The biochemical content and antioxidant capacities of endemic Tanacetum densum (Lab.) Schultz Bip. subsp. laxum , and Tanacetum densum (Lab.) Schultz Bip. Heywood growing in Turkey

Medicinal plants have a significant role in preventing and curing several diseases, and Tanacetum L. is one of these plants. The aim of the present study is to determine the fatty acid, lipid-soluble vitamin, sterol, phenolic content, and antioxidant capacity of Tanacetum densum subsp. laxum and Tanacetum densum subsp. amani, to compare the effect of altitude on the biochemical content and to compare systematically by using fatty acids and phenolics. This study showed that palmitic acid (C16:0) and stearic acid (C18:0) are major sources of saturated fatty acid and oleic acid (C18:1 n9), and linoleic acid (18:2 n6c) and a-linolenic acid (C18:3 n3) are the principal unsaturated fatty acids in the two endemic Tanacetum densum taxa. Also, this study found that the unsaturated fatty acid content (60.11±1.61%) of Tanacetum densum subsp. laxum was higher than the unsaturated fatty acid content (44.13±1.28%) of Tanacetum densum subsp. amani . And also, the ω6/ω3 ratio of Tanacetum densum subsp. laxum (1.74) and Tanacetum densum subsp. amani (1.60) was found to be similar. However, this study determined that the lipid soluble vitamin and sterol content of two endemic Tanacetum taxa are low except for stigmasterol. Present study showed that catechin is principal phenolic in the Tanacetum densum taxa. This study also found that Tanacetum densum subsp. laxum and Tanacetum densum subsp. amani had the highest levels of catechin, vanillic acid, and caffeic acid content though the phenolic amounts, particularly catechin and quercetin, were dissimilar in the T. densum taxa. This study suggested that ecological conditions such as altitude may affect the biochemical content of two endemic Tanacetum densum taxa. Furthermore, the current study determined that two endemic Tanacetum L. taxa had potent radical scavenging capacities and found a correlation between total phenolics and antioxidant activity.


Introduction
The Asteraceae family includes about 23.000 species throughout the world, and Tanacetum L. is the third biggest genus in the family (Yur et al., 2017). Tanacetum L. is found extensively in the northern hemisphere, especially in Europe, western Asia, North America, and north Africa include approximately 200 species (Oberprieler et al., 2007;Baranauskienė et al., 2014;Korkmaz et al., 2015;Bączek et al., 2017). The members of the genus generally are perennial and vary from herbs to subshrubs (Oberprieler et al., 2007).
Tanacetum L. has significant biological effects including antimicrobial, antioxidant, anti-inflammatory, and anticancer, and the genus has been used in folk medicine since ancient times for the treatment of migraine, stomach ache, toothache, insect bites, cancer, ulcers, high fever, arthritis, and vertigo (Marete et al., 2009;Marzouk et al., 2016;Mot et al., 2018;Coban et al., 2019). Additionally, Tanacetum, which is grown in gardens, is used in cosmetics and as a spicy food additive (Maxia et al., 2015;Mot et al., 2018). It has been demonstrated that essential oils, sterols, flavonoids, and sesquiterpenes are commonly found in this genus (Kilic, 2014;Marzouk et al., 2016). The genus is represented in Turkey by sixty taxa, and the endemism ratio is 43% (Orhan et al., 2015). T. densum, one of the endemic species, has four subspecies growing in Turkey: subsp. sivasicum, subsp. laxum, subsp. amani and subsp. eginense (Goren et al., 1995). Tanacetum densum is perennial, endemic, erect, or ascending subshrubs, the habitat is the limestone rocks and screes (1500-2500), and flowering time period is between June and August (Davis, 1988). The aim of the present study was to analyze the fatty acids, lipid-soluble vitamins, sterols, total phenolics, flavonoids, and phenolic acids as well as the radical scavenging and FRAP activities of plant extracts of endemic T. densum subsp. laxum and T. densum subsp. amani in order to contribute to knowledge of the medicinal properties of two Tanacetum taxa. Another aim of this study was to examine the effect of altitude on fatty acid and phenolic content and to systematically evaluate the fatty acid composition and phenolic content.

Material and Methods
All chemicals were purchased from Sigma-Aldrich. The plant materials were collected from natural habitats in 2014 July, and samples were stored at the Firat University Herbarium (FUH). The localities of the two taxa under examination are given Table 1.

Extraction of plant materials
2.1.1. Analysis for fatty acids, lipid soluble vitamins, and sterols Two g of plant materials was ground in a mill, and isopropanol/hexane (2:3 v/v) was added to analyze fatty acid, sterol, and lipid soluble vitamins (Hara & Radin, 1978). The lipid extract was centrifuged at 10.000 g for five minutes and filtered. The solvent was then removed by using a rotary evaporator at 40°C. The samples were left at -25°C.

Analysis for fatty acids
To obtain the fatty acid methyl esters, 2% sulphuric acid (v/v) in methanol was used (Christie, 1990). N-hexane was added to the fatty acid methyl esters and isolated by gas chromatography and flame-ionization detection (Shimadzu GC 17 Ver.3) coupled with a Glass GC 10 software. Nitrogen was used as a carrier gas with a flow ratio of 0.8 ml/min. and a capillary column of 25 m in length and 0.25 mm in diameter; Permabound 25 (Macherey-Nagel, Germany) was used to conduct the chromatographic separation.

Chromatographic analysis and quantification of lipid-soluble vitamins and sterols.
Lipid-soluble vitamins and phytosterols were obtained from the lipid fraction based on the method of Sánchez-Machado et al. (2002). The samples were suspended in acetonitrile/methanol (75/25 v/v), and 50 mL was added to the HPLC (Shimadzu, Japan). A Supelcosil TM LC18 (250 x 4.6 mm, 5 mm, Sigma, USA) was used as column and acetonitrile/methanol (75/25, v/v) for the mobile phase. The temperature of the column was kept at 40°C. The wavelength was 320 nm for retinol (vitamin A) and retinol acetate; 215 nm for d-tocopherol, vitamin D, a-tocopherol, and a-tocopherol acetate; 202 nm for phytosterols; and 265 nm for vitamin K1 (López-Cervantes et al., 2006). The results of the analyses were measured as μg/g.

Extraction of plant material for phenolics
Five ml 80% methanol was used to homogenize the flavonoid and phenolic acids. The extracts were centrifuged at 5000 rpm at +4°C, and dimethyl sulfoxide (DMSO) was used to provide a reserve solution (Kursat et al., 2011). Total phenolics were evaluated using the Folin-Ciocalteu method (Singleton et al., 1999). 100 µl of the extracts was added to the mixture that included 200 µl of Folin-Ciocalteu reagent and 3.16 ml of H 2 O. The samples were kept at room temperature for three min. The extracts were left at room temperature for two hours after anhydrous sodium carbonate (20%; w/v) was added to the mixture. The absorbance of the samples was analyzed at 765 nm (Sarhan et al., 2013).

DPPH radical scavenging capacity
The DPPH radical was prepared afresh based on the method by Liyana-Pathirana & Shahidi (2005). A 4. 0 ml DPPH solution was added to 25, 50, 100, 150, and 250 µL of the extract. The complex was kept in the dark for 30 minutes. The absorbance at a wavelength of 517 nm was measured spectrophotometrically. 1 µM quercetin was used as a reference (Liyana-Pathirana & Shahidi, 2005). The results were measured by using the following formula: DPPH radical scavenging activity (%)=[(Abs_control-Abs_sample)]/(Abs control)] x 100. The abs control is the absorbance of DPPH radical + methanol; the Abs_sample is the absorbance of DPPH radical + sample extract/standard.

Ferric-reducing antioxidant power assay (FRAP)
The FRAP method was performed using the procedure of Benzie & Strain (1996). A sodium acetate buffer (300 mmol/l), a TPTZ solution in 40 mmol/l, and 20 mmol/l FeCl 3 (10:1:1; v/v) were used to prepare the FRAP reagent. The absorbance was measured at 593 nm after 10 min. The FeSO 4 solution (100-2000 mmol/L) was used to form the standard curve. The results were evaluated as mM Fe (II)/g.

Statistical analysis
All analyses were done using the SPSS 21.0 statistical program. The simple linear regression model was used to determine the correlation between antioxidant capacity (ABTS and DPPH) and total phenolic contents. Data taken from the present study was represented as mean values ± standard deviation.
The present study demonstrated that T. densum subsp. laxum and T. densum subsp. amani had the lowest lipid-soluble vitamins and ergosterol and ß-sitosterol content (Table 3). However, the stigmasterol content of the two Tanacetum taxa studied was found to be between 59.45±3.11 µg/mg and 63.55±3.21 µg/mg. Plant sterols are important elements that include the interaction between the free hydroxyl group protein and phospholipids and they protect the cells against cancer and cardiovascular diseases (Tosun et al., 2018;Beyzi et al., 2019). Furthermore, stigmasterol is an unsaturated plant sterol found in several medicinal plants and plays a significant role in the biosynthesis of androgens, estrogens, vitamin D3, and corticoids (Kaur et al., 2011). Azizudin & Choudhary (2008) showed that β-sitosterol and stigmasterol were found in T. polycephalum. In another study, Ivanescu et al. (2018) showed that all three Tanacetum species (T. vulgare, T. macrophyllum, and T. corymbosum) contain beta-sitosterol, stigmasterol and campesterol, and traces of ergosterol. They found a high β-sitosterol content (530.78 µg/g dw -696.32 µg/g dw) compared to the present study (Ivanescu et al., 2018).  Chandler et al. (1982) also found β -sitosterol to be the major sterol in Tanacetum. This research may be the first report regarding the lipid-soluble vitamin content of the two endemic Tanacetum taxa. The studies demonstrated that phenolic content is responsible for the antioxidant activities of plants (Wojdylo et al., 2007;Arituluk et al., 2016). Phenolic content plays a preventive role against diabetes, cancer, cardiovascular disease, and Alzheimer's disease as well as in inducing DNA, cell adhesion, cell proliferation, and blocking signaling pathways (Huang et al., 2010;Gutierrez-Grijalva et al., 2016). Because synthetic antioxidants have harmful effects on health, there is a growing interest in natural antioxidants (Ahmed et al., 2011). This study found that the total phenolic contents of T. densum subsp. laxum and T. densum subsp. amani was 188.94±3.24 µg/mg and 137.01±2.47 µg/mg, respectively (Table 4). Tepe&Sokmen (2007) found that the total phenolic content of T. densum subsp. amani was 158.44 ± 2.17 µg/mg. However, Caniklioglu et al. (2018) determined that the total phenolic amount was 84.94±0.009 mg/g in the ethanol extracts of T. densum subsp. eginense. Also, catechin amounts were found at high levels in the present study (1,299.4±7.52 µg/mg-5,796.9±8.12 µg/mg). Though Michel et al. (2020) indicated that kaempherol glycosides are the main phenolic in the Asteracea, but present study showed that the major phenolic compound of T. densum taxa is catechin. Similarly, Gecibesler et al. (2016) found that catechin was a principal component of Tanacetum. The other phenolics studied were in low amounts or absent (Table 4). Zengin et al. (2019) indicated in their study that Tanacetum had various phenolics, but most of them were small amounts. On the other hand, the current study determined that T. densum subsp. laxum and T. densum subsp. amani had the highest vanillic acid (1,543.7±6.12 µg/mg and 1070.3±4.91 µg/mg, respectively) and caffeic acid (1,234.9±5.64 µg/mg and 790±3.89 µg/mg, respectively) contents (Table 5). Cinnamic acid was found trace amounts in the present study. It was also found that the rosmarinic acid amounts in T. densum taxa was the lowest (Table 5). Muresan et al. (2015) showed that Tanacetum had caffeic acid, ferulic acids, chlorogenic acid, rutin and quercetin, and kaempferol. They also showed that Tanacetum had good antioxidant activity (Muresan et al., 2015). Esmaeili et al. (2010) determined that the six Tanacetum species except for T. densum exhibited antioxidant activity. They also found that caffeic acid, ferulic acid, luteolin, apigenin, and rutin were major phenolic compounds in the Tanacetum species (Esmaeili et al., 2010). The differences in the phenolic amounts may originate from the growing conditions of the taxa because environmental factors including climate, weather, and sunlight exposure effect the phenolic content (Bahukhandi et al., 2013). Hashim et al. (2020) also indicated that ecological conditions affect antioxidant activity and the number of dominant constituents in endemic medical plants. In addition, it was reported that collection time has an important effect on the phenolic content (Varga et al., 2016). Climatic conditions, specifically the altitude of growing area, also effect the quantitative content of secondary metabolites such as phenolics at flowering time (Spitaler et al., 2008). The present results may suggest that the quantitative differences in the total phenolics, quercetin, catechin, caffeic acid, rosmarinic acid, and vanillic acid content of two endemic T. densum taxa exist because of the altitude. On the other hand, it has been suggested that qualitative and quantitative differences in the flavonoid and total phenolics could be used as taxonomical markers for tribes and subtribes of Asteraceae (Emerenciano et al., 2001;Sytar et al., 2018). In this study, while the phenolic content is similar in the two taxa studied, the phenolic amounts are different. The present study demonstrated that the two studied endemic taxa of T. densum had high DPPH (except for 25 µl) and ABTS radical scavenging activities (Tables 6, 7). The present DPPH results agreed with the study by Yur et al. (2017) who indicated that the methanol extracts from Tanacetum represented the highest DPPH activity. Also, Zengin et al. (2019) showed that the water and methanol extracts from Tanacetum had high phenolic content and high ABTS and DPPH radical scavenging capacities. However, Caniklioglu et al. (2018) found that the DPPH (20.64±0.26%-48.13±1.37%) and the ABTS radical scavenging activities (12.65±0.23% and

Conclusion
This study found that palmitic acid (C16:0) and stearic acid (C18:0) were the major saturated fatty acids and oleic acid (C18:1 n9), linoleic acid (18:2 n6), and a-linolenic acid (C18:3 n3) were the dominant unsaturated fatty acids. Also, the present study showed that the polyunsaturated fatty acid composition of T. densum subsp. laxum was higher (48.76±1.49%) than the poly unsaturated fatty acid composition (32.76±1.25%) of T. densum subsp. amani, but the mono unsaturated fatty acid compositions of T. densum taxa was similar. Besides, the ω6/ω3 of T. densum subsp. laxum (1.74) and T. densum subsp. amani (1.60) was determined to be at a similar ratio. However, this study also determined that the lipid soluble vitamin and sterol content of two endemic Tanacetum taxa were low except for the stigmasterol contents of the studied Tanacetum taxa. Current study demonstrated that catechin is main phenolic compound in the two endemic T. densum taxa. It was shown in the present study that T. densum subsp. laxum and T. densum subsp. amani had the highest catechin, vanillic acid and caffeic acid contents but the phenolic amounts, particularly catechin and quercetin, were different in the T. densum taxa. This study proposed that altitude may affect the biochemical contents of two endemic Tanacetum densum taxa. Moreover, this study determined that two endemic Tanacetum taxa had strong DPPH and ABTS radical scavenging capacities, and T. densum subsp. laxum, in particular, had the highest FRAP activity. This study found that there was a correlation between total phenolics and antioxidant capacity.