Cytotoxic evaluation and LC-MS/MS analysis of aerial parts of Eryngium kotschyi Boiss. grown in Turkey

Increasing biological activity and phytochemical investigations on Eryngium species showed its potential as pharmaceutical approach. Eryngium kotschyi Boiss. is one of the species of Eryngium genus and is endemic to Turkey. It is known that this plant is traditionally used in the South-western part of Turkey for the treatment of various diseases. This study focuses on cytotoxic activities of methanol extract and ethyl acetate, n -butanol and water sub-extracts from E. kotschyi in A549, COLO 205 and MDA-MB-231 cell lines by Sulforhodamin B assay and qualitative and quantitative determination of phytochemical constituents in active extract by LC-MS/MS. From the result of the study, it was seen that E. kotschyi ethyl acetate (EKE) sub-extract showed the strongest cytotoxic effect with the low IC 50 values (50.00; 31.96 and 22.26 μg/mL in A549; COLO 205 and MDA-MB-231 cells at 48 h, respectively). Preliminary examination of the mass spectrums revealed the presence of 15 phytochemical compounds in active sub- extract and 7 of them was quantiﬁed. According to quantitative analyses the main compounds of EKE sub-extract were rosmarinic acid (485.603 µg/mg extract ), chlorogenic acid (62.355 µg/mg extract ) and caffeic acid (59.266 µg/mg extract ). Moreover, this preliminary study on inhibitory activity of EKE sub-extract suggests further toxicologic investigations and detailed investigation on cytotoxic effect of various combinations of determined compounds.


INTRODUCTION
Cancer is one of the leading causes of mortality among the world today. Due to be an exceedingly complex disease, treating cancer has become a major challenge. Moreover, incidence and death rates are still important for several cancer types, including lung, colon and breast. Advancing the fight against cancer will require continued clinical and basic research (Siegel, Miller, Jemal, 2016). Herbal drugs have been used for the treatment of many diseases, including cancer and plants have recently been identified as the best source for clinically useful cytotoxic agents (Safarzadeh, Shotorbani, Baradaran, 2014).
The genus of Eryngium L. is widely distributed in the world and used in traditional medicine for different therapeutic purposes. In Turkish folk medicine, various species of the plant are used for a wide range of ailments; particularly, roots are used against various inflammatory disorders, edema, sinusitis, urinary infections or inflammations and snake or scorpion bites or goiter; roots and leaves for infertility and herbs for wound healing (Küpeli et al., 2006).

Plant material
Eryngium kotschyi Boiss. was harvested from Konya; South Hadim at 1600 m altitude of steppe areas in 2015 year. Plant samples was deposited in the Herbarium of Science Faculty at Selcuk University (Herbarium No: KNYA 26907). In this study dried flowering aerial parts of plants have been used.

Preparation of extracts
The aerial parts of E. kotschyi (500 g) were dried in well ventilated rooms and were powdered and extracted three times with methanol by maceration, at room temperature. Combined macerates were filtered and evaporated to dryness under reduced pressure at 37°C using a rotary evaporator. E. kotschyi methanol extract (EKM) was dispersed with water and partitioned with ethyl acetate (EKE) and n-butanol (EKB) sequentially. The crude extracts were stored in dark at -20°C. A total 3 sub-extracts were obtained from EKM extract. Yields of extract and sub-extracts were given in Table I.

LC-MS/MS Analyses
Compounds in active sub-extract were determined by using liquid chromatography-electrospray ionizationmass spectrometry/mass spectrometry (LC-ESI-MS/ MS, Shimadzu 8040). Mass spectrometry was conducted using a Shimadzu LCMS-8040 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface. 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.

Preparation of standard and sample solutions
Stock solutions of the malic acid, caffeic acid, quinic acid, chlorogenic acid, rutin, isorhamnetin 3-O-rutinoside and rosmarinic acid were prepared in methanol at 8 µg/ mL concentrations. The extract and sub-extracts solutions were prepared in methanol at 10 µg/mL.

Determination of cell viability
The effect of the extracts on the viability of A549, COLO 205 and MDA-MB-231 cells was determined by Sulforhodamine B (SRB) assay as described previously (Vichai, Kirtikara, 2006). The extracts were dissolved in DMSO (10 mg/mL). The final DMSO concentration in the medium was less than 0.1%. The cells were seeded 12,500 (for A549); 25,000 (for COLO 205) and 1000 (for MDA-MB-231) cells/wells and after 24 h incubation cells treated for different final concentrations (1; 2; 4; 8; 16; 65; 125; 250 and 500 µg/mL) of the extracts for 24 h and 48 h, followed by fixing the cells in 10% (v/v) of trichloroacetic acid (TCA) for 1 h at 4°C. After washing 5 times, cells were exposed to 0.5% (w/v) SRB solution for 30 min in a dark place and subsequently washed with 1% (v/v) acetic acid. After drying, 10 mM (pH 10.5) Tris base solution was used to dissolve the SRB-stained cells using a plate-shaker (PST-60 HL plus Biosan) and the absorbance was measured at 510 nm using a microplate reader (BiotEKM Synergy HT). Data are represented as a percentage of control cells. The measurement of the "half maximal inhibitory concentration" (IC 50 ) values were calculated with GraphPad Prism Software Version 7.01 (La Jolla, CA, USA). Mean values were calculated in three experiments using 4 wells per condition. Results were given as the mean ± SD of independent experiments.

Statistical analysis
Statistical analysis was performed by using GraphPad Prism Software Version 7.01 (La Jolla, CA, USA) to compare differences in values between the control and experimental group. The results are expressed as the mean ± standard deviation (S.D.  columns vs. control and p-values of less than 0.05 were considered statistically significant. *p < 0.05, **p <0 .001 and ***p <0 .0001 were considered as compared to the untreated control.

Cell viability is decreased by E. kotschyi
In order to determine the cytotoxic efficacy of EKB, EKE, EKM and EKW extracts, the SRB assay was carried out using A549, COLO 205 and MDA-MB-231 cells. All the cell lines were treated with extracts of E. kotschyi at different concentrations ranging from 1 to 500 μg/mL for 24 and 48 h.
As shown in Figure 1b and 1c, the viability of treated A549 cancer cells with EKE and EKM was significantly reduced in a time-and dose-dependent manner compared to untreated cancer cells. Also, the IC 50 values (minimum concentration of extract to reduce cell viability to 50 %) of ethyl acetate and methanol extracts of E. kotschyi after incubation for 24 and 48 h are reported in Table II. The inhibitory effect of the EKE extract on cell proliferation was significantly superior to that of EKM.
Page 5/16 Cytotoxic evaluation and LC-MS/MS analysis of aerial parts of Eryngium kotschyi Boiss. grown in Turkey The concentrations of EKB at 250 µg/mL and lower did not show decrease in the cell viability on COLO 205 cells (Figure 2a). The COLO 205 cells exposed to EKE at 65 µg/mL and above concentrations for 24 and 48 h were found to be cytotoxic (Figure 2b). COLO 205 cells exposed to EKE for 24 and 48 h also showed the statistically significant (p<0.001) decrease in the cell viability ( Figure 2b). The cell viability at 65, 125, 250, 500 µg/mL was recorded to be 70.84%, 39.22%, 16.84% and 32.65% (Figure 2b The concentrations of EKB at 65 µg/mL and higher proliferated COLO 205 cells (Figure 3a). The MDA-MB-231 cell line was more sensitive to EKE, which significantly reduced its viability (Figure 3b) (Figure 3b). The MDA-MB-231 cells exposed to EKM at 16 µg/mL and above concentrations for 48 h were found to be cytotoxic ( Figure  3c). The percent cell viability was recorded to be 66.82%, 74.79%, 10.48%, 11.75%, 16.25% and 15.69% at 16, 32, 65, 125, 250 and 500 µg/mL of EKE, respectively for 48 h (Figure 3c). The IC 50 values strongly indicated that the effective doses of EKE for COLO 205 were lower when compared to A549 cells after different incubation times (Table II). EKE extract also showed a marked cytotoxic activity against to the three cell lines (Figure 1b, 2b and 3b) and this cytotoxicity was cell-, dose-and time-dependent.
SRB results indicated that various concentrations of EKW had no cytotoxic effect on the tested cell lines after incubations of 24 and 48 h (Figure 1d, 2d and 3d).

Quantitative analyses of compounds
The compounds were subsequently analyzed in Q1Scan (Product Ion Scan) mode, using [M−H]ions as precursors. Obtained MS2 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.  (Barros, et al., 2013).  The other f lavonol diglycoside isorhamnetin 3-O-rutinoside represents specific fragmentation with the loss of CH 3 radical from the deprotonated aglycone, thus giving m/z 315→ m/z 300 and the m/z 285 pattern (Martucci, et al., 2014).
The tentative mass spectrum for rosmarinic acid ([M-H]ion at m/z 359.08) showed the caffeic acid at m/z 179.0 and m/z 161.0, m/z 135.0 corresponding to loss of water and carbon dioxide molecules respectively from the precursor ion (Hossain, et al., 2010). The obtained LC-MS/MS chromatogram and mass spectrum of compounds are presented in Figure 6.

DISCUSSION
Cancer is one of the main causes of death worldwide. Natural products and their secondary metabolites have a considerable significance to be investigated for possible anticancer agents considering their major toxicity to cancer cells (Cabral, et al., 2018). Assessment of the toxic effect of plant extracts is indispensable in cancer research. It allows identification of the intrinsic toxicity of the plant (Lagarto Parra, et al., 2001).
It is well known that the phenolic compounds are potential substances against oxidative and DNA damage, apoptosis induction in transformed cells or tumors (Chen, et al., 2008, Duthie, Duthie, Kyle, et al., 2000. These compounds are important because most of them have proven antitumor activity and may also act synergistically. In this study, the initial cytotoxicity screening showed that EKE was effective against all the cell lines but especially MDA-MB-231. IC 50 values is relatively low among all the cell lines for EKE extract especially in MDA-MB-231 cells. EKE also showed a marked cytotoxic activity against to the three cell lines and this cytotoxicity was cell-, dose-and time-dependent. The results indicated that EKE was generally more effective than EKM. The results demonstrated that these extracts do not have the same effects exactly due to the properties of cell lines and the presence of various potential bioactive molecules in extracts. Since the cell line has a well-known high aggressive and drug-resistant phenotype (Pillé, et al., 2005), the following experiments could be performed with MDA-MB-231 cancer cells. Therefore, the effective inhibition of MDA-MB-231 cancer cells suggests that ethyl acetate extract from E. kotschyi may be potentially promising anticancer agent for the effective treatment of breast cancer cells. The anti-proliferative effect of E. kotschyi was recognized in some cancer cell lines including hepatocellular, laryngeal epidermoid and glioma (Yurdakök, Baydan, 2013) but its molecular mechanisms of action on cancer cells are not yet established.
The criterion of cytotoxicity established by the US National Cancer Institute (NCI) for crude extracts was determined as IC 50 ˂ 30 μg/mL (Suffness 1990). Following this fact, extracts examined in this study with observed IC 50 values lower than 30 μg/mL were considered to have significant activity. EKE has more potential to be explored as novel anticancer agent. In contrast, the EKW did not show cytotoxicity against cancer cell lines. Furthermore, we also found that the selective cytotoxicity of these extracts against cancer cell lines was closely related to their chemical content. The cytotoxic effect of EKE may be related to its higher content of caffeic acid, chlorogenic acid and rosmarinic acid components. According to the literature, caffeic acid (Rzepecka-Stojko, et al., 2015), chlorogenic acid Page 13/16 Cytotoxic evaluation and LC-MS/MS analysis of aerial parts of Eryngium kotschyi Boiss. grown in Turkey (Sadeghi Ekbatan, et al., 2018) and rosmarinic acid (Jang, Hwang, Choi, 2018) presented inhibitory activities against different type of cancer cells.
To the best of our knowledge, this is the first report of the phytochemical analysis and in vitro cytotoxic activities of the aerial parts of E. kotschyi. In conclusion, our data illustrated that various cytotoxic effect of EKM and EKE related to different amounts of phytochemical compounds. The EKE exerted a higher level of cytotoxic compounds seems to be more effective against proliferation of cancer cells. In vitro SRB assay indicated that EKE exhibited the strongest inhibitory effect on A549, COLO 205 and MDA-MB-231 cancer cell lines. According to these results we can consider that the potent cytotoxic activity of EKE on A549, COLO 205 and MDA-MB-231 cells may be explained as the presence of anticancer compounds. As our results agree with previously reported studies, we can say that this work has revealed further potentials of this plant in the area of pharmacology for cancer research. Furthermore, the identified compounds are the possible contributors to the antiproliferative and cytotoxic effects of EKE and EKM suggesting an interesting potential for the pharmacotherapy of cancer. Based on these results, it is suggested that further toxicologic investigations with EKE and EKM should be carried out. On the other hand, this preliminary research suggests detailed investigations on cytotoxic effect of various combinations of determined compounds.