Untargeted Metabolomics Analysis of Crocus cancellatus subsp. damascenus (Herb.) B. Mathew Stigmas and Their Anticarcinogenic Effect on Breast Cancer Cells

Safranal, crocin, crocetin, and picrocrocin are major known compounds in the stigma extract of Crocus sativus with various medicinal properties. Crocus cancellatus is another Crocus species that grows extensively in Iran's various regions, such as the Kurdistan province. The predominant metabolites and biological properties of C. cancellatus have not yet been investigated. The ingredients of the stigma ethanol extract of C. cancellatus were investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS). The ROIMCR approach was performed to analyze the LC-MS full scan data sets. This method searches the MS regions of interest (ROI) data in the m/z domain and analyses the results using the multivariate curve-resolution alternating least squares (MCR-ALS) chemometrics technique for simultaneous resolution of two extracts. Also, the antiproliferative properties of C. cancellatus against MDA-MB-231 and MCF-7 cancer cells were examined by MTT, dual acridine orange/ethidium bromide test, Annexin V-FITC/PI, and zymography. The GC-MS and LC-MS untargeted metabolomics data analysis of the extract indicated the presence of cytotoxic agents including safranal, crocin, picrocrocin, and crocetin in the stigma ethanol extract of C. cancellatus. Biological tests showed that the viability of MDA-MB-231 and MCF-7 cancer cells is decreased following C. cancellatus treatment in a time- and dose-dependent way in both monolayer and 3D cell cultures. The MCF-7 cell spheroids had greater resistance to the cytotoxic activity of the extract in 3D cell culture than the MDA-MB-231 cell spheroids. The morphological changes of the cells treated with C. cancellatus stigmas extract were indicative of apoptosis. Zymography analysis revealed a similar trend of matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) activity in the treated cells with C. cancellatus extract in comparison with doxorubicin treatment as a positive control. The findings of this research indicate that the ethanolic extract of C. cancellatus stigmas was a good source of bioactive metabolites with anticancer activity.


Introduction
Over 80 species of the genus Crocus L. have been identified worldwide [1]. e most common species of this genus is Crocus sativus. Saffron, the most costly food colorant and flavor in the world, is derived from the dried stigmas of C. sativus, and it has been used in folk medicine to treat a range of disorders since ancient times [1][2][3][4]. Previous research has uncovered important information about the anti-cancer properties of saffron and its constituents [5]. However, there are no significant findings regarding the underlying compounds and anticancer characteristics of another Crocus species known as Crocus cancellatus subsp. damascenus [1]. e main bioactive constituents present in saffron are crocetin, crocin, safranal, and picrocrocin. Crocetin and crocin (a diester of crocetin with gentiobiose) are carotenoid compounds that have been shown in cell culture systems and animal models to be efficient at preventing or treating cancer. Safranal is a cyclic terpenic aldehyde obtained from the enzymatic and thermal degradation of picrocrocin and zeaxanthin. Picrocrocin is a monoterpene glycoside [6,7]. Crocus cancellatus is abundant throughout Iran, especially in the province of Kurdistan [1]. e main phytochemicals and the cytotoxic properties of stigmas of this species have not been studied yet.
Chemometric methods can be used to analyze complex data in chemical and biological systems. Recently, a novel chemometrics method based on the combination of the region of interest (ROI) [8][9][10][11] and the multivariate curve resolution-alternating least squares (MCR-ALS) method [12][13][14] has been employed for filtering, compressing, and resolving the complex data sets that are gathered by LC-MS analysis. e ROI method reduces the raw MS data without considerable loss of helpful information or mass accuracy of the measured data by searching within the region of interest for the m/z domain [15,16]. e ROI method compresses a large amount of big complex data [9,10]. In the current study, ROI-MCR-ALS methodology based on LC-MS full scan data sets was performed to resolve and identify many compounds in C. cancellatus stigmas compared to C. sativus.
is research aimed to characterize the contents of the stigma ethanol extract of C. cancellatus and determine their effect on the growth of MDA-MB-231 and MCF-7 human breast cancer cells.
e Annexin V-FITC/Propidium iodide kit was obtained from MabTag's Company, Germany. e MDA-MB-231 (a highly invasive human breast cancer cell line) and MCF-7 (human breast adenocarcinoma cell line) were purchased from the Pasteur Institute of Iran (Tehran).

Plant Collection and Extraction.
e plant of C. cancellatus subsp. damascenus (voucher specimen number: 725) was collected in autumn 2019 from the areas between latitude 35°18′36″N and longitude 46°57′26″E at an elevation of 1916 meters above sea level in Sanandaj, Kurdistan province, Iran. It was authenticated at the Research Center of Agricultural and Natural Resources of Sanandaj, Kurdistan province, Iran by Hosein Maroofi. e stigmas of the plants were separated and dried in the shade, powdered, and macerated with ethanol for 24 h in a dark place at a powder: solvent ratio of 1 g : 100 mL at room temperature. e extract was collected and filtered. e same material was subsequently extracted for the second and third time. Consequently, the ethanol solvent in the collected extract was concentrated using rotary evaporation. e concentrated extract was dried to determine the exact dry weight [17].

Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis. GC/MS analysis of the extracts was carried out using an Agilent 6890 series gas chromatograph equipped with a column VARIAN, cp-sil 8cb-ms (50 m length, 0.25 mm inner diameter, 0.25 m film thickness) coupled with an Agilent 5973 Network, mass selective detector. e moving phase was 99.999 percent helium with a steady flow rate of 1 mL/min via the column. e temperature was set at 60°C, ascending to 280°C, and the injector temperature was 280°C.
e injector volume was 1 μL. A 70 eV and the electron beam was used to generate ions. Masses between m/ z 40 and 600 were identified, and the acceleration voltage was turned on after a 4-minute solvent delay. e ingredients of the extracts were recognized by computing retention times and fragmentation patterns of the related peak compared to those of the NIST/Wiley internal reference mass spectrum library.

Liquid Chromatography with Mass Spectrometry (LC-MS).
e separation was carried out chromatographically on an Atlantis T3-C18 analytical column (3.0 m, 2.1 100 mm). At a flow rate of 0.15 mL/min, the column was eluted with a mobile phase composed of 95% methanol and 5% water containing 0.1 percent formic acid. e column was kept at a temperature of 45°C. e injection volume was 10 μL, and the analysis duration was 30 min. Electrospray ionization (ESI) was used in conjunction with a triple quadrupole mass analyzer in the positive mode, with the spray voltage set at 4500 V. e nebulizer gas was nitrogen (N2 Garde 5), and the nebulizer pressure was adjusted to 40 psi with a source temperature of 120°C. e collision energy was 35 eV.

Chemometric Data Analysis.
In this work, the ROIMCR method combining the regions of interest and Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) methods has been used [8]. is method searches the regions of interest in the LC-MS full scan data (MS-ROI) that is applied for compressing and filtering data without losing essential information on mass accuracy and spectral resolution. All the chemometrics analyses were carried out in MATLAB 2016b [8,9,12].

Region of Interest (ROI).
Compressing a large amount of data without reducing the information is a significant step in data analysis. In the present work, the region of interest (ROI) has been performed for filtering and compressing the LC-MS full-scan data without losing significant information on data resolution and the mass accuracy in the m/z domain [8,9]. e ROI method was used to create a data matrix suitable for multivariate modeling. is matrix contains the mass traces as well as the intensities of the same m/z values of distinguished dimensions [8,9,18]. ree parameters have a significant effect on the resulting mass intensity matrix: (1) the threshold of the mass intensity (SNRthr), (2) the mass accuracy or m/z error of the spectrometer, and (3) the minimum number of retention times that must be included in an ROI. e mentioned parameters in this study were 0.1% of the maximum MS signal intensity measured on each sample, 1 Da/e, and 50, respectively. e output parameters of this algorithm are a vector including the average of all m/z data from the same ROI, a matrix of data comprising MS spectra at each retention time scan (MS-ROI), and a cell array that contains the data regarding the m/z values, retention time, mass intensities, scan numbers, and average m/z values of ROIs. Finally, to be appropriate for MCR-ALS analysis, all ROI m/z values with significant traces were organized in the MSROI data matrices [8].

MCR-ALS Analysis.
Using a bilinear decomposition approach, MCR-ALS can resolve the profiles (spectra, concentration, pH profiles, time profiles, elution profiles, etc.) of the components in mixtures without providing any information regarding their compositions. is algorithm decomposed the instrumental response matrix (D) to the C matrix (concentration/elution profiles) and the S T matrix (pure spectra) of the components present in a mixture (equation (1)). e residual and nonmodeled information is included in the matrix, and the symbol T indicates a transposed matrix: In the first step of the MCR-ALS method, the purest spectra derived by the SIMPLISMA (SIMPLe-to-use Interactive Self-modeling Mixture Analysis) algorithm served as the initial estimations for singular value decomposition (SVD) analysis. en, suitable constraints must be applied to reduce the ambiguities associated with the resolution of the D matrix. In this study, nonnegativity and unimodality constraints were performed to optimize ALS for elution and mass spectra profiles. Also, 60 components containing one elution and mass spectra profiles were resolved by MCR-ALS [12,19]. An example of the resolution of one of the components is shown in Figure 1. e peak area and heights of each elution profile retrieved by MCR-ALS can be related to the concentration of components. Finally, the spectral m/z mass values for each spectrum and their elution profiles recovered by MCR-ALS were performed to identify possible chemical compounds in the samples.

MTT Assay.
e MTT test was used to evaluate the cytotoxic potential of the extracts against cancer cells [20]. In 96-well plates, 15,000 cells were distributed in 200 µl of growth media and incubated with different concentrations of the extract for 48 and 72 hours in an incubator at 37°C under 5% CO 2 pressure after adhering to the bottom of the plate and taking morphology. To quantify the number of living and dead cells in each well, the culture medium was drained, and after being washed with PBS, the MTT solution (0.5 mg/mL) was added. e plate was incubated for four hours at 37°C with 5% carbon dioxide pressure. e viable cells transform the MTT solution into formazan crystals. To dissolve formazan crystals, the wells were depleted of the solution, and 100 mL of DMSO was added. As soon as the crystals were completely dissolved in DMSO, the 570 nm absorbance of each well was measured by a microplate reader (BioTek, USA). To determine the IC 50 value, first, the inhibition percentage of each extract was calculated; next, the IC 50 value (concentration equivalent to 50 percent survival of cancer cells) was obtained for each extract using regression analysis of the dose-response curve.

Fluorescence Labeling of Cells with Acridine Orange (AO) and Ethidium Bromide (EB).
e cancer cells were incubated with the corresponding IC 50 values of the extract for 12 h. Following trypsinization and washing with PBS, the cells were mixed with 100 μL of acridine orange and ethidium bromide (1 : 100 mg/mL) solution and observed under a fluorescent microscope (Zeiss, Germany) [21]. Green-colored cells are considered viable, while red/yellow-colored cells are considered dead. For quantitative measurement, a minimum of 200 cells were counted in different fields under a fluorescence microscope. e percentage of cells undergoing apoptosis was calculated according to the following equation: The percentage of cells undergoing apoptosis � Total apoptotic cells Total cells counted × 100. (2)

Flow Cytometric Analysis of Cells with Apoptotic Markers.
One of the markers of apoptotic cells is the translocation of phosphatidylserine from inside the cell membrane to the exterior, which can be detected by staining cells with Annexin V-FITC. Propidium iodide is another fluorescent dye used to identify necrotic and late apoptotic cells. For this Evidence-Based Complementary and Alternative Medicine analysis, the cancer cells were cultured with the IC 50 values of the extract. After 48 hours, the cells were trypsinized, washed using PBS, and resuspended in the binding buffer. 5 µL of each fluorescent dye were added to the cell suspension, kept in the dark at room temperature for 15 minutes, and analyzed using flow cytometry (Partec PAS, Germany) without delay.

Gelatin Zymography.
Gelatin zymography is one of the effective methods for measuring MMP-2 (EC 3.4.24.24) and MMP-9 (EC 3.4.24.35) activities in cells and growth medium [22]. In order to perform the analysis, the medium from the cells treated with extract without serum was sampled, and the Bradford method was used to measure the total protein concentrations [23]. 20 μg of each sample was mixed with 5X nonreducing sample buffer (including 125 mM Tris HCl (pH 6.8), 20% glycerol, 4% SDS, and 0.01% bromophenol blue) and was loaded into each well of 10% acrylamide gel containing gelatin. e gel was run at 100 V, and after band separation, it was separated from glass and washed for 2 × 30 min with washing buffer (50 mM Tris HCl (pH 7.5), 1 μM ZnCl 2 , 5 mM CaCl 2 , and 2.5% Triton X-100) at room temperature with agitation. After washing, the gel was immersed in incubation buffer (50 mM Tris HCl (pH 7.5), 5 mM CaCl 2 , 1% Triton X-100, 1 μM ZnCl 2 ) for 5-10 min at 37°C with shaking. en, the gel was incubated in a fresh incubation buffer for 48 h, stained with staining solution (40 mL methanol, 10 mL acetic acid, 0.5 g Coomassie Blue, and 50 mL H 2 O), and incubated with destaining solution (10 mL acetic acid, 40 mL methanol, and 50 mL H 2 O), respectively. e white bands on a uniformly blue background of gel are indicative of MMP activity. ImageJ was used to analyze the gel bands.

Statistical Analysis.
ree independent experiments were used to obtain the data. e results are reported as the mean standard deviation. A t-test was conducted independently to analyze the data using the SPSS software (SPSS Inc., Chicago, IL, USA). e difference between the two groups was considered significant at a p value <0.05.  Table 1 ( e chromatogram is shown in Figure S1, supplemental file).

MCR-ALS of Full Scan LC-MS Chromatograms.
e MCR-ALS technique was applied to analyze the LC-MS data sets of the C. sativus and C. cancellatus extracts in the positive and negative MS ionization modes (see Figure 1). e profiles of elution for 19 components have been shown in Figure 1(a). Regarding the peak area of this component in both samples, it is clear that the concentration of this compound for samples of C. sativus and C. cancellatus is different. Figure 1(b) shows the retrieved mass spectrum of this ingredient using the MCR-ALS. Upon examination of the ion with the highest abundance at m/z (299), this component has been identified as an important component in the C. sativus and C. cancellatus samples. e proportion of this compound in the sample of C. sativus is much higher than in the sample of C. cancellatus. MCR-ALS identified all 60 components using a similar approach. Finally, 24 and 14 common metabolites for C. sativus and C. cancellatus samples, respectively, were identified in both positive and negative ionization modes. Table 2 shows the components retrieved by the MCR-ALS method for C. sativus and C. cancellatus. In this table, the name of the compounds,   Evidence-Based Complementary and Alternative Medicine measured mass, identified adducts, retention times, and peak area for samples of C. sativus and C. cancellatus are given.

Cytotoxicity Evaluation of the Stigma Ethanolic Extract of C. cancellatus on MDA-MB-231 and MCF-7 Cell Spheroids.
Tumor cells generally exhibit less sensitivity to chemotherapeutic agents in 3D cell culture models than those in monolayer culture [24]. Cell viability of MDA-MB-231 and MCF-7 spheroids following exposure to the various concentrations of stigma ethanolic extract of C. cancellatus was evaluated by the MTT test. As shown in Figure 3, spheroids' viability declined as a function of dosage and time compared  (Table 3) of the extract against MDA-MB-231 spheroids was approximately 2-3 times higher than the corresponding value in monolayer culture. MCF-7 spheroids had more resistance to the extract in comparison to MCF-7 in monolayer cell culture.

Discussion
Over the last decades, there has been a trend to find new sources of natural anticancer. Due to the side effects of synthetic anticancer agents, natural product-based drugs have been becoming more popular and have resulted in an increased interest in the search for chemotherapeutic agents in natural sources [25]. e dried stigmas of C. sativus (known as saffron) contain various anticancer agents, including safranal, crocin, picrocrocin, and crocetin [2]. C. cancellatus, a herbaceous plant of the family Iridaceae, is native to different areas of Iran, especially in Kurdistan province; however, no investigation has evaluated the main phytochemicals and the cytotoxic properties of stigmas of C. cancellatus [26]. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS) were used to determine the ingredients of the stigma ethanol extract of C. cancellatus. Chemometric analysis of the LC-MS full scan data was performed using the ROIMCR approach, which selects MS regions of interest (ROI) from full-scanning data sets and analyses them by the Multivariate Curve-Resolution Alternating Least Squares (MCR-ALS) chemometrics method for simultaneous resolution of the extract in comparison with C. sativus extract. Based on the most abundant ion at m/ z (299), safranal was identified as an important compound in the C. sativus and C. cancellatus samples. In total, 24 compounds were detected in negative ionization mode, among which 14 compounds were identified as ferulic acid, 3, 5, 5-trimethyl-4-hydroxy-1-cyclohexanon-2-ene, safranal, gamma-crocetin, nonanoate, carotene, crocusatin G, (all-E)crocetin, picrocrocin, 5-hydroxymethyl-2-furancarboxaldehyde, rhamnetin, beta-Sitosterol, norathyriol, and riboflavin. In positive ionization mode, 14 compounds were  Evidence-Based Complementary and Alternative Medicine detected in both samples, among which 9 compounds were identified as isopimpinellin, 4H-pyran-4-one-2, 3-dihydro-3, 5-dihydroxy-6-methyl, Osthol, 3, 5, 5-trimethyl-2-hydroxy-1, 4-cyclohexadion-2-ene, 2, 4, 4-trimethyl-3-formyl-6-hydroxy-2, 5-cyclohexadiene-1-one, Methyl arachidate, kaempferol 3-O-alpha-L-rhamnofuranoside, isophorone, and crocin 4. In saffron, the four most important bioactive substances are crocin, crocetin, picrocrocin, and safranal. Saffron's color, aroma, and flavor are determined by the crocin, safranal, and picrocrocin, respectively. Picrocrocin is a monoterpene glycoside, and the isolation of sugars from picrocrocin yields safranal, the essential oil of saffron. Glucosidic derivatives of crocetin are called crocin and are categorized according to the number of attached sugars [27,28]. Crocin 4 is a monoglucosyl ester of crocetin that was identified in C. cancellatus at m/z (169) and a retention time of 15.40 minutes. 3, 5, 5-Trimethyl-4-hydroxy-1cyclohexanon-2-ene, 2, 4, 4-trimethyl-3-formyl-6-hydroxy-2, 5-cyclohexadien-1-one, and isophorone are 6-membered ring compounds which have been previously reported as volatile constituents of saffron [29]. Kaempferol 3-O-alpha-L-rhamnofuranoside is a type of flavonoid found in herbs and spices [30]. It was determined through GC-MS analysis that the main compounds in the ethanolic stigmas extract of C. cancellatus are fatty acids (lauric acid, octanoic anhydride, myristic acid, pentadecanoic acid, palmitic acid, palmitic acid ethyl ester, margaric acid, linoleic acid, and stearic acid). Furthermore, GC-MS analysis revealed that the extract contained safranal. GC-MS analysis of the C. cancellatus extract by Loizzo et al. revealed that the most abundant compounds are 5-(hydroxymethyl)-2-furancarboxaldehyde, methyl oleate, methyl stearate, and osthol [31]. In recent years, C. sativus stigmas have been widely researched for their potential effects on cancer and other diseases [4,32]. According to the identification of the main bioactive constituents of saffron, the antiproliferative effect of C. cancellatus stigmas extract in both 2D monolayer and 3D cell cultures of MDA-MB-231 and MCF-7 human breast   [33], cytotoxicity results indicated that the antiproliferative activity of C. cancellatus is not dependent on the existence of estrogen receptors. is is the first report on the cytotoxic potential of C. cancellatus stigmas. e cytotoxicity effect of stigmas ethanolic extract on both breast cancer cells in spheroids was also validated. Spheroids are excellent models for tumor studying and cytotoxic agent screening [34]. C. cancellatus also exhibited cytotoxic activity against MDA-MB-231 and MCF-7 spheroids in a concentration-and time-dependent manner. Our findings suggest that spheroids are less susceptible to the cytotoxic activity of the extract than 2D cultures. Unlike 2D culture, the resistance of MCF-7 spheroids to the toxic effect of the extract was higher than that of MDA-MB-231 spheroids.
One of the major ways anticancer drugs suppress cancer cells is the induction of apoptosis [35]. e results of the AO/ EB double staining assay displayed that the morphological changes observed in cells treated with C. cancellatus are attributed to apoptosis. Apoptosis is distinguished from other types of cell death by specific morphological changes such as shrinkage of nuclei and cells, blebbing of the plasma membrane, and fragmentation of oligo-nucleosomal DNA [36]. Unlike apoptosis, loss of cell membrane integrity is a typical feature of necrosis. e accompanying cell membrane changes of apoptosis can be differentiated from necrosis by the Dual AO/EB fluorescent staining test. AO can pass through the intact cell membrane, bind to DNA, and emit green fluorescence. EB has only been permitted to cross damaged membranes and emit orange-red fluorescence upon binding to DNA. So, cells that are in the early stages of apoptosis, as well as viable cells, appear green, while those in the late stages of apoptosis, as well as necrotic cells, appear orange-red. Early and late apoptotic cells exhibit bright green and orange fluorescent spots within the nuclei, respectively, as a result of nuclear fragmentation and chromatin condensation; these phenomena do not occur in the nuclei of necrotic cells [37]. e stages of apoptosis and cell death in single-cell suspensions were analyzed by Annexin-V-FITC and propidium iodide (PI). A unique biochemical event in apoptotic cells is the translocation of phosphatidylserine (PS) from the inner side of the cytoplasmic membrane to the outer surface of the cell membrane, a process that Annexin-V-FITC can monitor. During this reaction, Annexin-V-FITC is specifically bound to PS and detects early apoptotic cells. As PI (an intercalating agent) is unable to penetrate healthy cell membranes, cells in the late stages of apoptosis and necrotic cells are stained by it due to the loss of cell membrane integrity [38]. Annexin V-FITC/PI staining exhibited that stigma ethanol extract of C. cancellatus promoted early apoptosis in MDA-MB-231 and MCF-7 cells after 48 h of incubation. It has been shown that overexpression of matrix metalloproteinases (MMPs) by tumor and stromal cells has been linked to cell migration and metastasis [39]. MMPs are secreted and zinc-dependent endoproteinases that break down extracellular matrix (ECM) proteins (collagen, gelatin, fibronectin, vitronectin, and laminin) [40].  [41]. In an attempt to study the antimetastatic potential of the stigmas ethanol extract of C. cancellatus, the zymography method was used to determine the activity of MMP-2 and MMP-9 in the ECM of treated cells. e findings suggest that the behavior of MMP-2 and MMP-9 in the treated cells with the extract is similar to that of the treated cells with doxorubicin as a positive control.

Conclusion
is work has shown that stigma ethanolic extract of C. cancellatus inhibited the proliferation of MDA-MB-231 and MCF-7 human breast cancer cell lines. e AO/EB double staining assay and Annexin V-FITC/PI validated that the treated cells were undergoing apoptosis. e antimetastatic effect of the stigma ethanol extract of C. cancellatus was similar to doxorubicin. Analyses by GC-MS showed safranal to be present in C. cancellatus extract. A potent strategy based on selecting the LC-MS regions of interest (ROI) and multivariate curve resolution (MCR-ALS) was carried out to analyze C. cancellatus LC-MS data compared to C. sativus. e metabolites and compounds present in the C. sativus and C. cancellatus samples were analyzed, and the concentration differences between the two samples were evaluated. e current investigation provides essential intuitions into the antiproliferative effect of C. cancellatus, which may be related to the presence of safranal, crocin, crocetin, and picrocrocin.

Data Availability
e research data used to support the findings of this study are included in the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.