LC-ESI QToF MS Non-Targeted Screening of Latex Extracts of Euphorbia seguieriana ssp. seguieriana Necker and Euphorbia cyparissias and Determination of Their Potential Anticancer Activity

Euphorbia seguieriana ssp. seguieriana Necker (ES) and Euphorbia cyparissias (EC) with a habitat in the Deliblato Sands were the subject of this examination. The latexes of these so far insufficiently investigated species of the Euphorbia genus are used in traditional medicine for the treatment of wounds and warts on the skin. To determine their chemical composition, non-targeted screening of the latexes’ chloroform extracts was performed using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry employing an electrospray ionization source (LC-ESI QTOF MS). The analysis of the obtained results showed that the latexes of ES and EC represent rich sources of diterpenes, tentatively identified as jatrophanes, ingenanes, tiglianes, myrsinanes, premyrsinanes, and others. Examination of the anticancer activity of the ES and EC latex extracts showed that both extracts significantly inhibited the growth of the non-small cell lung carcinoma NCI-H460 and glioblastoma U87 cell lines as well as of their corresponding multi-drug resistant (MDR) cell lines, NCI-H460/R and U87-TxR. The obtained results also revealed that the ES and EC extracts inhibited the function of P-glycoprotein (P-gp) in MDR cancer cells, whose overexpression is one of the main mechanisms underlying MDR.


Introduction
Cancer is the second leading cause of mortality in the world.Many natural compounds such as anthracyclines (e.g., doxorubicin, DOX), vinca alkaloids (e.g., vincristine), podophyllotoxins (e.g., etoposide), and taxanes (e.g., taxol) are used for cancer therapy [1].However, the main cause of unsuccessful cancer treatment is the development of multidrug resistance (MDR) [2].MDR is a phenomenon that indicates that cancer cells exhibit resistance to a number of chemotherapeutic agents with different structure and mode of action.One of the most relevant mechanisms underlying MDR is a decrease in the intracellular drug concentration due to the over-expression of the membrane transporter Pglycoprotein (P-gp) [3].Thus, P-gp has become a significant target for overcoming MDR [4].Many natural compounds from various sources possess the potential to modulate MDR [5].Different metabolites isolated from Euphorbia ssp., besides antiproliferative and cytotoxic effects, showed potential to overcome MDR by P-gp inhibition [6].
The Euphorbia genus consists of over 2000 species of annual, biennial, or perennial flowering herbaceous plants, shrubs, trees, as well as cactus-like plants.Members of the genus are spread throughout the terrestrial part of the globe and grow in almost all habitats, in very different climatic conditions and soils of different quality.As a result of their great diversity in morphology, geographical distribution and habitat, Euphorbia species synthesize the most diverse metabolites, many of which are found in their milky latex.Latex is produced by all Euphorbia species in specialized laticifer cells and has a defensive role-it protects the plant from both mechanical injuries and injuries caused by herbivores (insects and mammals) [7] and various microorganisms.Latex was found to contain a broad range of specialized metabolites, different from those found in the corresponding plants, such as terpenoids, cardenolides, cerebrosides, alkaloids, and phenolics [8][9][10], which are partly responsible for their antibacterial, antifungal, anthelmintic, cytotoxic, and insectrepellent activities [11].Latexes have also been recognized as reservoirs of defense-related proteins [7,12].
The cypress spurge E. cyparissias L. is a hardy perennial, herbaceous plant growing in a wide range of habitats, from lowland areas to alpine locations.It is widely distributed in Europe (including in the Balkan Peninsula and Serbia) and Asia Minor, but it also occurs as an introduced plant in North America, Australia, Japan, and Hawaii.When the plant is cut, it secretes a white, bitter, and very spicy milk that causes inflammation and blisters on the skin and ocular inflammation [29].The seeds are also pungent and poisonous, as is the whole plant.The roots of the plant were once used as a purgative.In people, the plant is still used for external treatments-removal of warts-while it is rarely used for its internal effects (inducing vomiting and purging).In previous investigations, ingenanes [30] and jatrophanes [31] were isolated from the roots and whole plant, respectively.In plant material other than latex, triterpenes [32][33][34][35], glycolipids [36], and flavonoids [37,38] were identified.For latex, only the identification of serine proteases [39] and invertase [40] has been reported.
The aim of the present work was to examine the chemical profiles of chloroform extracts of the latexes of Euphorbia seguieriana ssp.segiueriana Necker (ES) and Euphorbia cyparissias L. (EC) as sources of bioactive chemicals and whether these extracts can inhibit cancer cell growth and modulate P-gp function.

Non-Targeted Screening of the Latex Chloroform Extracts Using Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Employing an Electrospray Ionization Source
During the search for new sources of bioactive compounds, the chemical profiles of chloroform extracts of the latexes of ES and EC were investigated.For that purpose, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-ESI QToF MS) in positive ion mode was employed.The total ion chromatograms of the chloroform extracts of the ES and EC latexes, obtained as a result of the analysis, are shown in Figures 1 and 2, respectively.

Non-Targeted Screening of the Latex Chloroform Extracts Using Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Employing an Electrospray Ionization Source
During the search for new sources of bioactive compounds, the chemical profiles of chloroform extracts of the latexes of ES and EC were investigated.For that purpose, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-ESI QToF MS) in positive ion mode was employed.The total ion chromatograms of the chloroform extracts of the ES and EC latexes, obtained as a result of the analysis, are shown in Figures 1 and 2, respectively.The non-targeted screening of the ES extract allowed the detection of a total of 31 components, while a total of 49 metabolites were detected in the EC extract (Tables 1 and  2, respectively).The chemical formulas of these components were determined based on mass accuracy, the number of double bond equivalents, the valency based on the nitrogen rule, and the isotopic pattern match of the suggested formula with the observed mass spectrum, as well chemical expertise.For a tentative identification of the metabolites, an extensive online literature search was conducted using the terms "Euphorbia, Euphorbiaceae" on SciFinder, an online database, for each proposed chemical formula.Also, the characteristic fragmentation pattern observed in the mass spectra of some of the detected

Non-Targeted Screening of the Latex Chloroform Extracts Using Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Employing an Electrospray Ionization Source
During the search for new sources of bioactive compounds, the chemical profiles of chloroform extracts of the latexes of ES and EC were investigated.For that purpose, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-ESI QToF MS) in positive ion mode was employed.The total ion chromatograms of the chloroform extracts of the ES and EC latexes, obtained as a result of the analysis, are shown in Figures 1 and 2, respectively.The non-targeted screening of the ES extract allowed the detection of a total of 31 components, while a total of 49 metabolites were detected in the EC extract (Tables 1 and  2, respectively).The chemical formulas of these components were determined based on mass accuracy, the number of double bond equivalents, the valency based on the nitrogen rule, and the isotopic pattern match of the suggested formula with the observed mass spectrum, as well chemical expertise.For a tentative identification of the metabolites, an extensive online literature search was conducted using the terms "Euphorbia, Euphorbiaceae" on SciFinder, an online database, for each proposed chemical formula.Also, the characteristic fragmentation pattern observed in the mass spectra of some of the detected The non-targeted screening of the ES extract allowed the detection of a total of 31 components, while a total of 49 metabolites were detected in the EC extract (Tables 1 and 2, respectively).The chemical formulas of these components were determined based on mass accuracy, the number of double bond equivalents, the valency based on the nitrogen rule, and the isotopic pattern match of the suggested formula with the observed mass spectrum, as well chemical expertise.For a tentative identification of the metabolites, an extensive online literature search was conducted using the terms "Euphorbia, Euphorbiaceae" on SciFinder, an online database, for each proposed chemical formula.Also, the characteristic fragmentation pattern observed in the mass spectra of some of the detected metabolites allowed their closer class determination (Figures S1-S79, Supplementary Materials).Diterpenoids were found to represent the most predominant chemical class in the examined extracts, but a smaller number of triterpene derivatives (in the EC extract) were also identified.LC-ESI QToF MS is more suitable for the analysis of diterpenes and other highly oxygenated molecules than for that of triterpene derivatives, which contain a small number of centers that can be ionized under soft ionization conditions.The weak ionization of triterpene derivatives can lead to the wrong conclusion that the presence of these compounds in the tested sample is small or negligible; however, our experience has shown that triterpenes are generally more abundant than expected, especially in non-polar extracts.
In soft ionization conditions, such as those used for recording the mass spectra of the components of the examined extracts, without additional collision energy, some compounds generate only quasimolecular ions, while other compounds spontaneously fragment (Figures S1-S79, Supplementary Materials), which indicates differences in the stability of their skeletons.Some diterpene esters produce fragment ions resulting from the neutral loss of water or acyl chains, which are not informative on the diterpene skeleton, but others, due to the presence of a different number of oxygenated groups, produce different characteristic fragment ions that could provide indications about the diterpene skeleton.

Examination of the Anticancer Activity of the ES and EC Latex Extracts
To evaluate the impact of the EC and ES extracts on the growth of human cell lines, including both normal and cancerous ones, we conducted an MTT assay.Our study included five different human cell lines, comprising two pairs of sensitive and MDR cancer cell lines (non-small cell lung carcinoma NCI-H460 and NCI-H460/R and glioblastoma U87 and U87-TxR cell lines) and normal human embryonic pulmonary fibroblasts (MRC-5).The results of the assay, which are outlined in Table 3, revealed that both EC and ES extracts had a significant impact on cancer cell growth, with IC50 values below 40 µg mL −1 .However, we also observed that the efficacy of the extracts was affected by the presence of the MDR phenotype in NCI-H460/R cells.This was evidenced by a significant increase in the IC50 values for the MDR cells compared to those determined for the corresponding, sensitive NCI-H460 cells.It was also noted that this resistant profile was more pronounced in the case of the EC extract.Interestingly, both extracts were found to be almost equally effective in the sensitive U87 and MDR U87-TxR glioblastoma cells.Our analysis also indicated that the extracts were not selective towards cancer cells, as the normal MRC-5 cells exhibited lower IC50 values compared to those obtained for the cancer cells.To investigate whether the ES and EC extracts affect the function of the P-gp pump in MDR cancer cells, the intracellular accumulation of the P-gp substrate Rho 123 was analyzed by flow cytometry after a 30 min treatment (Figure 3).Both extracts were applied at 20 µg mL −1 .As shown by a marked increase in Rho 123 intracellular accumulation, the ES and EC extracts significantly inhibited P-gp function in both MDR cancer cell lines.
U87 and U87-TxR cell lines) and normal human embryonic pulmonary fibroblasts (MRC-5).The results of the assay, which are outlined in Table 3, revealed that both EC and ES extracts had a significant impact on cancer cell growth, with IC50 values below 40 µg mL −1 .However, we also observed that the efficacy of the extracts was affected by the presence of the MDR phenotype in NCI-H460/R cells.This was evidenced by a significant increase in the IC50 values for the MDR cells compared to those determined for the corresponding, sensitive NCI-H460 cells.It was also noted that this resistant profile was more pronounced in the case of the EC extract.Interestingly, both extracts were found to be almost equally effective in the sensitive U87 and MDR U87-TxR glioblastoma cells.Our analysis also indicated that the extracts were not selective towards cancer cells, as the normal MRC-5 cells exhibited lower IC50 values compared to those obtained for the cancer cells.To investigate whether the ES and EC extracts affect the function of the P-gp pump in MDR cancer cells, the intracellular accumulation of the P-gp substrate Rho 123 was analyzed by flow cytometry after a 30 min treatment (Figure 3).Both extracts were applied at 20 µg mL −1 .As shown by a marked increase in Rho 123 intracellular accumulation, the ES and EC extracts significantly inhibited P-gp function in both MDR cancer cell lines.

Non-Targeted Screening of the Latex Chloroform Extracts Using Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Employing an Electrospray Ionization Source
The soft ionization conditions applied for the LC-ESI QToF MS analysis in positive ion mode allowed, based on the precisely measured mass of molecular ions, the determination of the molecular formula of the components present in the tested latex chloroform extracts of ES and EC, while an extensive online literature search using the terms "Euphorbia, Euphorbiaceae" in SciFinder, an online database, and the characteristic fragmentation pattern observed in the corresponding mass spectra enabled the tentative identification and chemical class determination of the majority of the components (Tables 1 and 2, Figures S1-S79, Supplementary Materials).In total, twenty components could not be tentatively identified in this way, seven of which were in the ES extract ( Diterpenoids were found to represent the most predominant chemical class in the examined extracts, but triterpene derivatives (in the EC extract) were also identified.
The compound with molecular formula C 39 H 45 NO 12 was detected in both extracts, but at different retention times in the chromatograms (1: t R = 5.26 min in the ES extract, and 43: t R = 7.76 min in the EC extract), indicating the existence of two different metabolites.Almost half of the detected metabolites in the ES extract appeared to contain nitrogen, while in the EC extract, only three metabolites, including amino acid 32 (C 7 H 15 NO 2 at t R = 1.31 min), were shown to contain nitrogen, thus indicating the presence or absence of a nicotinoyl ester group in their structures.Only three metabolites detected in the ES extract showed the same molecular formulas as myrsinanes isolated and characterized in previous research on E. seguieriana [14]; those metabolites are 4: C 39 H 43 NO 11 , t R = 6.52 min, 9: C 35 H 43 NO 11 , t R = 7.28 min, and 17: C 40 H 45 NO 11 , t R = 9.36 min.Ingenanes contained in the latex of E. seguierina [25,26] were not detected in our study in the ES extract.In the EC extract, only four metabolites, i.e., three ingenanes ( , t R = 16.18 min), showed the same molecular formulas as those of compounds isolated and characterized in previous research on E. cyparissias [30,34].However, two jatrophane diterpenes (cyparissins A and B) with molecular formula C 38 H 42 O 12 , previously isolated from E. cyparissias [31], were not detected in the examined EC extract.These findings indicate the ecological importance of the collection site.
Fragment ions 311, 293, and 265, characteristic of phorbol esters (PEs) [160] and some ingenanes [161], could be observed in the mass spectrum of compound 28, while, according to the literature data, the only compound with molecular formula C 38 H 50 O 9 so far identified in the genus Euphorbia belong to the dPE type of diterpenes [68,69].Similarly, the same fragment ions occurred in the mass spectrum of compound 67, while the only compound with molecular formula C 37 H 50 O 8 so far identified in the Euphorbiacea family belong to the daphnane type of diterpenes [129].
In the ES extract, four pairs of isobaric compounds were detected: two compounds with molecular formula C 36 H 46 O 12 -7 at t R = 7.12 and 13 at t R = 8.39 min-and two compounds with molecular formula C 41 H 48 O 12 -18 at t R = 9.62 and 22 at t R = 10.69 min-while only one Euphorbia/Euphorbiaceae premyrsinane with a corresponding molecular formula has been identified from each pair so far [44,[51][52][53]61], in addition to two compounds with molecular formula C 36 H 48 O 12 -10 at t R = 7.33 and 14 at t R = 8.77 min-corresponding to two known premyrsinanes [46,47,57], and two compounds with molecular formula C 36 H 50 O 8 -29 at t R = 13.92 min and 30 at t R = 14.13 min-whose mass spectra showed fragment ions corresponding to the loss of a water molecule, as well as fragment ions 313, 295, and 267.The only compound with the same molecular formula so far identified in the genus Euphorbia belongs to the PE type of diterpene esters [70][71][72][73].
In the EC extract, five pairs of isobaric compounds were detected: two compounds with molecular formula C 38 H 44 O 12 -38 at t R = 5.94 min and 40 at t R = 6.17 min-in whose mass spectra, fragment ions corresponding to the loss of a water molecule and a benzoic acid molecule could be observed, as occurs with four known jatrophans with the same formula [77, 102,103]; two compounds with molecular formula C 38 H 42 O 11 -44 at t R = 7.88 min and 47 at t R = 8.70 min-with the observation, in the mass spectrum of the latter, of a fragment ion characteristic of the loss of benzoic acid, which is a substituent in three ingols [88,101,105] and one jatrophane [86]; two compounds with molecular formula C 40 H 44 O 12 -48 at t R = 9.26 min and 50 at t R = 9.47 min-in whose mass spectra, fragment ions corresponding to the loss of a benzoic acid molecule, present as a substituent in two known ingols [88] and one known jatrophane [86,93,107], could be observed; two compounds with molecular formula C 38 H 48 O 12 -54 at t R = 10.19 min and 56 at t R = 10.33 min-corresponding to two known jatropahanes [114,115] and one known myrsinane [57]; and two compounds with molecular formula C 39 H 54 O 8 -69 at t R = 14.22 min and 72 at t R = 15.63 min-corresponding to two known ingenanes [138].
Compounds 27 and 61, as well compounds 64 and 70, so far undescribed in the genus Euphorbia and Euphorbiaceae family, produced fragment ions 313, 295, and 267, characteristic of the IE/dPE type of diterpenes [160], and fragment ions 311, 293, and 265, characteristic of the PE type of diterpenes and of some ingenanes [160,161].
The incomplete identification of the components present in the investigated extracts is the main drawback of this study and reflects the limitations of LC-ESI QToF MS in the annotation of compounds such as diterpene esters.For the complete identification of the components present in the examined extracts, the isolation and characterization of the compounds are required.

Examination of the Anticancer Activity of the ES and EC Latex Extracts
As shown by the analysis of the data available in the literature on the biological activities of the classes of molecules detected in the ES and EC extracts by LC-ESI QToF MS, the results obtained in this research confirmed the literature data.Our research indicated that both extracts of EC and ES have the potential to inhibit the growth of cancer cells.However, their effectiveness may be reduced in the case of MDR cancer cells, especially that of the EC extract.We discovered that both extracts could increase the accumulation of the P-gp substrate Rho123, which suggests that some compounds present in the extracts may be P-gp substrates that can also competitively inhibit P-gp activity.This is likely the reason for the decreased efficacy of the extracts in MDR cancer cells, such as MDR non-small cell carcinoma cells.Additionally, some components of the extracts are toxic to normal cells, which raises concerns about their use as anticancer agents.Nevertheless, the presence of different bioactive compounds suggests that some of them may be selective against cancer cells, while others are not.Therefore, further testing of isolated compounds is necessary to identify the best candidates as anticancer agents and lead compounds.
The potential of ES and EC to inhibit P-gp could be attributed to jatrophane derivatives identified in both extracts.In fact, the largest number of identified metabolites in the EC extract belong to the jatrophane class, while in the ES extract, jatrophane derivatives appeared to be the second most abundant metabolites.Our previous study demonstrated that jatrophane diterpenoids isolated from the latex of Euphorbia dendroides were able to modify P-gp function in three different human MDR cancer cell lines, i.e., non-small cell lung carcinoma, colorectal carcinoma, and glioblastoma cell lines [162].Further study also showed that jatrophane diterpenoids isolated from the latex of Euphorbia nicaeensis collected in Serbia possessed P-gp-inhibiting activity in two MDR cancer cells of different origin [58].Also, other compounds detected in the EC and ES extracts, such as lathyranes, are known as potent P-glycoprotein inhibitors in the treatment of multidrug-resistant (MDR) cancers [88,163,164].Jo et al. determined the anti-proliferative potential of daphnane derivatives in lung cancer cells, finding IC 50 values in the nM range [165].At the same time, the tested compounds showed selectivity towards carcinoma cells compared to MRC-5 cells [165].The difference in the IC 50 values of the examined extracts for the NCI-H640 cell line and the stronger anti-cancer activity of the EC extract compared to the ES extract can be explained by the potential presence of daphnane diterpenes in the EC extract.Strong inhibitory activity against the human glioblastoma cell line U87 was demonstrated for triterpene lanostane derivatives isolated from the fungus Naematoloma fasciculare [166].Lanostane derivatives are frequent metabolites in the Euphorbia genus; so, additional experiments and compound isolation are necessary to determine whether lanostane derivatives are responsible for the inhibitory activity of the extracts in the U87 cell line [134,167].
of the solvent under a mild nitrogen stream, the solid residue was dissolved in 1 mL of a mixture of dichloromethane and acetonitrile (1:5, v/v), filtered through Captiva RC 0.45 mm filters (Agilent Technologies, Waldbronn, Germany), and analyzed by liquid chromatography-electrospray quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS), as described below.For the untargeted analysis, the prepared samples were injected into the analyzing system, including a liquid chromatograph (1290 Infinity LC system; Agilent Technologies, Waldbronn, Germany) with a quaternary pump, a column oven, and an autosampler, connected to a quadrupole time-of-flight mass detector (6550 iFunnel Q-TOF MS, Agilent Technologies; Santa Clara, CA, USA) equipped with a dual-spray Agilent Jet Stream (AJS) electrospray ion source [168,169].In this case, the separation of the compounds was performed using a Zorbax Eclipse XDB-C18 RRHT column (100 × 4.6 mm, 1.8 µm, Agilent Technologies).The mobile phase was composed of solvents A (water containing both 0.1% formic acid and 5 mM ammonium formate) and B (ACN containing 0.1% formic acid).The following gradient program was used: 0-2 min 60% B, 2-12 min 60-95% B, 12-18 min 95% B, and 5 min 60% B. The mobile phase flow rate was 0.60 mL min −1 , the column temperature was 50 • C, and the injection volume of the samples was 0.1 µL.After separation, the compounds were analyzed using a mass detector.Positive ion mode was used, and the instrument was operated in accurate TOF/MS scanning mode in the m/z range of 100-2000, under the following conditions: capillary voltage, 3500 V, fragmentor voltage, 70 V, nozzle voltage, 1000 V, skimmer 1, 65 V, octupole RF peak, 750 V, desolvation gas (nitrogen) temperature, 200 • C, desolvation gas (nitrogen) flow, 14 L min −1 , nebulizer pressure, 35 psi, sheath gas (nitrogen) temperature, 350 • C, and sheath gas (nitrogen) flow, 11 L min −1 .A calibrating solution containing internal reference masses at m/z 121.0508 and 922.0098 was used in conjunction with an automated calibration delivery system to obtain accurate mass measurements for each peak in the total ion chromatogram.A personal computer system running Agilent MassHunter software (revisions B.06.01 and B.07.00) was used for data acquisition and processing.Extraction of the raw data (d) using both the find-by-molecular-feature (MFE) and the find-by-formula algorithms (FBF) in Agilent MassHunter Qual.software (revision B.07.00) allowed the detection of compounds in the tested samples.

Drugs
The extracts of EC and ES were kept as 20 mg mL −1 stocks in 100% ethanol at −20 • C. Working solutions were prepared in deionized water.

Cells and Cell Culture
The NCI-H460 and U87 cell lines were bought from the American Type Culture Collection, Manassas, VA, USA, while the MRC-5 cell line was obtained from the European Collection of Authenticated Cell Cultures, Salisbury, UK.NCI-H460/R and U87-TxR cells were created by exposing NCI-H460 and U87 cells to increasing concentrations of doxorubicin and paclitaxel, respectively, in order to kill sensitive cells and obtain cells resistant to many structurally and functionally unrelated drugs [170,171].NCI-H460 and NCI-H460/R cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine, and an antibiotic-antimycotic mixture, U87 and U87-TxR cells were cultured in MEM medium supplemented with 10% fetal bovine serum, L-glutamine, antibiotics, and non-essential amino acids, and MRC-5 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 4 g L −1 of glucose, L-glutamine, and an antibiotic-antimycotic mixture.The cells were sub-cultured twice a week and seeded into fresh medium at a density of 8000 cells cm −2 (NCI-H460 and NCI-H460/R cells) or 16,000 cells cm −2 (U87, U87-TxR, and MRC-5 cells).

Cell Viability Assay
To determine cell viability, we employed the MTT assay, which is based on the ability of active mitochondria in living cells to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide into a formazan dye [172].We initially seeded the cells in 96-well tissue culture plates, seeding 2000 cells/well for NCI-H460 and NCI-H460/R cells and 4000 cells/well for U87, U87-TxR, and MRC-5 cells, and incubated them overnight in appropriate medium.We then treated the cells with varying concentrations of the EC and ES extracts-1, 5, 10, 25, and 50 µg mL −1 -for 72 h.
Following the treatment, we added MTT to each well at a final concentration of 0.2 mg mL −1 and left it for 4 h.We subsequently dissolved the formazan product in dimethyl sulfoxide and measured the absorbance at 570 nm using an automatic microplate reader (Multiskan Sky from Thermo Scientific, Waltham, MA, USA).Using non-linear regression analysis in GraphPad Prism 8 software, San Diego, CA, USA, we calculated the IC50 values, which represent the concentration of each extract that inhibited cell growth by 50%.

Rhodamine 123 Accumulation Assay
We conducted an investigation using flow cytometry to examine the function of Pgp, a protein that transports substances out of cells.Specifically, we wanted to see how the EC and ES extracts affected the accumulation of the P-gp substrate rhodamine 123 (Rho123) [173] in two types of P-gp-overexpressing cells (NCI-H460/R and U87-TxR) and compared the results with those from control cells (NCI-H460 and U87).To carry out the experiment, we grew all cell lines to 80% confluence in 25 cm 2 flasks, collected the cells, and put them in a solution containing Rho123 (2.5 µmol L −1 ).We immediately treated the MDR cells with the EC and ES extracts (20 µg mL −1 , the average IC50 calculated for all tested cancer cell lines) and incubated them at 37 • C in 5% CO 2 for 30 min.After the accumulation period, we washed the samples twice, collected the cells, and analyzed them using a CytoFLEX flow cytometer (Beckman Coulter, IN, USA).The orange fluorescence of Rho123 was measured on fluorescence channel 1 (FL1) at 525 nm.We tested at least 20,000 events for each sample, and the mean fluorescence intensities were analyzed using Summit v4.3 software (Dako Colorado Inc., Fort Collins, CO, USA).We analyzed the mean ± SEM values from three independent experiments using GraphPad Prism 8 (San Diego, CA, USA) and used Sidak's multiple comparison test for two-way ANOVA for the statistical analysis.

Conclusions
The selected plant species proved to be a rich source of biologically active compounds, primarily from the class of diterpenes.The small number of references on the chemical composition of these plant species, as well as the very limited number of ambiguous literature data on the mass spectra of Euphorbia diterpenes indicate the necessity of a detailed examination of the numerous compounds of this class that we detected.From the available literature data, it is known that, from E. cyparissias, two jatrophane diterpenes (cyparissins A and B) with the molecular formula C 38 H 42 O 12 were isolated, which were not detected in the examined extract, which further indicates the need to investigate this plant species in more detail because habitat conditions can also significantly affect the metabolites synthesized by the plant.
Another important result from this study is the finding that the extracts obtained from E. seguieriana and E. cyparissias showed the ability to inhibit P-gp function.The results of our study may contribute to the development of more effective cancer treatments in the future.

Figure 1 .
Figure 1.Total ion chromatogram of the chloroform extract of the latex of E. seguieriana ssp.seguieriana Necker (ES).

Figure 2 .
Figure 2. Total ion chromatogram of the chloroform extract of the latex of E. cyparissias (EC).

Figure 1 .
Figure 1.Total ion chromatogram of the chloroform extract of the latex of E. seguieriana ssp.seguieriana Necker (ES).

Figure 1 .
Figure 1.Total ion chromatogram of the chloroform extract of the latex of E. seguieriana ssp.seguieriana Necker (ES).

Figure 2 .
Figure 2. Total ion chromatogram of the chloroform extract of the latex of E. cyparissias (EC).

Figure 2 .
Figure 2. Total ion chromatogram of the chloroform extract of the latex of E. cyparissias (EC).

Figure 3 .Figure 3 .
Figure 3. Flow cytometric profiles of Rho123 accumulation in NCI-H460/R (a) and U87-TxR (b) cells untreated and treated with 20 µg mL −1 of the ES and EC extracts.Sensitive NCI-H460 and U87 cells Figure 3. Flow cytometric profiles of Rho123 accumulation in NCI-H460/R (a) and U87-TxR (b) cells untreated and treated with 20 µg mL −1 of the ES and EC extracts.Sensitive NCI-H460 and U87 cells were used as a positive control for Rho 123 accumulation.Two independent experiments were performed (a minimum of 10,000 events were collected for each experimental sample).

Table 1 .
Tentative identification of the components of the chloroform extract of the latex of E. seguieriana ssp.seguieriana Necker (ES) by LC-QToF MS according to the literature data available in SciFinder, an online database.
* Components identified and confirmed using the molecular feature extraction (MFE) and find by formula algorithms of the MassHunter software (revision B.07.00), respectively./ Components that could not be tentatively identified by online literature search using the terms "Euphorbia, Euphorbiaceae" in SciFinder, an online database.

Table 2 .
Tentative identification of the components of the chloroform extract of the latex of E. cyparissias (EC) by LC-QToF MS according to the literature data available in SciFinder, an online database.No.
* Components identified using the molecular feature extraction (MFE) and find by formula algorithms of the MassHunter software (revision B.07.00), respectively./ Components that could not be tentatively identified by online literature search using the terms "Euphorbia, Euphorbiaceae" in SciFinder, an online database.

Table 3 .
Cell growth inhibition induced by the EC and ES extracts.