Antifungal and Cytotoxic Activity of Diterpenes and Bisnorsesquiterpenoides from the Latex of Euphorbia resinifera Berg

Euphorbia resinifera latex has been extensively utilized in traditional medicine due to its range of bioactivities. Chromatographic separations on silica gel of ethanol extract of E. resinifera latex led to the development of a new procedure for isolating resiniferatoxin (4) via dried E. resinifera latex and the identification of nine compounds. Among these, catechol (7), protocatechuic acid (8) and 3,4-dihydroxyphenylacetic acid (9), known phenolic compounds, were identified for the first time in E. resinifera latex. Herein we investigated the effects of major compounds of the latex of E. resinifera on the yeast Saccharomyces cerevisiae, on the growth of Aspergillus carbonarius, a widespread fungal contaminant, and on the breast cancer cell line MCF7 as well as on MCF10A normal breast cells. 12-deoxyphorbol-13-isobutyrate-20-acetate (2) had an inhibiting effect on the growth of A. carbonarius, and 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a negative effect on yeast cell growth and also a cytotoxic effect on breast cancer cell line MCF7, but not on MCF10A cells. Deglucosyl euphorbioside A (5) and euphorbioside A (6) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and also cytotoxicity only on the cancer cell line that was tested. Interestingly, treatment of MCF7 cells with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) not only led to a specific cytotoxic effect but also to the increase in the level of intracellular ROS.


Introduction
According to the World Health Organization, cancer was responsible for approximatively 9.6 million deaths worldwide, with 18.1 million new cases recorded in 2018 [1]; among those new cases, lung and breast cancers are prevalent. Breast cancer is the most frequently diagnosed and its incidence far exceeds that of other cancers in both developed and developing countries [2]. One major form of cancer treatment involves chemotherapy by means of synthetic drugs [3]. However, critical side-effects (from vomiting and diarrhea, 2 of 15 to more major complications e.g., neurological, cardiac, pulmonary, and renal toxicity) and the development of multi-drug resistance (MDR), are often associated with these drugs and can lead to poor therapeutic efficiency. Likewise, the incidence of fungal infections is a major cause of morbidity and mortality in immunocompromised patients worldwide [2]. The relative lack of available antifungal compound classes and the resistance of certain fungal species to the available drugs are obstacles to treating these infections [4]. Thus, there is a significant demand for new drugs with improved potency and lower toxicity and there is continuous research to find new anticancer and antifungal agents in both academic and industrial settings [3].
Natural molecules with antifungal and anticancer properties have been a key research focus over the past few years. Recently, natural products have proven to be a key source of new drugs with a wide range of biological and pharmacological applications [5,6]. The Euphorbiaceae family is ubiquitous and pharmaceutically relevant. In particular, the genus Euphorbia is the most important genus of the Euphorbiaceae family, with more than 2000 species worldwide, all of which are characterized by the presence of milky irritant latex [7].
Euphorbia resinifera Berg. is a plant that is endemic to Morocco, where it typically grows on the slopes of the anti-Atlas Mountains [8]. Euphorbia resinifera, and specifically its latex, has been studied primarily due to the presence of diverse phytochemicals such as polycyclic and macrocyclic, diterpenes [8][9][10][11][12], triterpenes [13][14][15], sesquiterpenoids [8], and phenolic acids [16]. These chemical constituents are providing leading compounds for drug discovery due to their therapeutic applications deriving from their cytotoxic [15,[17][18][19], anti-neurodegenerative [20], antiviral [21,22], antimicrobial [23], and antiparasitic [24] properties and anti-inflammatory activities [25,26]. Euphorbium-i.e., the dried latex of E. resinifera contains a daphnane diterpene (+)-Resiniferatoxin (RTX), one of the most ancient drugs that is still being used as a starting point in the development of a novel class of analgesics [27][28][29]. RTX is used in the treatment of pain that is associated with diabetic polyneuropathy and in the desensitization of nociceptive neurons [30][31][32]. This compound also shows a varied biological response and has powerful analgesic effects that are more potent than capsaicin (at least 10 3 -10 5 times) [33]. Furthermore, Euphorbium is used in Moroccan traditional medicine to suppress chronic pain, to mitigate pain for dental cavities and tooth aches, and treat articular tuberculosis [34]. As part of our continuing valorization of Euphorbia species that are native to Morocco, we have investigated the effects of the major compounds of the latex of E. resinifera on the growth of the yeast Saccharomyces cerevisiae and of Aspergillus carbonarius, a widespread fungal contaminant; furthermore, the cytotoxic effect was evaluated on breast cancer cell line MCF7, as well as on normal breast cells MCF10A.

Chemical Results
Within the context of our search for the chemical constituents of Euphorbia resinifera latex and their antifungal and cytotoxic activities, the dried E. resinifera latex was extracted with ethanol, employing a Soxhlet apparatus. The combined extracts were concentrated to produce the ethanol extract. The extract was processed with increasing polarity liquid-liquid partition and continuous conventional chromatographic technique combination, such as normal-phase silica gel column chromatography and preparative normalphase thin-layer chromatography (TLC). A total of nine compounds were characterized and among them certain phenolic compounds, catechol (7), protocatechuic acid (8), and 3,4 dihydroxy-phenylacetic acid (9) (Figure 1) were identified for the first time in E. resinifera latex. Other known compounds were also identified based on the spectral and chemical evidence, which were in agreement with those that were reported in the literature [7].

Evaluation of the Effect of Compounds on the Yeast S. cerevisiae
The yeast cells were treated with the considered compounds at 400 µM (Figure 2A) [41]; the concentration of 400 µM was selected after carrying out a dose response curve with concentrations from 10 µM to 500 µM (not shown). The 7-p-metoxyphenylacetate-3,8,12triacetate ingol (3) was found to exert a negative effect on yeast growth. On the contrary, 12-deoxyphorbol-13-angelate-20-acetate (1) showed a positive effect. The observation of the culture showed that the cells that were treated with 7-p-metoxyphenylacetate-3,8,12triacetate ingol (3) evidenced a bigger cell size with swollen vacuoles, compared to untreated yeast cells (W303 NT) ( Figure 2B).

Evaluation of the Effect of Compounds on the Yeast S. cerevisiae
The yeast cells were treated with the considered compounds at 400 μM ( Figure 2A) [41]; the concentration of 400 μM was selected after carrying out a dose response curve with concentrations from 10 μM to 500 μM (not shown). The 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) was found to exert a negative effect on yeast growth. On the contrary, 12-deoxyphorbol-13-angelate-20-acetate (1) showed a positive effect. The observation of the culture showed that the cells that were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) evidenced a bigger cell size with swollen vacuoles, compared to untreated yeast cells (W303 NT) ( Figure 2B). To verify if the cells that were treated with the compounds overproduced ROS, cells were stained with dihydrorodamine123. In the tested conditions, the compounds did not induce oxidative damage in the yeast cells, because no evident overproduction of ROS was visualized. In fact, most of the treated cells in the logarithmic phase showed a limited fluorescence, reflecting the presence of normal ROS background (data not shown). We then evaluated by halo test the sensitivity of the treated cells to hydrogen peroxide (H2O2). The yeast cells were treated with the compounds at 400 μM and plated in YPD medium. A paper disk was soaked with 5 μL of H2O2 9.2 M. After 24 h, the diameter of the halo was measured ( Figure 3). In the cells that were treated with deglucosyl euphorbioside A (5) and euphorbioside A (6), a higher sensitivity to H2O2 compared to the cells that were challenged only with dimethyl sulfoxide (DMSO) was observed, with a halo production of 3.7 cm and 4 cm, respectively, instead of 3 cm ( Figure 3). It is also interesting to note the loss of the red color of the yeast cells that were treated with these compounds, see "Discussion". To verify if the cells that were treated with the compounds overproduced ROS, cells were stained with dihydrorodamine123. In the tested conditions, the compounds did not induce oxidative damage in the yeast cells, because no evident overproduction of ROS was visualized. In fact, most of the treated cells in the logarithmic phase showed a limited fluorescence, reflecting the presence of normal ROS background (data not shown). We then evaluated by halo test the sensitivity of the treated cells to hydrogen peroxide (H 2 O 2 ). The yeast cells were treated with the compounds at 400 µM and plated in YPD medium. A paper disk was soaked with 5 µL of H 2 O 2 9.2 M. After 24 h, the diameter of the halo was measured (Figure 3). In the cells that were treated with deglucosyl euphorbioside A (5) and euphorbioside A (6), a higher sensitivity to H 2 O 2 compared to the cells that were challenged only with dimethyl sulfoxide (DMSO) was observed, with a halo production of 3.7 cm and 4 cm, respectively, instead of 3 cm ( Figure 3). It is also interesting to note the loss of the red color of the yeast cells that were treated with these compounds, see "Discussion".

Evaluation of the Compounds' Antifungal Effect
The considered compounds were also assayed on the fungus A. carbonarius at 10 µM, 50 µM, and 100 µM ( Figure 4). The growth of the fungus was investigated after 3, 6, 11, and 16 days of incubation, i.e., between the beginning of the exponential growth phase and the plateau phase. Adding 12-deoxyphorbol-13-isobutyrate-20-acetate (2) at all the tested concentrations to the fungal culture had the effect of decreasing its growth by 25%. This effect, evident after 3 days of incubation, was lost after longer incubation periods. The remaining compounds that were tested did not display any significant effect on fungal growth.
Molecules 2022, 27, 5234 5 of 16 Figure 3. H2O2 sensitivity of S. cerevisiae that was induced by the tested compounds. The sensitivity was visualized with a growth inhibition test. Deglucosyl euphorbioside A (5) and euphorbioside A (6) induced a slight sensitivity to H2O2 producing a halo of 3.7 cm and 4 cm, respectively, bigger than the halo that was produced with cells that were treated with DMSO (3 cm).

Evaluation of the Compounds' Antifungal Effect
The considered compounds were also assayed on the fungus A. carbonarius at 10 μM 50 μM, and 100 μM (Figure 4). The growth of the fungus was investigated after 3, 6, 11 and 16 days of incubation, i.e., between the beginning of the exponential growth phase and the plateau phase. Adding 12-deoxyphorbol-13-isobutyrate-20-acetate (2) at all the tested concentrations to the fungal culture had the effect of decreasing its growth by 25% This effect, evident after 3 days of incubation, was lost after longer incubation periods The remaining compounds that were tested did not display any significant effect on fun gal growth.   . H2O2 sensitivity of S. cerevisiae that was induced by the tested compounds. The sensitivity was visualized with a growth inhibition test. Deglucosyl euphorbioside A (5) and euphorbioside A (6) induced a slight sensitivity to H2O2 producing a halo of 3.7 cm and 4 cm, respectively, bigger than the halo that was produced with cells that were treated with DMSO (3 cm).

Evaluation of the Compounds' Antifungal Effect
The considered compounds were also assayed on the fungus A. carbonarius at 10 μM, 50 μM, and 100 μM ( Figure 4). The growth of the fungus was investigated after 3, 6, 11, and 16 days of incubation, i.e., between the beginning of the exponential growth phase and the plateau phase. Adding 12-deoxyphorbol-13-isobutyrate-20-acetate (2) at all the tested concentrations to the fungal culture had the effect of decreasing its growth by 25%. This effect, evident after 3 days of incubation, was lost after longer incubation periods.
The remaining compounds that were tested did not display any significant effect on fungal growth.

Evaluation of the Compounds' Cytotoxicity
To explore the effect of the main compounds that were obtained from the E. resinifera extracts on breast cancer and normal cell growth, the cells were exposed to different concentrations (0.001 µM, 1 µM, 10 µM, and 100 µM) of 12-deoxyphorbol-13-angelate-20-acetate (1), 12-deoxyphorbol-13-isobutyrate-20-acetate (2), 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3), deglucosyl euphorbioside A (5), and euphorbioside A (6) for 72 h, and the cell viability was assessed as described in Figure 5. Our results have shown a significant decrease in the percentage of viable breast cancer cells MCF7 that were treated with increasing concentrations of the considered molecules ( Figure 5). This result was particularly evident for 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3). To investigate whether the cytotoxicity of the compounds is specific for breast cancer cells, we treated with the same doses also normal breast cells MCF10A. The subsequent cytotoxic effect was significantly lower for 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) at 10 µM and 100 µM, compared to the breast cancer cells MCF7, suggesting that these compounds are more selective to cancer cells than normal ones ( Figure 5).

Evaluation of the Compounds' Cytotoxicity
To explore the effect of the main compounds that were obtained from the E. resinifera extracts on breast cancer and normal cell growth, the cells were exposed to different concentrations (0.001 μM, 1 μM, 10 μM, and 100 μM) of 12-deoxyphorbol-13-angelate-20-acetate (1), 12-deoxyphorbol-13-isobutyrate-20-acetate (2), 7-p-metoxyphenylacetate-3,8,12triacetate ingol (3), deglucosyl euphorbioside A (5), and euphorbioside A (6) for 72 h, and the cell viability was assessed as described in Figure 5. Our results have shown a significant decrease in the percentage of viable breast cancer cells MCF7 that were treated with increasing concentrations of the considered molecules ( Figure 5). This result was particularly evident for 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3). To investigate whether the cytotoxicity of the compounds is specific for breast cancer cells, we treated with the same doses also normal breast cells MCF10A. The subsequent cytotoxic effect was significantly lower for 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) at 10 μM and 100 μM, compared to the breast cancer cells MCF7, suggesting that these compounds are more selective to cancer cells than normal ones (Figure 5).

Evaluation of Mitochondrial ROS Formation in Human Cell Lines
To investigate the possible generation of ROS in response to the tested compounds, MCF7 and MCF10A cells were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) to obtain 10 μΜ concentration and the level of ROS production was assessed accordingly (Figure 6). A significant increase in the level of intracellular ROS was observed in MCF7 cells that were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5). However, the level of ROS production remained approximately unchanged in MCF10A cells (fold change compared to the untreated cells) (Figure 6).

Evaluation of Mitochondrial ROS Formation in Human Cell Lines
To investigate the possible generation of ROS in response to the tested compounds, MCF7 and MCF10A cells were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) to obtain 10 µM concentration and the level of ROS production was assessed accordingly (Figure 6). A significant increase in the level of intracellular ROS was observed in MCF7 cells that were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5). However, the level of ROS production remained approximately unchanged in MCF10A cells (fold change compared to the untreated cells) (Figure 6).

Discussion
The main compounds that were purified from the Euphorbium (Figure 1) were tested in different contexts: yeast cells, a representative of the mold species contaminating various foodstuffs, and cancerous and normal cells. Each one of the tested organisms and cell lines has its own specificity. In particular, S. cerevisiae constitutes a valid model for evaluating the effects of molecules that are endowed with biological activity and it is essential to investigate the effects on the mitochondrial function because the mitochondrial respiration is dispensable in S. cerevisiae [42][43][44].
The considered compounds were tested at 400 μM because this was the minimal concentration showing a toxic effect. The use of compounds at the concentration of 400 uM is not unusual for natural compounds, also given the presence of the thick, rigid cell wall of the yeast S. cerevisiae [41,43].
The effect of the tested compounds on the yeast S. cerevisiae revealed that the 7-pmetoxyphenylacetate-3,8,12-triacetate ingol (3) exerted a negative effect on yeast growth inducing a bigger cell size with swollen vacuoles (Figure 2), while deglucosyl euphorbioside A (5) and euphorbioside A (6) induced sensitivity to H2O2. It is interesting to note that deglucosyl euphorbioside A (5) was also active in MCF7 cells, inducing ROS production. Moreover, deglucosyl euphorbioside A (5) and euphorbioside A (6) negatively influenced the accumulation of an adenine biosynthetic pathway intermediate because the treated cells lost the red color (Figure 3) characteristic of a defective adenine biosynthesis in S. cerevisiae cells [45]. The loss of the red color could also indicate a mitochondrial dysfunction because the W303 wild-type yeast colonies are red because the ade2 -mutation results in the accumulation of the substrate of the Ade2, an enzyme of the adenine biosynthetic pathway. The formation of this red intermediate is produced in the mitochondria. If the mitochondria are not functional, the intermediate is not produced, and the colonies result in white colonies. Thus, yeast cells that were treated with 7-p-metoxyphenylacetate-3,8,12triacetate ingol (3), deglucosyl euphorbioside A (5), and euphorbioside A (6) deserve further analysis.

Discussion
The main compounds that were purified from the Euphorbium (Figure 1) were tested in different contexts: yeast cells, a representative of the mold species contaminating various foodstuffs, and cancerous and normal cells. Each one of the tested organisms and cell lines has its own specificity. In particular, S. cerevisiae constitutes a valid model for evaluating the effects of molecules that are endowed with biological activity and it is essential to investigate the effects on the mitochondrial function because the mitochondrial respiration is dispensable in S. cerevisiae [42][43][44].
The considered compounds were tested at 400 µM because this was the minimal concentration showing a toxic effect. The use of compounds at the concentration of 400 uM is not unusual for natural compounds, also given the presence of the thick, rigid cell wall of the yeast S. cerevisiae [41,43].
The effect of the tested compounds on the yeast S. cerevisiae revealed that the 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) exerted a negative effect on yeast growth inducing a bigger cell size with swollen vacuoles (Figure 2), while deglucosyl euphorbioside A (5) and euphorbioside A (6) induced sensitivity to H 2 O 2 . It is interesting to note that deglucosyl euphorbioside A (5) was also active in MCF7 cells, inducing ROS production. Moreover, deglucosyl euphorbioside A (5) and euphorbioside A (6) negatively influenced the accumulation of an adenine biosynthetic pathway intermediate because the treated cells lost the red color (Figure 3) characteristic of a defective adenine biosynthesis in S. cerevisiae cells [45]. The loss of the red color could also indicate a mitochondrial dysfunction because the W303 wild-type yeast colonies are red because the ade2mutation results in the accumulation of the substrate of the Ade2, an enzyme of the adenine biosynthetic pathway. The formation of this red intermediate is produced in the mitochondria. If the mitochondria are not functional, the intermediate is not produced, and the colonies result in white colonies. Thus, yeast cells that were treated with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3), deglucosyl euphorbioside A (5), and euphorbioside A (6) deserve further analysis.
The test of the molecules that were considered on the fungus A. carbonarius allowed to evaluate the possible effect on the growth of this widespread contaminant. In fact, the inhibitory effect of plant extracts from the genus Euphorbia against Aspergillus niger, a fungal contaminant belonging to the "black Aspergilli" group as well as A. carbonarius, has been demonstrated [46,47]. The only compound that had an inhibitory effect on the growth of A. carbonarius was 12-deoxyphorbol-13-isobutyrate-20-acetate (2) (Figure 4). This molecule only differs from the ineffective compound 12-deoxyphorbol-13-angelate-20-acetate (1) in the ester group chain, which makes molecule 1 larger and planar. We can hypothesize that the general structure of the molecule is a good starting point for the search for compounds that are capable of inhibiting fungal growth. It is possible that the lack of effect of molecule 1 on fungal growth may be due to its steric hindrance, which does not allow the interaction with the membrane receptors or the passage of the membrane itself.
It has been reported that some species of Euphorbia may have cytotoxic effects against different cell lines [48]. In particular, the in vitro anti-cancer and genotoxic activity of methanolic extract of Euphorbia triaculeata were previously evaluated using the MTT assay in human breast cancer cell line MCF7 and normal breast epithelial cell line MCF10A [49]. Thus, we decided to use these cell lines to test the cytotoxic effects of our compounds by comparing the behavior of tumor and normal cell lines ( Figure 5). A panel of concentrations was tested to obtain a general evaluation of the anti-cancer effects of our compounds. Future experiments, in which we will compare our compounds with well-known anti-cancer compounds, could clarify their anti-cancer activity.
This kind of testing leads to the selection of compounds to be sent for further experimentation and finally to a possible use as therapy.
An increase in intracellular ROS level in cancer cells sustains stressful conditions [50]. The role of ROS in breast cancer is very controversial. It is well known that breast cancer cell lines MCF7 cells and non-transformed breast cell lines MCF10A express low levels for ROS [48]. Interestingly, the induction of ROS in breast cancer cell lines MCF7 by several drug treatments is correlated with a loss of cell viability (Figure 5), suggesting a role of ROS induction as a mechanism of apoptosis induction in cancer cells [51]. Consistently, we found that 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) were able to increase mitochondrial ROS levels in MCF7 cells, but not in the nontransformed MCF10A cells (Figure 6), suggesting that the loss of viability that is induced by these two drugs is related to the difference in ROS induction, but only at the highest assayed doses. These results point out that these compounds are cytotoxic on cancer cells, albeit only at high doses, suggesting that they are not useful as such for anti-cancer therapies. Further experiments will clarify this point.
It is interesting to note how 12-deoxyphorbol-13-isobutyrate-20-acetate (2) has shown activity on both A. carbonarius growth and on higher cells, despite the diversity of these biological systems. Furthermore, the loss of the red color of the yeast cells that were treated with deglucosyl euphorbioside A (5) and euphorbioside A (6) is a clue that encourages studies on the effect of these compounds on mitochondrial functionality.
Overall, our data characterized for the first time the effects of these natural compounds in different systems.

General Experimental Procedures
Analytical and preparative normal-phase thin-layer chromatography (TLC) were performed on 100 mm × 200 mm glass that was pre-coated with 0.25 mm layer of silica plates Kiesilgel 60F254 (Merck, Darmstadt, Germany) for analytical TLC, and with 0.5 mm layer of silica gel for preparative TLC. Spots were visualized using short (254 nm) UV light before using an ethanolic solution of phosphomolybdic acid (heating). Purifications by column chromatography were performed using silica gel Kiesgel 60, 40−63 µm. The melting points (mp) were measured by a Kofler hot bench or Reichert plate-heating microscope and are reported uncorrected. The IR spectra were obtained using IRAffinity-1S FTIR Infrared spectrophotometer (Shimadzu, Kyoto, JP). The measurements were made by loading the sample directly onto a diamond cell. The measurements are reported on the wavenumber scale (cm −1 ). 1 H and 13 C NMR spectra were recorded on a BrükerAvance spectrometer (Billerica, MA, USA) at 400.13 and 100.61 MHz, respectively, using CDCl 3 and Molecules 2022, 27, 5234 9 of 15 CD 3 OD-d4 as solvent. Chemical shifts ( 1 H and 13 C) were reported in ppm relative to the solvent residual signals (e.g., chloroform: δ 1 H = 7.26 ppm and δ 13 C = 77.16 ppm; methanol: δ 1 H = 4.87 ppm and δ 13 C = 49.00 ppm). The spectra were processed with Mnova program from MestRelab Research. LC−MS was recorded a Q Exactive Quadrupole-Orbitrap mass spectrometer that was coupled to an HPLC Ultimate 3000 that was equipped with a DAD UV/vis 3000 RS detector (Thermo Fisher Scientific, Waltham, MA, USA. The column was a Kinetex EVO C18; 1.7 µm; 100 mm × 2.1 mm. A flow rate of 0.45 mL min −1 was applied with the following linear gradient: solvent B from 5% to 95% over 7.5 min (solvent A = H 2 O + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid).

Plant Material
Latex from E. resinifera Berg. was collected in October 2016, from plants in the area of Azilal, Morocco, by making repeated cuts along the stems of the plants with a knife and collecting the white milky exudates. A voucher specimen (20161023) deposited at the herbarium of Laboratory of Molecular Chemistry, Materials, and Catalysis, Sultan Moulay Slimane University, Faculty of Science and Technology, BP 523, 23,000 Beni-Mellal.

Extraction and Isolation
The latex (1.5 L) from E. resinifera Berg. was air-dried at room temperature in a dark room, and the resulting coagulum (600 g) was extracted with 96% EtOH (3 L), employing a Soxhlet apparatus. After 24 h, ethanolic extract was concentrated to obtain a crude gummy material (88.8 g). This crude extract was then dissolved in water and extracted three times with increasing polarity solvent to give five fractions: n-hexane (23.5%), CH 2 Cl 2 (24%), EtOAc (1.7%) n-butanol (2.3%), and water (48.5%).

Fungal Culture
The yeast strain that was used in this study was Saccharomyces cerevisiae strain W303 (MATa leu2-3112, trp1-1, can1-100, ura3-1, ade2-1, his3-11,15). A pre-culture of the yeast strain was grown in YPD, a culture-rich medium (1% bactopeptone, 1% yeast extract, and 2% glucose) for 24 h, then tubes with fresh medium were inoculated from the pre-culture (2 mL of YPD liquid medium) in which the compounds were added at the final concentration of 400 µM. The cultures were grown overnight. The cells were then counted, and serial dilutions (1 × 10 7 , 1 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1 × 10 3 /mL) were spotted on YPD plates and grown at 28 • C for 24 h. To monitor the intra-cellular ROS levels, we used dihydrorodamine123, a dye that reacts with ROS to generate a fluorophore. The ROS levels were detected in exponential phase cultures that were treated with the compounds. Cell visualization was performed using the Axio Observer (ApoTome-Zeiss, Oberkochen, Germany). H 2 O 2 sensitivity was tested plating the treated cells on YPD plates and spotting 5 µL of H 2 O 2 9,2 M on a sterile paper disk. After 24 h, the diameter of the halo was measured.
A black Aspergillus isolate (A. carbonarius) from wine grapes that were grown in the countryside of Manduria (Taranto, Italy) was used. The isolate was unambiguously identified by molecular techniques and maintained in CDA (Czapek-Dox Agar, Difco-BD, Franklin Lakes, N J, USA) slants at 25 • C.

Antifungal Assay
Aspergillus carbonarius conidia from a culture tube that were incubated for 10 days at 25 • C were scraped off and put in sterile distilled water. The conidia suspension was counted by a Thoma counting chamber (Merck, Darmstadt, Germany) and diluted with sterile distilled water to 3 × 10 5 in 0.1 mL. That quantity was introduced into each one of the 50 mL-Erlenmeyer flasks that was filled with 25 mL of Czapek-Dox-Yeast 0.2% (CDA + Yeast extract 0.2%), a medium conducive for A. carbonarius growth. Each compound was dissolved in DMSO, and 50 µL of solutions subsequently diluted to obtain 10 µM, 50 µM, and 100 µM in the Erlenmeyer flask were added at the same time as the fungal inoculum. The fungal growth was evaluated after 3, 6, 11, and 16 days of incubation at 25 • C, by weighing the mycelial part of the cultures, that were previously filtered through filter paper on a separatory funnel, and dried at 80 • C for 48 h.

Cell Culture
Cell lines MCF7 and MCF10A were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA). Human breast cancer cells MCF7 were cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum (Invitrogen, Waltham, MA, USA), The MCF10A normal breast cells were cultured in Dulbecco's modified Eagle's medium and F12 medium (DMEM-F12) which was supplemented with horse serum (5%), hydrocortisone (0.5 µg/mL), EGF (20 ng/mL), and insulin (10 µg/mL), [46,47]. The maintained conditions were provided at 37 • C with an atmosphere of 5% CO 2 and 100% humidity. The statistical significance was calculated by comparing the two groups: MCF7 and MCF10A cells that were treated at 100 µM, and supports our conclusions.

Cytotoxicity Assay
Cells' viability in the normal and treatment conditions was measured by the MTS assay (Promega, Wisconsin, IA, USA). In brief, the cells were cultured in adhesion conditions in TC-treated well at density 1000 cells/100 µL medium and treated with different compounds. After 24 h, the MTS solution was added per well and incubated at 37 • C for 3 h. Finally, the optical density (OD) was measured under the 492-nm wavelength, and the survival rates were calculated.

Mitochondrial ROS Formation in Human Cell Lines
Mitochondrial ROS formation was detected using MitoSOX™ Red (Thermo Fisher Scientific, Waltham, MA, USA), a highly selective indicator for the detection of superoxide in the mitochondria of living cells. Briefly, the cells were seeded in 96-well black plates at a density of 10 × 10 3 cells per well and left to grow overnight. The cells were treated with the tested compounds for 1 h. The cells were then further incubated at 37 • C with 5 µM MitoSOX for 15 min in the dark. The fluorescence intensity of MitoSOX™ Red was recorded by using an Infinite M200 plate reader at an emission wavelength of 580 nm and at an excitation wavelength of 510 nm.

Conclusions
In conclusion, nine compounds were isolated from the dried E. resinifera latex. Among these, catechol (7), protocatechuic acid (8), and 3,4-dihydroxyphenylacetic acid (9), known phenolic compounds, were identified for the first time in E. resinifera latex. Other known compounds were also identified (1-6). Extensive NMR and mass spectroscopic analysis were used to elucidate their structures. Most of the isolated compounds were tested for their antifungal and cytotoxic activities. We demonstrated that 12-deoxyphorbol-13isobutyrate-20-acetate (2) had an inhibiting effect on the growth of A. carbonarius, and 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a cytotoxic effect on breast cancer cell line MCF7. Furthermore, deglucosyl euphorbioside A (5) and euphorbioside A (6) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and compound 5 also showed a specific cytotoxic effect on the cancer cell lines MCF7. The results suggest that E. resinifera latex may contain compounds with control activity against cancerous cells and contaminant molds. Further investigations will be carried out on the most active compounds in order to study their mechanisms of action.