Phytochemical Content of Centaurea polypodiifolia boiss. var. polypodiifolia

Various solvents including hexane, CHCl3, EtOAc, and MeOH were used to extract the aerial parts of C. polypodiifolia Boiss. var. polypodiifolia. Hexane extract was analyzed by GC-MS and nonacosane and octacosane were determined as the main constituents. HPLC-TOF/MS analyses were carried out on CHCl3, EtOAc, and MeOH extracts. The main constituents of CHCl3 extract were detected as 4-hydroxybenzoic acid, vanilic acid, and gentisic acid. Gentisic acid, fumaric acid, chlorogenic acid, and vanilic acid were determined as the main compounds of EtOAc extract. Finally, main constituents of MeOH extract were found as fumaric acid, chlorogenic acid, quercetin-3-β-D-glucoside, gentisic acid and diosmin.


INTRODUCTION
The genus Centaurea which belongs to Asteraceae family, comprises nearly 500 species around the World [1]. Centaurea species are generally used in folk medicine due to their pharmacological properties such as fever treatment, diabetes, hemoroid, and peptid ulcer [2]. Biological activity studies of Centaurea genus revealed that they have exhibited antioxidant, antimicrobial and antipyretic activities [3].
Phytochemical studies of Centaurea species showed that the main compound groups of different Centaurea species were sesquiterpenes [4][5][6][7][8], lignin compounds [9][10][11], flavonoids [12][13][14], and their glycosides. Secondary metabolites especially phenolic compounds are responsible from the biological [15][16][17][18] activities. Phenolic and flavonoid compounds, as important phytochemicals, are present in vegetables, fruits and cereal grains. Phytochemicals such as phenolic and flavonoid compounds commonly found in plants have multiple biological effects and play an important role in the defense against cardiovascular disease, aging and cancer [19]. In recent years, the studies related with plant-derived phytochemicals and their biological activities have gained interest that they give natural solutions for health-care [20]. Therefore, determination of the phytochemical content of the plants is an important step for the treatment and prevention of some diseases.
The objective of the current study is to determine the phytochemical content of different extracts of Centaurea polypodiifolia boiss. var. polypodiifolia. So, it facilitates the future investigations related with biological activity.

General Experimental Procedures
GC-MS analysis was performed with a Perkin Elmer Clarus 500 Series GC system. Phenolic constituents of the extracts were determined using an Agilent Technologies 6210 Time-of-Flight LC-MS. All solvents used in HPLC analysis were of HPLC grade and purchased from Merck. The extraction solvents were distilled before the extraction processes.

Extraction Procedure
Flowers of CPP (100 g) were extracted with hexane (1 L x 3) for 24 h. After the process, it was filtered and evaporated. The residue was re-extracted with the same procedure for the following solvents CHCl3, EtOAc, and MeOH, respectively.

Methylation Procedure
The fatty acid composition of hexane extract was determined by using methylation of the lipid extracts to form fatty acid methyl esters (FAME) for gas chromatography (GC) analysis. For this purpose, 40 mg of extracted oil was dissolved in a tube which contained n-heptane. Then, 2 M KOH (2 mL) was added to the tube, the mixture was shaken vigorously and allowed to reach formation of phases. The upper layer, which contained the FAME was transferred to a vial and diluted with n-hexane for GC analyses [21].

Gas Chromatography (GC) Analysis
FAME analyses were carried out with a Perkin Elmer Clarus 500 Series GC system equipped with flame ionization detector (FID) and a TR-FAME apolar capillary column (30 m x 0.25 mm and 0.25 m ID). The split ratio was 50:1 port. Helium was used as the carrier gas at a flow rate of 0.5 mL/min. The temperatures of the injector and detector were set to 250 and 260 °C, respectively. An initial column oven temperature of 100 °C was raised to 220 °C at a rate of 2 °C/min. Identification of the FAME peaks was performed by comparing the retention times of each peak with those of authentic standards (Supelco 37 Comp. Fatty acid Mix, 18919) and their mass spectral

Fatty Acid Content
GC-MS analysis results of the hexane extract of CPP was given in Table 1. Main constituent of the hexane extract of CPP was assigned as nonacosane (43.53%).

CONCLUSION
The presence of the phytochemicals plays an important role in the treatment or prevention of many diseases due to their potential biological activities. Therefore, it is important to know the phytochemical content of plants. In this study, different extracts of the flowers of Centaurea polypodiifolia Boiss. var. polypodiifolia were screened for their secondary metabolite contents using GC-MS and HPLC-TOF/MS, respectively. The results demonstrated that the main constituent of the hexane extract was nonacosane. Main phenolic compounds of CHCl3, EtOAc, and MeOH extracts were determined as 4-hydroxybenzoic acid, gentisic acid, and fumaric acid, respectively.