while Dent et al. [49] tested Salvia officinalis from the island of Pag (Croatia). These authors also detected phenolic acids such as p-hydroxybenzoic acid, caffeic acid, ferulic acid, gallic acid, p-coumaric acid, syringic acid, and vanillic acid, as well as flavonoids, mainly luteolin and apigenin (and their derivatives), hispidulin, and naringin. Additionally, Farhat et al. [52] and Hamrouni-Sellami et al. [53] identified the phenolic diterpenes carnosic acid and carnosol. Amato et al. [27], who for the first time showed a qualitative profile of the polyphenols in Salvia hispanica leaves, also detected compounds such as caffeic acid, chlorogenic acid, ferulic acid, rosmarinic acid, apigenin, luteolin, kaempferol, quercetin, and others. To the best of our knowledge, the polyphenol profile of the Salvia sclarea herb and the quantitative analysis of polyphenolic compounds in Salvia hispanica have not yet been evaluated.
The obtained results indicated that Salvia hispanica and Salvia officinalis, after freezedrying, were characterized by the highest content of total polyphenols. Similar observations for Salvia officinalis were made by Sadowska et al. [31]. It is known that the low temperature of this process can protect polyphenolic compounds. Additionally, limited oxygen access can minimize the oxidation of these compounds [54]. Due to modification of the microstructure of freeze-dried plant tissues, the dried product is more porous, and thus the extraction process is more effective [55]. Furthermore, this process can inhibit the activity of some enzymes such as polyphenol oxidases, resulting in the protection of polyphenolic compounds [56]. Different polyphenol profiles in dried herbs, in comparison to fresh ones, can be due to the release of compounds after thermal treatment. The elevated temperature can influence the cell wall structure, promoting the release of internal compounds from the plant cells [57]. The greatest loss of polyphenols was found in samples of sage after drying at 50 • C, caused by the use of the highest temperature. Moreover, during natural drying (in comparison to freeze-drying) the degradation of polyphenol oxidases was not immediate; therefore, oxidation of these compounds may occur during the process [56]. The obtained results do not allow an indication of the drying method that protects polyphenols during storage to the highest extent.
Our research indicated that Salvia hispanica and Salvia officinalis were characterized by higher antioxidant activity compared to Salvia sclarea. These results were in agreement with the data reported by Jeshvaghani et al. [47], who studied various species of sage. However, Stagos et al. [6] indicated that the herb Salvia sclarea had the highest antioxidant activity among all the tested sage samples. The results of our study showed that the herb Salvia hispanica was characterized by a fairly high antioxidant activity.

Conclusions
The obtained results showed that the herb of a barely investigated species of sage, Salvia hispanica, was rich in polyphenolic compounds and showed high antioxidant activity. Both the polyphenol content and the antioxidant capacity were on the same level as those determined in the well-known Salvia officinalis, and were significantly higher compared to those found in Salvia sclarea. In all tested species of sage, rosmarinic acid was the most abundant polyphenolic compound. Furthermore, Salvia hispanica was rich in sinapinic acid, naringin, rutin, and carnosol. The use of different drying methods allowed us to indicate freeze-drying as the most effective for preserving polyphenols and carotenoids in the studied species of sage. Long-term storage up to 12 months resulted in a gradual reduction in antioxidant activity as well as in the content of polyphenols and carotenoids. However, even after such a long period of storage the herbs of all tested species of sage showed strong antioxidant activity and a high content of bioactive compounds. To conclude, the herb Salvia hispanica showed a high potential to be used as a rich source of bioactive compounds, including antioxidants, in the food industry, cosmetics, biomaterials, and active food packaging, similarly to the well-known Salvia officinalis.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.