Characterization of Secondary Metabolites in Two Cousinia species

Cousinia is one of the widespread genera of Asteraceae family. According to previous studies on some Cousinia species it was found that these species are rich of triterpenes, sesquiterpenes, flavonoids, acetylenes and steroids. According to our knowledge, there are no published reports on the chemical composition of C. iconica Hub. Mor. and C. aintabensis Boiss. & Hausskn., thus we aimed to investigate secondary metabolites of these species. In this study, the phytochemical constituents of these species were evaluated. Seven of identified compounds were quantified. The quantitative and qualitative determination of compounds within the extracts was carried out by LC-MS/MS. Phytochemical analyses revealed the presence of flavonoids, saponins, terpenes and steroids. Preliminary examination of the mass spectrums revealed the presence of phenolic acids and derivatives and flavonoid compounds in extracts. According to quantitative analyses the main compound of C. iconica (CI) and C. aintabensis (CA) extracts was rutin with the highest contents (169.779 μg/mgextract and 161.638 μg/mgextract). Moreover, qualitative and quantitative study combined with different biological activities will shed new lights to the advanced studies. ARTICLE HISTORY Received: July 21, 2019 Revised: November 16, 2019 Accepted: December 23, 2019


INTRODUCTION
Cousinia Cass. is one of the widespread genera of Asteraceae family with 600-700 species distributed in Central and South-West Asia. There are 39 species and 6 sections of Cousinia genus in Turkey [1]. In the literature, taxonomic and systematic studies are generally performed on the genus of Cousinia, but phytochemical and activity studies are rarely seen. Numerous studies have shown that plants of the genus are rich in triterpenes, sesquiterpenes, flavonoids, acetylenes and steroids [2][3][4][5][6][7][8]. In phytochemical studies it was reported isolation of guianolide type sesquiterpenes from Cousina picheriana Bornm. ex Rech.f., C. piptocephala Bunge. and C. canescens DC. [2], oxygenated bisabolene derivatives from C. canescens DC., phenolic and triterpenic compounds from C. adenostica Bornm., C. aitchisonii Boiss. [5,[9][10][11][12] and fatty acids from C. aurea C.Winkl., C. seversovii Regel, C. umbrosa Bunge. [13,14]. In a study, ethanol extracts from different Cousinia species were subjected to cytotoxic screening on the fibrocarcinoma cell line. The highest activity was observed in C. verbascifolia Bunge. (IC50 = 18.4 ± 0.59 μg / mL) [15]. According to Iranshahy et al. sesquiterpene compounds namely desoxyjanerin and raserolit obtained from the dichloromethane extract of C. aitchisonii were subjected cytotoxic screening on five different cell lines. As a result, both compounds showed significant cytotoxic effect on breast cancer MCF-7 cell line (IC50 = 4.5 μg / mL and 4.6 μg / mL, respectively) [10]. In another study MMP inhibitor effect of C. shulabadensis Attar & Ghahr. was investigated and reported to have a considerable inhibitory effect (IC50 = 49.2 ± 0.51 μg / mL) [16]. In a study by Shahverdi et al. the antibacterial effects of ethanol extracts of seven different Cousinia species was investigated by disk diffusion method in different Gram (+) and Gram (-) strains and the highest effect was determined in C. phyllocephala Bornm. & Gauba extract against Staphylococcus aereus and Bacillus subtilis (MIC = 4 mg / disc) [15]. Cousinia iconica Hub.-Mor. from Cousinia section is endemic to Turkey and distributed in open areas, scrublands and stony slopes. Cousinia aintabesis Boiss. & Hausskn. is a perennial herb with purple flowers from Cynaroidae Bunge. section [1]. Because a few studies were reported on these species we aimed to investigate the phytochemical properties of these species. In this study, phytochemical profile of methanol extracts and quantitative analyses of determined compounds were quantified.

Chemicals
All chemicals, standarts and reagents were analytical or HPLC grade and purchased from Sigma-Aldrich.

Plant material and preparation of extracts
The flowering aerial parts of C. iconica was collected from Konya and C. aintabensis from Mardin in July 2013. The voucher specimens were deposited at the Herbarium unit of the Science Faculty, Selcuk University, Konya, Turkey (Voucher No. 1, KNYA 11.040; Voucher No. 2, KNYA 77.81 respectively). Air dried aerial parts of C. iconica (500 g) and C. aintabensis (500g) were powdered and extracted three times with methanol by maceration, at room temperature. Combined macerates filtered and evaporated to dryness under reduced pressure at 37°C using a rotary evaporator. The crude extracts were stored in a dark at -20°C. Yields of methanol extracts of CA and CI were %10 and %15 respectively.

Preliminary phytochemical analysis
The secondary metobilites of CA and CI extracts were evaluated by following standard methods [17][18][19].

Test for carbohydrates
Fehling's test: 2 mL of Fehling A and 2 mL of Fehling B was added to 1 mL of test solution in a test tube and carefully heated in a water bath. Precipitation of red Cu2O indicated the presence of reducing sugars.
Benedict's test: 1 mL of test solution was taken in a test tube and 2mL of Benedict's reagent was added to test solution. The mixture was boiled, and a reddish-brown precipitate was occurred. This result indicated the presence of the carbohydrates.

Test for flavonoids (Shinoda test)
The crude extract was taken in a capsule and 5 mL of a mixture of ethanolic hydrochloric acid (ethanol-HCl-water 1: 1: 1 v / v) was added. Finally, 5-6 magnesium ribbon was added in this mixture. Appeared Pink scarlet color indicated the presence of flavonoids.

Test for saponins
Crude extract was shaked with 5mL of distilled water in a test tube. The formation of stable foam was indicated the presence of saponins. Liebermann's test: Crude extract which mixed about 2 mL of chloroform is evaporated to dryness on a water bath in a porcelain capsule. Then the residue was dissolved by the addition of 1 mL of glacial acetic acid. About1-2 mL of concentrated H2SO4 carefully added. A color change from violet to blue to green represented the presence of steroidal saponins.
Salkowski's test: For preparation test solution crude extract was mixed with 2mL of chloroform. Then about 2 mL of concentrated H2SO4 was added and shaken gently. A reddish brown colour remarked the presence of steroidal ring.

Cardiac glycosides
Keller-Killiani test: Crude extract was dissolved in 2mL of glacial acetic acid (containing 1-2 drops of 2% FeCl3 solution. Then 2mL of concentrated H2SO4 was added. a brown ring at the interphase indicated the presence of cardiac glycosides. Baljet test: Crude extract was dissolved with chloroform ethanol mixture (4:1). Following this sodium picrate reagent and 2 drops of 20% NaOH was added to mixture. If cardiac aglycon is present yellow to orange color will be seen.
Kedde test: Crude extract is treated with a small amount of Kedde reagent (Mix equal volumes of a 2% solution of 3,5-dinitrobenzoic acid in menthol and a 7.5% aqueous solution of KOH) and 2 drops of 20% NaOH solution. Development of a blue or violet color showed presence of cardiac aglycon.

Test for alkaloids
Crude extract was mixed with 2 mL of 1% HCl and heated gently. Then reagents of Mayer and Wagner were added to the test solution. Turbidity of the resulting precipitate was showed the presence of alkaloids.

Test for tannins
Crude extract was boiled with 20 mL distilled water for 5 min and filtered while hot. Then 1 ml of cool filtrate was diluted to 5 mL with distilled water and a few drops (2-3) of 10% ferric chloride were added and observed for the formation of precipitates and any color change. A bluish-black or brownish-green precipitate indicated the presence of tannins.

Test for combined anthraquinones
Powdered sample (1 g) was boiled with 2 mL of 10% hydrochloric acid for 5 min. Then the mixture was filtered while hot, cooled and partitioned with the equal volume of chloroform. The chloroform layer was taken into test tube and an equal volume of 10% ammonia solution was added, shaken and allowed to separate. Rose pink color in separated aqueous layer indicated the presence of anthraquinones.

Qualitative and Quantitative LC-MS/MS Assay
Compounds in CA and CI extracts were determined qualitative and quantitative by using liquid chromatography-electrospray ionization-mass spectrometry/ mass spectrometry (LC-ESI-MS/MS, Shimadzu 8040). The liquid chromatograph was a Shimadzu (Kyoto, Japan) Nexera XR system with an SIL-20AC autosampler, an LC-20AD high-pressure gradient pump system (20-μL mixer), a DGU-20A3R vacuum degasser, and a CTO-10AS VP column oven. Mass spectrometry was conducted using a Shimadzu LCMS-8040 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface in the negative-ion mode.

Preliminary phytochemical analysis
The phytochemical characteristics of two extracts were summarized in the Table 1. From the results, it was found that, carbohydrates, flavonoids, steroids and saponins were present, but alkaloids, anthraquinones and cardiac glycosides were absent in the plant extracts. Although, tannins were not detected in CI extract, but CA extract showed positive result for this secondary metabolite. The preliminary phytochemical tests are helpful in finding chemical constituents in the plant material that may lead to their quantitative estimation and also in locating the source of pharmacologically active chemical compound.

Qualitative analysis of chemical compounds
The identification of chemical compounds in methanol extracts was evaluated on the basis of the accurate mass, the registered mass spectra fragmentation patterns and literature data. The mass spectrometric behavior of the compounds was studied using both positive-ion, and negative-ion mode. But negative-ion mode provided a better sensitivity than the other for these compounds due to more efficient ionization, simpler fragmentation, and lower baseline noise. Total ion chromatograms (TIC) of extracts were shown in Figure 1. The mass spectrums of extracts revealed the presence of 3 phenolic acids (vanilic acid, chlorogenic acid and caffeic acid), 2 organic acids (quinic acid and malic acid) and 2 flavonoid (rutin and isorhamnetin 3-O-rutinoside) compounds in methanol extract of C. iconica and C. aintabensis ( Table 2). The mass spectra of extracts were shown in Figure 2.

Optimisation of LC-MS/MS Condition
The mass spectrometric behavior of compounds was studied using both positive-ion and negative-ion mode. Negative-ion mode provided a better sensitivity for these compounds due to more efficient ionization, simpler fragmentation, and lower baseline noise. These compounds were subsequently analyzed in Q1Scan (Product Ion Scan) mode, using [M−H]ions as precursors. Obtained MS 2 spectras were used to select the optimal product ions. The MRM parameters, such as the precursor ion m/z, collision energy, and product ion m/z for compounds were optimized by an automatic MRM optimization function. Isorhamnetin represents specific fragmentation with the loss of CH3 radical from the deprotonated aglycone, thus giving m/z 315→ m/z 300 and the m/z 285 pattern as a result of fragmentation in C-ring [22]. With the loss of CO2 providing an intense ion at m/z 123 for vanilic acid [23]. The obtained LC-MS/MS chromatogram and mass spectrum of compounds are presented in Figure 3.

Preparation of Standard and Sample Solutions
Stock solutions of compounds were prepared in methanol at 8 µg/ mL concentrations. The extracts solutions were prepared in methanol at 10 µg/mL.

CONCLUSION
This is the first report on the phytochemical characterization of these species from Cousinia genus. Moreover, it was thought that chemical compounds identified in this genus could represent a chemical marker of the Cousinia genus as contributing to the chemotaxonomy.