An In-Depth Study of Metabolite Profile and Biological Potential of Tanacetum balsamita L. (Costmary)

Asteraceae species Tanacetum balsamita L. (costmary) is renowned for its traditional usage as an aromatic, carminative and tonic plant. This work aimed at in-depth study of the phytochemical and in vitro biological profilings of methanol–aqueous extracts from the costmary leaves, flower heads and roots. An UHPLC-HRMS analysis revealed more than 100 secondary metabolites including 24 acylquinic acids, 43 flavonoid glycosides, aglycones and methoxylated derivatives together with 15 phenolic acids glycosides. For the first time, 91 compounds are reported in the costmary. The flower heads extract possessing the highest content of total phenolics and flavonoids, actively scavenged DPPH (84.54 ± 3.35 mgTE/g) and ABTS radicals (96.35 ± 2.22 mgTE/g), and showed the highest reducing potential (151.20 and 93.22 mg TE/g for CUPRAC and FRAP, respectively). The leaves extract exhibited the highest inhibition towards acetyl- and butyrylcholinesterase (2.11 and 2.43 mg GALAE/g, respectively) and tyrosinase (54.65 mg KAE/g). The root extract inhibited α-glucosidase (0.71 ± 0.07 mmol ACAE/g), α-amylase (0.43 ± 0.02 mmol ACAE/g) and lipase (8.15 ± 1.00 mg OE/g). At a concentration >2 µg/mL, a significant dose dependent reduction of cell viability towards THP-1 monocyte leukemic cells was observed. Costmary could be recommended for raw material production with antioxidant and enzyme inhibitory properties.


Introduction
The use of plants as sources of drugs and secondary metabolites has been attracting scientific attention over the past decades, considering not only the well-known medicinal species but also plants used in traditional medicines and a variety of edible plants. Tanacetum balsamita L. (costmary) is renowned for its traditional use as flavor, carminative and cardiotonic in the Mediterranean, Balkan and South American countries [1]. The species is distributed in the South-East of Europe and South-West of Asia but has also been widely naturalized throughout the whole world [2,3]. T. balsamita is commonly referred to as: costmary, balsam herb, alecost, sweet tongue and bible leaf. The plant is cultivated in Iran, Turkey, Romania, Germany, Italy, Spain and England [1]. It has a traditional usage as aromatic plant in Europe and Asia. Fresh and dried costmary leaves possess a strong lemony-minty flavor and a sweet astringent taste. The dried leaves have a long history of application as flavorings in soups and meats, sausages and cakes, as well as for making tonic tea. Costmary leaves have been used in ethnopharmacological approach as a hepatoprotective, tonic, sedative, pain relief and astringent agent [1]. The analgesic, anti-inflammatory, antimicrobial and antioxidant activity of the essential oil and extracts (59.75 ± 0.66 mg GAE/g and 41.02 ± 0.50 mg QE, respectively), while the lowest values were determined in the leaves (30.82 ± 0.16 mg GAE/g for TPC) and roots (3.74 ± 0.07 mg QE for TFC). It is worth noting that the results revealed a higher amount in both studied classes compared to those in Chrysanthemum balsamita var. tanacetoides aerial parts, where 2.92 g GAE% and 1.19 g RE% were evidenced [16]. Overall, our results were in the same order of magnitude or lower than TPC and TFC reported in flower heads and aerial parts from T. poterifolim, T. macrophyllum, T. vulgare and T. parthenium with a Turkish and a Bulgarian provenance [20,21,25,26] and higher than those in T. corymbosum, T. vulgare and T. macrophyllum from Romanian origin [27]. In line with the results, the accumulation of phenolic compounds could be firmly connected to the plant ontogenetic development and they could be related differently to each plant organs [28]. Therefore, the diverse qualitative and quantitative content of the bioactive compounds reveal different potential and effects. As it was previously reported, Tanacetum species contain a wide range of hydroxycinnamic esters and flavonoid derivatives [20,21,[24][25][26]29]. To assess the secondary metabolites, non-targeted metabolic profiling of the hydroxycinnamic acids, flavones, flavonols and flavanones of each methanol-aqueous extract was carried out by UHPLC-Orbitrap-HRMS. The parameters of Full-scan ddMS 2 mode were adjusted to favor the formation of diagnostic fragment ions for the subclasses of phenolic acids derivatives, acylquinic acids (AQA) and flavonoids. Fragmentation patterns along with exact masses of precursor ions in negative and positive (for flavonoids) ionization mode were depicted in Tables 2 and S1. Thus, based on the fragmentation patterns and characteristic ions and authentic standards, fragmentation keys for recognition of AQA and methoxylated flavonoids were generated.
Previously, cichoric acid was determined as prevailing compound in the costmary aerial parts, being present at 3.33 g/100 g extract [14]. In contrast, cichoric acid was not found in this study. Ethanol-aqueous extracts of T. vulgare leaves and flower heads were especially rich in protocatechuic acid and its hexoside along with caffeic and salicylic acid and caffeic acid-O-(salicyl)-hexoside [20]. In line with these findings, T. parthenium aerial parts were rich in p-hydroxyphenylacetic and caffeic acid, being present in 280.4 and 129.8 mg/kg extract, respectively [25]. On the other hand, T. macrophyllum was distinguished by the phenylpropanoid glycosides caffeic acid-O-(hydroxybutanoyl)-hexoside and vanillic/gentisic acid-O-(caffeoyl)-hexoside, together with two caffeoyl-(syringic) acid isomers.
Hexuronides of luteolin, isorhamnetin, apigenin, chrysoeriol and jaceosidin were exclusively produced by T. balsamita leaves ( Figure S3), Flavonoid glycosides profile of both leaves and flower heads extracts were dominated by rutin; the former was also characterized by luteolin/jaceosidin-hexuronide, while the latter was rich in isoquercitrin and hyperoside. Previously, apigenin-and luteolin 7-glucoside were determined in T. balsamita aerial parts, being present in 1099.3 and 725.7 mg/100 g extract [14].

Heatmap Analysis
To gain an intuitive viewing of the metabolite contrast among the different extracts of Tanacetum balsamita, a heatmap was generated. Figure 1 displays the outcomes and highlights the arrangements of the groups of metabolites characterizing each extract. The red and blue color in the plot specify higher and lower metabolite amounts than the mean, respectively. As observed, the metabolites within group C were abundant in the leaves extract. In contrast, the lowest concentration of the metabolites within group A1 was exhibited by the leaves extract. Similarly, higher concentrations of the group A2 metabolites were found in the flower heads extract, while the group E metabolites were found at lower levels. On the other hand, the metabolites of the root were of low concentrations and were consolidated in the group B. A few studies have reported that the concentration of metabolites is varying in the different parts of the same species. High concentration of phenolics in leaves versus that of roots may be resulted to the presence or absence of light that impacts the phenolic contents of organs [38]. Furthermore, variation in the amount of various phenolic molecules in plants during its phenological cycle is reported by Çirak et al. [39].

Antioxidant Properties
Antioxidants impair the oxidative damage in foods and herbs by delaying or inhibiting oxidation, and expand the shelf-life and quality of these foods [40]. Thus, their consumption could be of help in the treatment of diseases correlated with oxidative damage, as cardiac vascular diseases, inflammations, diabetes and cancer [41]. Therefore, in the present work, the in vitro antioxidant properties of T. balsamita extracts were assayed, and the results are depicted in Table 3.

Antioxidant Properties
Antioxidants impair the oxidative damage in foods and herbs by delaying or inhibiting oxidation, and expand the shelf-life and quality of these foods [40]. Thus, their consumption could be of help in the treatment of diseases correlated with oxidative damage, as cardiac vascular diseases, inflammations, diabetes and cancer [41]. Therefore, in the present work, the in vitro antioxidant properties of T. balsamita extracts were assayed, and the results are depicted in Table 3.
The collected data are consistent with the highest values of TPC and TFC (Table 1). The total antioxidant capacity (TAC) of the T. balsamita extracts was evaluated by the phosphomolybdenum assay, where the highest values, up to 1.48 ± 0.01 mmol TE/g were detected in the root extract, with the highest values, followed by flower heads and leaf extracts (Table 3). Regarding TAC, our results were comparable to those obtained in T. poterifolim, T. macrophyllum, T. vulgare and T. parthenium [20,21,25,26]. Additional antioxidant assays were carried out to provide insights into the antioxidant properties of the assayed T. balsamita extracts. The flower heads extract had the most pronounced radical scavenging activity in DPPH (84.54 ± 3.35 mg TE/g) and ABTS (96.35 ± 2.22 mg TE/g) assays. Reducing power is an important way to evaluate electron-donating ability of antioxidants. Thus, the reducing power of the assayed extracts was investigated by FRAP (from Fe 3+ to Fe 2+ ) and CUPRAC (from Cu 2+ to Cu + ) assays. The highest reducing power in both assays was found in the flower heads extract (93.22 ± 1.59 mg TE/g for FRAP and 151.20 ± 0.22 mg TE/g for CUPRAC). The results compare favorably with our previous study on Tanacetum species using the same assays. Generally, the received data for radical scavenging activity are consistent with those previously recorded in T. parthenium, T. poteriifolium and T. vulgare extracts and substantially lower in comparison with T. macrophyllum extracts [20,21,25,26]. The aforementioned Tanacetum species revealed higher reducing power activity than costmary extracts. T. balsamita leaves extract possessed strong chelating ability being more potent compared to T. parthenium, T. poteriifolium and T. vulgare extracts.

Enzyme Inhibitory Activity
The inhibitory ability of extracts prepared from the T. balsamita flower heads, leaves and roots against enzymes targeted in the management of type II diabetes mellitus, Alzheimer's disease, lipid metabolism and skin hyperpigmentation problems were investigated.
The highest AChE and BChE inhibitory potential were observed for the leaf extract (2.11 ± 0.04 mg GALAE/g and 2.43 ± 0.04 mg GALAE/g, respectively) ( Table 4). The same sample was found to have the highest tyrosinase and α-amylase inhibitory activity (54.65 ± 1.30 mg KAE/g and 0.44 ± 0.01 mmol ACAE/g, respectively). α-Glucosidase inhibitory potential did not exceed 0.71 ± 0.07 mmolACAE/g, while lipase inhibition was up to 8.15 ± 1.00 mg OE/g, whre both were for the root extract (Table 4). Aforementioned results were consistent with those previously recorded in T. vulgare except for the lower α-glucosidase and the higher tyrosinase inhibitory potential [20]. It's worth noting that T. poteriifolium aerial parts demonstrated remarkable tyrosinase and α-glucosidase inhibition among the assayed Tanacetum species [26].
These findings could be related to the extracts chemical profiling (Table 2). For instance, flavonoids and acylquinic acids have been shown as inhibitors of the studied enzymes. However, the enzyme inhibitory potential is not directly related to TPC and TFC as seen in T. vulgare and T. macrophyllum [20,21]. It appears that the enzyme inhibition could be ascribed to the sesquiterpene lactones. Hence, it may be assumed that sesquiterpene lactones act in a synergistic way in AChE related disorders [42]. Furthermore, the germacranolide parthenolid and monoterpene thujone have been already reported as cholinesterase inhibitors [43][44][45]. Orhan et al. (2015) hypothesized that parthenolode plays a role in AChE inhibition in a synergistic manner together with other compounds (monoterpenes). Thus, the leaves extract of Tanacetum argenteum subsp. flabellifolium had the highest AChE inhibitory effect (96.68 ± 0.35%). C-flavonoid glycoside homoorientin, identified in costmary leaves extract, was previously reported to inhibit AChE in an in silico and in vivo study [46]. At 100 mg/kg for 3 weeks homoorientin inhibited the activity of AChE in rats with experimentally induced Alzheimer's disease. Hispidulin, identified in the Phyla nodiflora extracts, was previously reported to inhibit tyrosinase with an IC 50 value of 146 µM [47]. In addition, chlorogenic acid and its derivatives have potential as cholinesterase and glucosidase inhibitors [48][49][50]. Thus, chlorogenic acid inhibited AChE and BChE and pro-oxidant-induced lipid peroxidation in rat brain in vitro (IC 50 value of 8.01 mg/mL and 6.3 mg/mL, respectively) [49]. At 5 mg/kg 3,5-dicaffeoylquinic acid reduced significantly the blood glucose levels and ameliorate the oxidative stress biomarkers reduced glutathione, malondialdehyde and serum biochemical parameters [50].

PLS-DA Analysis
Based on the antioxidant, enzyme inhibitory, a supervised partial least square discriminant analysis (PLS-DA) was simulated considering parts as class membership criteria, and the outcomes are summarized in Figure 2.

PLS-DA Analysis
Based on the antioxidant, enzyme inhibitory, a supervised partial least square discriminant analysis (PLS-DA) was simulated considering parts as class membership criteria, and the outcomes are summarized in Figure 2. The discriminant analysis resulted in a good segregation of the three parts ( Figure  2A). Figure 2B. showed the performance of the model evaluated through the Area Under the Curve (AUC) average using one-vs-all comparisons. An AUC value of 1 was obtained when taking account 2 function, suggesting the great segregation between the three parts along the first two function. By referring to Figure 2C, it appears that the first function The discriminant analysis resulted in a good segregation of the three parts (Figure 2A). Figure 2B. showed the performance of the model evaluated through the Area Under the Curve (AUC) average using one-vs-all comparisons. An AUC value of 1 was obtained when taking account 2 function, suggesting the great segregation between the three parts along the first two function. By referring to Figure 2C, it appears that the first function separated the samples based on DPPH, ABTS, CUPRAC, FRAP, MCA, AChE, BChE, tyrosinase and amylase activities, while the second function separated the samples according to PBD, glucosidase and lipase activities. Regarding Figure 2D, the strongest activity recorded by the flower heads extract were DDPH, FRAP, CUPRAC and ABTS. Similarly, the roots proved to be the most effective plant part to give better PBD, anti-glucosidase and antilipase properties, while the higher anti-BChE, anti-AChE and anti-tyrosinase activity were recorded by the leaves extract. Furthermore, the contribution of the metabolites in the biological activities was evaluated through the Pearson's correlation analysis. As reported in Tables S2 and S3, several metabolites seem to be involved in various biological activities, since a positive Pearson coefficient higher than 0.7 was obtained. Some metabolites are well known in the literature for their various properties; protocatechuic acid, syringic acid, isorhamnetin and quercetin have been reported for potential action, such as antioxidant activity [51][52][53][54]. In addition, neochlorogenic was reported to be the predominant antioxidant compound in Polygonum cuspidatum leaves [55]. Further, experimental studies support the effectiveness of protocatechuic acid and vanillic acid in the prevention of diabetes diseases and neurodegenerative processes, including Alzheimer's [56,57]. In addition, p-coumaric acid is well known for its antioxidant activity, prevention and improvement of diabetes and neuroprotection [58].

Cytotoxicity Assay
To assess the cytotoxicity of the extracts, we used the common THP-1 cells, a human monocytic cell line that mimics the behavior of the costmary extracts towards the immune system ( Figure 3). After 24 h of incubation of macrophage cell line THP-1 with flower heads, roots, and leaves extracts, we observe 25% toxicity for the three extracts at concentration of 2 µg mL −1 . A toxicity of 50% was reached at 200 µg mL −1 for flower heads extract, 1000 µg mL −1 and 2000 µg mL −1 for leaves and roots extracts, respectively, which may be regarded as a very high concentration, without any biological meaning. At a concentration of 3000 µg mL −1 , a 100% toxicity for flower heads extract was observed.
Plants 2022, 11, x to be involved in various biological activities, since a positive Pearson coefficient higher than 0.7 was obtained. Some metabolites are well known in the literature for their various properties; protocatechuic acid, syringic acid, isorhamnetin and quercetin have been reported for potential action, such as antioxidant activity [51][52][53][54]. In addition, neochlorogenic was reported to be the predominant antioxidant compound in Polygonum cuspidatum leaves [55]. Further, experimental studies support the effectiveness of protocatechuic acid and vanillic acid in the prevention of diabetes diseases and neurodegenerative processes, including Alzheimer's [56,57]. In addition, p-coumaric acid is well known for its antioxidant activity, prevention and improvement of diabetes and neuroprotection [58].

Cytotoxicity Assay
To assess the cytotoxicity of the extracts, we used the common THP-1 cells, a human monocytic cell line that mimics the behavior of the costmary extracts towards the immune system ( Figure 3). After 24 h of incubation of macrophage cell line THP-1 with flower heads, roots, and leaves extracts, we observe 25% toxicity for the three extracts at concentration of 2 µg mL −1 . A toxicity of 50% was reached at 200 µg mL −1 for flower heads extract, 1000 µg mL −1 and 2000 µg mL −1 for leaves and roots extracts, respectively, which may be regarded as a very high concentration, without any biological meaning. At a concentration of 3000 µg mL −1 , a 100% toxicity for flower heads extract was observed.

Plant Material
The leaves, flower heads and roots of T. balsamita were collected from herbal garden

Plant Material
The leaves, flower heads and roots of T. balsamita were collected from herbal garden (Belopoptsi village, Gorna Malina region) in Bulgaria at 700 m a.s.l. (42.67 • N 23.77 • E), derivatives and flavonoids) compounds. The screening was achieved by selecting spectra based on the following criteria: m/z error of the molecular ion <15 ppm, retention time error <2%, number of fragment ions match >2/3, absolute error of the percentage intensity of matched fragment ions <15. Spectra identified as the same reference compound found in the same chromatographic peak were grouped, i.e., the spectra were summed, the m/z were adjusted by weight averaging where is the recalculated m/z value and int i are the m/z and the intensity of the ith fragment ion, respectively.

Total Phenolic and Flavonoid Contents
Total phenols and flavonoids were measured as gallic acid (GAE) and rutin (RE) equivalents respectively, through validated spectrophotometric methods. The experiments were carried out as reported in previous studies [60][61][62]. The detailed protocols are given in Supplementary Materials.

Cell Line and Culture
The human monocytic THP-1 (TIB-202) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells grown in RPMI 1640 were supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin and 0.5% Amphotericin B. Cells were cultured in a humidified atmosphere at 37 • C under a 5% CO 2 atmosphere.

Cytotoxicity Assay
THP-1 cells (at 1.10 5 cells/mL) in RPMI medium (Thermo-Fisher) supplemented with 10% FBS were seeded in each well of a 48-well plate (n = 4). Cells were permitted to adhere for 24 h, and then treated with roots, flowers and leaves extracts of T. balsamita in a medium for 24 h. Then, 40 µL of WST-1 testing solutions (Sigma-Aldrich) was added to each well and the plate incubated at 37 • C for 2 h. The contents of each well were laid down in 3 wells of a 96-well plate [65]. The absorbances were measured at 350 and 630 nm with an Omega StarLab spectrophotometer (Omega, Ortenberg, Germany).

Statistical Analysis
In the antioxidant and enzyme inhibitory assays, the values are expressed as mean ± SD of three parallel experiments.
In terms of antioxidant and enzyme inhibitory abilities, one way ANOVA with Tukey's assay was performed to determine differences between the tested extracts. The statistical analysis was performed using XlStat 16.0 software. Clustered Image Maps (CIM) were used to visualize metabolite variation among the extracts. Prior to CIM analyses, data were normalized and centered. Afterwards, a supervised Partial Least-Square discriminant analysis (PLS-DA) was done to discriminate the different parts regarding their biological activities. Then, CIM was applied on PLS-DA outcomes to characterize each extract. Lastly, Pearson's correlation coefficients were calculated to evaluate the relationship between secondary metabolites and the biological activities, respectively.

Conclusions
More than 100 secondary metabolites, including methoxylated flavonols and flavones, acylquinic acids analogues, hydroxybenzoic and hydroxycinnamic acids derivatives, and their glycosides, were annotated/dereplicated in the costmary leaves, flower heads and roots extracts. Ninety-one compounds are reported in the species for the first time. Chlorogenic, 3, 5-diCQA and 4, 5-diCQA acid dominated the leaves and roots extracts profiles. Despite the previously published data on the high concentration of cichoric acid in the costmary aerial parts extract, we were not able to confirm the presence of either cichoric acid or any esters of tartaric acid and hydroxycinnamic acid. According to this study, the presence of 6-methoxylated flavones and flavonols, dicaffeoylquinic acids and their hexosides and phenolic acids glycosides could be considered significant in the chemotaxonomy of the Tanacetum genus. To understand the relationship between plant parts and biological activity, multivariate statistical analyses were performed. The strongest antioxidant activity (DDPH, FRAP, CUPRAC and ABTS) of the flower heads extract could be related to the presence of rutin, isoquercitrin and hyperoside, and the corresponding aglycone. A variety of acylquinic acids, flavoneshexuronides and methoxylated aglycones in the leaves extract could be associated with its anti-BChE, anti-AChE and anti-tyrosinase activity. Phenolic acidshexosides, di-and tri-caffeoylquinic acids accounted for the stronger α-glucosidase and α-lipase inhibitory activity of the roots extracts. The assayed extracts expressed low cytotoxicity towards THP-1 viability. In addition to evoking an antioxidant response, costmary extracts display in vitro enzyme inhibitory effects, which generate interest in the plant as a valuable herbal drug. Moreover, this study advocates further work geared towards additional in vivo studies.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12010022/s1: Figure S1: Extracted ion chromatogram of hydroxybenzoic and hydroxycinamic acids and derivatives in negative ion mode of methanol-aqueous extracts from Tanacetum balsamita. Figure S2: Extracted ion chromatogram of acylquinic acids in negative ion mode of methanol-aqueous extracts from Tanacetum balsamita. Figure S3: Extracted ion chromatogram of flavonoid glycosides in negative ion mode of methanol-aqueous extracts from Tanacetum balsamita. Figure S4: Extracted ion chromatogram of flavonoid aglycones in the negative ion mode of methanol-aqueous extracts from Tanacetum balsamita. Table S1: Flavonoids in Tanacetum balsamita extracts assayed by UHPLC-ESI-MS/MS in positive ion mode. Assays for the total phenolic and flavonoid content. Determination of antioxidant and enzyme inhibitory effects. Table S2: Correlation antioxidant activity. Table S3: Correlation enzyme inhibition.