NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines

In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines. The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive (Caco-2, GEO, SW48), intrinsic (HT-29 and HCT-116), and acquired (GEO-CR, SW48-CR) cetuximab-resistant cell lines. Ononis diffusa showed remarkable cytotoxic activity, especially on the cetuximab-resistant cell lines. The active extract composition was determined by NMR analysis. Given its complexity, a partial purification was then carried out. The fractions obtained were again tested for their biological activity and their metabolite content was determined by 1D and 2D NMR analysis. The study led to the identification of a fraction enriched in oxylipins that showed a 92% growth inhibition of the HT-29 cell line at a concentration of 50 µg/mL.


Introduction
Natural products play a crucial role in the discovery of anticancer compounds even today, when new technologies are available to easily obtain a wide range of drug candidates. The success of natural products is inherent to their structure: they possess a well-defined three-dimensional scaffold with functional groups precisely oriented in space. Furthermore, they are characterized by an enormous structural and functional diversity [1,2]. These features confer them with extraordinary selectivity and specificity, when compared to artificially designed molecules [3]. Plants, in particular, are a unique source of chemicals since they use specialized metabolites to interact with the environment and in response to several biotic and abiotic stresses [4,5]. Humankind has, therefore, always exploited this ability to produce a wide and diversified array of chemicals [6].
In recent years, the need for anticancer compounds has become particularly pressing and, in this context, colorectal cancer is definitely under the spotlight. This cancer is indeed one of the leading causes of cancer-related death worldwide and one of the most frequently diagnosed malignant diseases in Europe [7]. Although the outcomes of patients with metastatic colorectal cancer (mCRC) has improved in recent years [8], new challenges are on the horizon. Nowadays, resistance to both chemotherapy and molecularly targeted therapies represents a major problem for setting up effective treatments. Specifically, Cell growth is expressed as percentage from control, and is plotted on the vertical axis, while doses of plant extracts are reported on the horizontal axis.

Cytotoxic Activity of the Crude Extract against Colon Cancer Cell Lines with Acquired Resistance to Cetuximab
Based on the significant activity shown by O. diffusa extract on the three colon cancer cell lines, including the intrinsically cetuximab-resistant ones, this extract was further explored for its activity against both cetuximab-sensitive (GEO and SW48) and secondary cetuximab-resistant cancer cell lines (GEO-CR and SW48-CR). The extract showed a remarkable inhibition of the proliferation of the acquired resistant cell lines ( Figure 2). Indeed, while a growth inhibition of 50% on SW48 cells was observed at the highest tested concentration (250 µ g/mL), a 50% inhibition of the corresponding secondary resistant cell line was observed at a dose as low as 50 µ g/mL. On the other hand, the activity on the GEO and GEO-CR cell lines was comparable; important effects were observed starting from a concentration of 150 µ g/mL of the extract.

NMR Profiling of the Extracts
NMR spectra were obtained for the extracts of the two Ononis species. The plant material was extracted with a mixture of methanol-d4 and phosphate buffer in D2O (1:1) and the solution thereby obtained was analyzed by NMR.
Although O. variegata showed no activity in the biological tests, a comparison of the profiles of the two species helped us narrow down the set of the possible bioactive com-  Cell growth is expressed as percentage from control, and is plotted on the vertical axis, while doses of plant extracts are reported on the horizontal axis.
2.1.2. Cytotoxic Activity of the Crude Extract against Colon Cancer Cell Lines with Acquired Resistance to Cetuximab Based on the significant activity shown by O. diffusa extract on the three colon cancer cell lines, including the intrinsically cetuximab-resistant ones, this extract was further explored for its activity against both cetuximab-sensitive (GEO and SW48) and secondary cetuximab-resistant cancer cell lines (GEO-CR and SW48-CR). The extract showed a remarkable inhibition of the proliferation of the acquired resistant cell lines ( Figure 2). Indeed, while a growth inhibition of 50% on SW48 cells was observed at the highest tested concentration (250 µ g/mL), a 50% inhibition of the corresponding secondary resistant cell line was observed at a dose as low as 50 µ g/mL. On the other hand, the activity on the GEO and GEO-CR cell lines was comparable; important effects were observed starting from a concentration of 150 µ g/mL of the extract.

NMR Profiling of the Extracts
NMR spectra were obtained for the extracts of the two Ononis species. The plant material was extracted with a mixture of methanol-d4 and phosphate buffer in D2O (1:1) and the solution thereby obtained was analyzed by NMR.
Although O. variegata showed no activity in the biological tests, a comparison of the profiles of the two species helped us narrow down the set of the possible bioactive compounds. It was clear, indeed, from the comparison of the 1 H-NMR spectra (Figure 3 On the other hand, the activity on the GEO and GEO-CR cell lines was comparable; important effects were observed starting from a concentration of 150 µg/mL of the extract.

NMR Profiling of the Extracts
NMR spectra were obtained for the extracts of the two Ononis species. The plant material was extracted with a mixture of methanol-d 4 and phosphate buffer in D 2 O (1:1) and the solution thereby obtained was analyzed by NMR.
Although O. variegata showed no activity in the biological tests, a comparison of the profiles of the two species helped us narrow down the set of the possible bioactive compounds. It was clear, indeed, from the comparison of the 1 H-NMR spectra (Figure 3), that there were metabolites present in both species that, furthermore, were the main components of O. variegata. These metabolites could not be considered responsible for the activity observed for O. diffusa extract. Besides several sugars, amino acids, and organic acids identified on the basis of literature data [19,20], O. variegata also showed signals of caffeic acid and of trigonelline ( Figure 3) [20,21].
there were metabolites present in both species that, furthermore, were the main components of O. variegata. These metabolites could not be considered responsible for the activity observed for O. diffusa extract. Besides several sugars, amino acids, and organic acids identified on the basis of literature data [19,20], O. variegata also showed signals of caffeic acid and of trigonelline ( Figure 3) [20,21]. . Spectra were acquired at 300 MHz, in 1:1 methanol-d4: buffer. Diagnostic signals of the main metabolites detected in both extracts are indicated on O. variegata spectrum by the following abbreviations: aa = amino acids, ala = alanine, asn = asparagine, ca = caffeic acid, ci = citric acid, glc = glucose, sucr = sucrose, tr = trigonelline.
All these metabolites were also detected in the O. diffusa extract. Other signals only present in O. variegata were very likely attributable to caffeoylquinic acids. However, as these signals were not detected in the active extract, the identification of the metabolites generating them was beyond the scope of the present work.
The aromatic and olefinic region of O. diffusa 1 H-NMR spectrum showed several peaks that were not detected in O. variegata ( Figure 3). Further differences were observed in the aliphatic region; protons at δH 1.23 and a triplet at δH 0.83 indicated the presence of alkyl chains. These signals are usually attributed to fatty acids. However, the solvent mixture herewith used would not explain the extraction of these compounds, which seem to be rather abundant in the extract.
In the attempt to assign signals to metabolites only present in O. diffusa, and therefore very likely responsible for the activity, an extensive 2D-NMR study of the extract was carried out. However, the main correlations detected for the signals in the aromatic region did not allow us to further identify the unknown metabolites. While the HSQC and COSY (Figures S1 and S2) experiments suggested the presence of further 1,2,4-trisubstituted ring systems, the long-range correlation experiments were not determining, since the main correlations detected were those of the already-known compounds (whose identity was therefore confirmed, Figure S3). Concerning the signals in the aliphatic region, the presence of an alkyl chain was undoubtful, based on the NMR data previously discussed. However, long-range correlations of protons resonating in this region with carbon signals  All these metabolites were also detected in the O. diffusa extract. Other signals only present in O. variegata were very likely attributable to caffeoylquinic acids. However, as these signals were not detected in the active extract, the identification of the metabolites generating them was beyond the scope of the present work.
The aromatic and olefinic region of O. diffusa 1 H-NMR spectrum showed several peaks that were not detected in O. variegata ( Figure 3). Further differences were observed in the aliphatic region; protons at δ H 1.23 and a triplet at δ H 0.83 indicated the presence of alkyl chains. These signals are usually attributed to fatty acids. However, the solvent mixture herewith used would not explain the extraction of these compounds, which seem to be rather abundant in the extract.
In the attempt to assign signals to metabolites only present in O. diffusa, and therefore very likely responsible for the activity, an extensive 2D-NMR study of the extract was carried out. However, the main correlations detected for the signals in the aromatic region did not allow us to further identify the unknown metabolites. While the HSQC and COSY ( Figures S1 and S2) experiments suggested the presence of further 1,2,4-trisubstituted ring systems, the long-range correlation experiments were not determining, since the main correlations detected were those of the already-known compounds (whose identity was therefore confirmed, Figure S3). Concerning the signals in the aliphatic region, the presence of an alkyl chain was undoubtful, based on the NMR data previously discussed. However, long-range correlations of protons resonating in this region with carbon signals in the range of 60-80 ppm suggested the possibility that the fatty acids present in the extracts could be oxygenated, as in the case of oxylipins.

Partial Purification of the Extract and Biological Activity
Since the 2D NMR of O. diffusa extract was dominated by the signals of the alreadyknown compounds, a partial purification of a larger quantity of extract was carried out. The extract was first partitioned with water and ethyl acetate. The water fraction obtained was chromatographed on amberlite (XAD-4 and XAD-7). The columns were eluted with methanol first, and then with water. The methanolic fractions were joined together due to their very similar TLC profiles. The fraction (OdM) thereby obtained was analyzed by NMR and the 1 H-NMR spectrum, along with the HSCQ experiment, clearly showed that the aim was achieved (Figure 4). The OdM spectra did not show signals of primary metabolites, caffeic acid, or trigonelline (eluting in the water fraction).
in the range of 60-80 ppm suggested the possibility that the fatty acids present in the extracts could be oxygenated, as in the case of oxylipins.

Partial Purification of the Extract and Biological Activity
Since the 2D NMR of O. diffusa extract was dominated by the signals of the alreadyknown compounds, a partial purification of a larger quantity of extract was carried out. The extract was first partitioned with water and ethyl acetate. The water fraction obtained was chromatographed on amberlite (XAD-4 and XAD-7). The columns were eluted with methanol first, and then with water. The methanolic fractions were joined together due to their very similar TLC profiles. The fraction (OdM) thereby obtained was analyzed by NMR and the 1 H-NMR spectrum, along with the HSCQ experiment, clearly showed that the aim was achieved (Figure 4). The OdM spectra did not show signals of primary metabolites, caffeic acid, or trigonelline (eluting in the water fraction). However, the spectrum was still rich in signals, especially in the aromatic region. The HSQC experiment ( Figure 4) and the other 2D NMR experiments revealed several correlations. In particular, it was observed that the singlet proton at δH 8.14 was not bound to a carbon atom; this value was in good agreement with a hydrogen atom of an amidic functional group. This signal heterocorrelated, in the CIGAR-HMBC experiment ( Figure S4), with a carbonyl carbon at δC 176.5. Furthermore, it also correlated, in the same experiment, with the quaternary carbon at δC 124.2, which was in turn correlated with two protons of a 1,2,4-tribubstituted aromatic system resonating at δH 6.90 (dd, J = 8.2; 2.0 Hz) and 6.97 (d, J =2.0 Hz). The latter signal showed cross peaks with two oxygenated aromatic carbons at δC 147.8 and 146.1, both of which correlated with a third aromatic proton at δH 6.76 (d, J = 8.2 Hz) and with a singlet methylene at δH 5.87 (δC 101.2), indicating the presence of a 3,4dioxymethylenephenyl group bound to the amide nitrogen. In addition, in the CIGAR experiment, the amidic proton correlated with a further aromatic carbon at δC 157.9, which in turn displayed cross-peaks with a proton at δH 8.01 (d, J = 9.0 Hz), as an indicator of a second 1,2,4-tribubstituted aromatic system. This proton correlated in the long-range experiment with the amide carbonyl and with a quaternary aromatic carbon at δC 118.8, However, the spectrum was still rich in signals, especially in the aromatic region. The HSQC experiment ( Figure 4) and the other 2D NMR experiments revealed several correlations. In particular, it was observed that the singlet proton at δ H 8.14 was not bound to a carbon atom; this value was in good agreement with a hydrogen atom of an amidic functional group. This signal heterocorrelated, in the CIGAR-HMBC experiment ( Figure S4), with a carbonyl carbon at δ C 176.5. Furthermore, it also correlated, in the same experiment, with the quaternary carbon at δ C 124.2, which was in turn correlated with two protons of a 1,2,4-tribubstituted aromatic system resonating at δ H 6.90 (dd, J = 8.2; 2.0 Hz) and 6.97 (d, J = 2.0 Hz). The latter signal showed cross peaks with two oxygenated aromatic carbons at δ C 147.8 and 146.1, both of which correlated with a third aromatic proton at δ H 6.76 (d, J = 8.2 Hz) and with a singlet methylene at δ H 5.87 (δ C 101.2), indicating the presence of a 3,4-dioxymethylenephenyl group bound to the amide nitrogen. In addition, in the CIGAR experiment, the amidic proton correlated with a further aromatic carbon at δ C 157.9, which in turn displayed cross-peaks with a proton at δ H 8.01 (d, J = 9.0 Hz), as an indicator of a second 1,2,4-tribubstituted aromatic system. This proton correlated in the long-range experiment with the amide carbonyl and with a quaternary aromatic carbon at δ C 118.8, which in turn correlated with other two aromatic protons resonating at δ H 7.07 (d, J = 2.4 Hz) and 7.22 (dd, J = 9.0; 2.4 Hz), showing cross-peaks with an oxygenated aromatic carbon at δ C 162.2. This carbon correlated with an anomeric proton at δ H 4.98, therefore suggesting the presence of a sugar moiety, putatively identified, on the basis of an HSQC-TOCSY experiment, as glucose. These data were in agreement with the presence of a glucopyranosyl-2-hydroxy-N-(3,4-dioxymethylenephenyl)benzamide. However, the isolation and complete structural elucidation of this compound will be needed to confirm the hypothesized structure.
More signals were detected in the aromatic region. Signals belonging to the A-ring of flavonoids were observed as two COSY-correlating doublets (J = 2.0 Hz) at δ H 6.20 and 6.39. These protons were correlated in the HSQC experiment to the carbons at δ C 93.1 and 98.4, respectively. Furthermore, the former proton also showed long-range correlations with the two carbons resonating at δ C 161.2 and 165.0. This second carbon was also correlated to the δ H 6.39, which showed further long-range correlations with the carbons at δ C 156.9 and 104.0. It was not possible to unambiguously identify these metabolites, due to the lack of further correlations, but the NMR data here described prompted us to hypothesize that the flavonoids were all characterized by a hydroxy function bound to the C-3 [21].
Finally, the signals in the range of 6.8-7.4 ppm were correlated, in the HSQC experiment, with the carbons in the range of 125-145 ppm, suggesting the presence of olefinic protons. These protons also showed COSY correlations with protons resonating in the range of 5.9-6.1 ppm bound to carbons in the range of 120-125 ppm ( Figure S5). The proton signals at 5.9-6.1 ppm showed further COSY correlations with signals in the aliphatic region (2.4-2.8 ppm), in turn correlating with protons geminal to oxygen. Since the mixture was complex and these compounds were minor components, it was not possible to definitely identify the compounds. However, it is plausible that these signals belonged to oxylipins.
The OdM fraction was tested on HT-29 cells ( Figure 5), but no activity was observed. Two possible explanations were then taken into consideration: (i) the occurrence of synergistic effects in the crude extract or (ii) the fact that the OdM fraction obtained from the amberlite chromatography, although enriched in several specialized metabolites, did not contain a significant amount of the active compounds. These active metabolites could be then found either in the water fraction obtained from the same chromatography, or in the ethyl acetate fraction (OdE) obtained in the previous step. By comparing the NMR profiles of the OdM and the crude extract (Figures 3 and 4), it was clear that, besides the signals belonging to metabolites also detected in the crude extract of the inactive species O. variegata, there was also a significant decrease in the intensity of signals so far putatively attributed to oxylipins. These oxylipins could have been in the OdE fraction, which was therefore assayed. The ethyl acetate fraction showed activity on HT-29 cells ( Figure 5). hypothesized structure.
More signals were detected in the aromatic region. Signals belonging to the A-rin flavonoids were observed as two COSY-correlating doublets (J =2.0 Hz) at δH 6.20 and 6 These protons were correlated in the HSQC experiment to the carbons at δC 93.1 and 9 respectively. Furthermore, the former proton also showed long-range correlations w the two carbons resonating at δC 161.2 and 165.0. This second carbon was also correla to the δH 6.39, which showed further long-range correlations with the carbons at δC 15 and 104.0. It was not possible to unambiguously identify these metabolites, due to the l of further correlations, but the NMR data here described prompted us to hypothesize t the flavonoids were all characterized by a hydroxy function bound to the C-3 [21].
Finally, the signals in the range of 6.8-7.4 ppm were correlated, in the HSQC exp ment, with the carbons in the range of 125-145 ppm, suggesting the presence of olef protons. These protons also showed COSY correlations with protons resonating in range of 5.9-6.1 ppm bound to carbons in the range of 120-125 ppm ( Figure S5). The p ton signals at 5.9-6.1 ppm showed further COSY correlations with signals in the aliph region (2.4-2.8 ppm), in turn correlating with protons geminal to oxygen. Since the m ture was complex and these compounds were minor components, it was not possibl definitely identify the compounds. However, it is plausible that these signals belonged oxylipins.
The OdM fraction was tested on HT-29 cells ( Figure 5), but no activity was observ Two possible explanations were then taken into consideration: i) the occurrence of syn gistic effects in the crude extract or ii) the fact that the OdM fraction obtained from amberlite chromatography, although enriched in several specialized metabolites, did contain a significant amount of the active compounds. These active metabolites could then found either in the water fraction obtained from the same chromatography, or in ethyl acetate fraction (OdE) obtained in the previous step. By comparing the NMR prof of the OdM and the crude extract (Figures 3 and 4), it was clear that, besides the sign belonging to metabolites also detected in the crude extract of the inactive species O. va gata, there was also a significant decrease in the intensity of signals so far putatively tributed to oxylipins. These oxylipins could have been in the OdE fraction, which w therefore assayed. The ethyl acetate fraction showed activity on HT-29 cells ( Figure 5)  The inhibition was of 92% at a concentration of 50 µg/mL, and around 40% at the concentration of 10 µg/mL. The 1 H-NMR of the ethyl acetate extract confirmed that this fraction was enriched in signals that were only detected in traces in the methanol fraction, and that could be attributed to oxylipins (Figure 6). The inhibition was of 92% at a concentration of 50 µ g/mL, and around 40% at the concentration of 10 µ g/mL. The 1 H-NMR of the ethyl acetate extract confirmed that this fraction was enriched in signals that were only detected in traces in the methanol fraction, and that could be attributed to oxylipins (Figure 6). Figure 6. 1 H-NMR spectrum of the OdE fraction. A tentative general structure for the oxylipins is proposed, and diagnostic signals are indicated by colored arrows.
Besides the triplet at δH 0.83 and the methylene signals at δH 1.23, signals belonging to two olefinic protons conjugated to a carboxylic function were detected at δH 5.90 and δH 7.00. Allylic protons were instead resonating in the range 2-3 ppm, while signals in the region between 3 and 5 ppm were attributable to protons geminal to hydroxyl groups. The hypothesized structure is reported in Figure 6. This hypothesis was drawn based on the analysis of the 2D-NMR spectra of the extract previously discussed.
A complete structural elucidation of these metabolites was not possible in mixture, because it is very likely to have been made up of compounds with variable chain lengths. However, it is also clear that compounds with different substitution patterns were present in the mixture, including compounds that might bear acetyl groups (as shown by singlets at around 2 ppm).

Discussion
The present study demonstrates that O. diffusa is a source of potential anticancer compounds acting on drug-resistant colon cancer cell lines. The aim of the present work was not only to identify cytotoxic extracts, but particularly sources of compounds able to overcome anti-EGFR therapy resistance in mCRC.
EGFR is a very important target in cancer therapy [11], being a central regulator of tumor progression in a variety of human cancers, including mCRC [22], one of the leading causes of cancer-related death worldwide. One way to inhibit the activation of EGFR is with monoclonal antibodies like cetuximab [23]. Unfortunately, primary resistance (due to specific mutations) to cetuximab in mCRC patients, who therefore do not respond to this treatment, has been reported [24]. Furthermore, it has been shown that one fourth of Besides the triplet at δ H 0.83 and the methylene signals at δ H 1.23, signals belonging to two olefinic protons conjugated to a carboxylic function were detected at δ H 5.90 and δ H 7.00. Allylic protons were instead resonating in the range 2-3 ppm, while signals in the region between 3 and 5 ppm were attributable to protons geminal to hydroxyl groups. The hypothesized structure is reported in Figure 6. This hypothesis was drawn based on the analysis of the 2D-NMR spectra of the extract previously discussed.
A complete structural elucidation of these metabolites was not possible in mixture, because it is very likely to have been made up of compounds with variable chain lengths. However, it is also clear that compounds with different substitution patterns were present in the mixture, including compounds that might bear acetyl groups (as shown by singlets at around 2 ppm).

Discussion
The present study demonstrates that O. diffusa is a source of potential anticancer compounds acting on drug-resistant colon cancer cell lines. The aim of the present work was not only to identify cytotoxic extracts, but particularly sources of compounds able to overcome anti-EGFR therapy resistance in mCRC.
EGFR is a very important target in cancer therapy [11], being a central regulator of tumor progression in a variety of human cancers, including mCRC [22], one of the leading causes of cancer-related death worldwide. One way to inhibit the activation of EGFR is with monoclonal antibodies like cetuximab [23]. Unfortunately, primary resistance (due to specific mutations) to cetuximab in mCRC patients, who therefore do not respond to this treatment, has been reported [24]. Furthermore, it has been shown that one fourth of cetuximab-sensitive mCRC patients develop secondary resistance [11,12]. The emergence of secondary resistance might be due to the selection of drug-insensitive subclones imposed by the continuous EGFR blockade [24]. Finding drugs acting with alternative modes of action, able to overcome or bypass these innate and acquired resistances is therefore crucial. In this study, we used Caco-2, GEO, and SW48 cells as cetuximab-sensitive models, HT-29 and HCT-116 as intrinsically cetuximab-resistant models and GEO-CR and SW48-CR as cell models with acquired resistance to cetuximab. Although the data here reported are preliminary and extensive tests on pure compounds are needed to assess their activity, toxicity, and modes of action, the extract herewith analyzed showed a strong inhibition of cell growth on all the cell lines. Of note, the activity on cetuximab-resistant human cancer cell lines was remarkable. In particular, the extract strongly inhibited the growth of HT-29, a cell line harboring a BRAF mutation, which is a strong negative prognostic biomarker for patients suffering from mCRC [12].
Based on the partial purification and on the fraction testing and profiling, we could identify the class of compounds potentially responsible for the activity exerted by the extracts. It was therefore suggested that the oxylipin components of the extract could be the compounds responsible for the biological activity. The crude extract demonstrated strong cytotoxicity (Figures 1 and 2). Based on a comparison to the inactive extract of the related plant O. variegata (Figure 3), which was also tested and analyzed, it was possible to exclude some of the metabolites from the list of candidate bioactive compounds. These compounds (caffeic acid, caffeoyl derivatives, trigonelline, and several primary metabolites) were, indeed, the main components of the O. variegata extract (Figure 3), which however showed no activity even at the highest tested concentration. A second level of selection was obtained after the partial purification of the crude extract: OdE and OdM fractions were obtained when the extract was partitioned with ethyl acetate/water and then the aqueous fraction therefrom was further purified on amberlite. Although the OdM fraction was particularly enriched in phenolic compounds, it did not show activity against HT-29 cells (Figures 4 and 5). On the other hand, the OdE fraction was very active and even more strongly inhibiting of HT-29 cell growth than the crude extract. Based on the NMR analysis, it was possible to tentatively identify the main compounds in this fraction as oxylipins ( Figure 6 and Figures S1-S3).
Oxylipins are oxidized fatty acids, used by plants mostly as signaling molecules [25,26], to the best of our knowledge. Jasmonic acid is by far the most studied oxylipin, due to its central role as a plant hormone involved in the regulation of developmental and defenserelated processes [27]. Given these roles in signaling and as plant hormones, oxylipins are usually present at low concentrations [25] and therefore have also been seldom studied for further biological activities. It has been suggested that these molecules promote apoptosis in animal cells by altering the intracellular calcium signaling and inducing cytoskeletal instability [28], although the molecular mechanism is not yet known. Besides jasmonates, different substitute oxylipins have been reported, including oxylipins with cytotoxic, antiinflammatory, and potential anticancer properties [29,30]. The oxylipins in O. diffusa extract seemed to be polyoxygenated compounds. This hypothesis was supported by their polarity and chromatographic behavior.
Although the oxylipins were the most abundant compounds in the active fraction, we cannot completely exclude the possibility that less-abundant compounds could have been responsible for the observed activity. However, a further effort to perform a complete isolation and structural elucidation of the pure compounds will be carried out as the object of future studies. The observed biological activity makes the OdE fraction a potential source of compounds that could be further explored with the aim of finding drug candidates able to overcome either intrinsic or acquired drug resistance in colorectal cancer cells.

Plant Sampling
a plate reader. Results were reported as mean +SD of % of cell growth respect to the control, from six replicates. The control was represented by 0.25% DMSO treatment, corresponding to the higher amount of DMSO used for the tests.

Statistical Analyses
Bioassays were carried out in six replicates. Statistical analyses were performed using Excel 2010 (Microsoft Corporation; Redmond, WA, USA). A Student's t test (p < 0.001) was used to determine the statistical significance of the experimental results.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors upon request, without undue reservation.