Chemical profile and cytotoxic evaluation of aerial parts of Euphorbia tirucalli L. on gastric adenocarcinoma (AGS cells)

Abstract Ethanol extract from the aerial parts of Euphorbia tirucalli L. as well as the latex of the plant suspended in water are used by the Brazilian population for the treatment of various diseases, including cancer. The purposes of this study were to determine if the ethanol extract is effective as cytotoxic agent against gastric adenocarcinoma cells (AGS) and its chemical composition by GC-MS, ESI-(-)-FT-ICR MS and (-)-ESI-LTQ-MS/MS. The results were compared with that of latex previously described by us. Hexane and aqueous fractions showed higher cytotoxicity on AGS cells. Nine triterpene compounds were detected by GC-MS in hexane fraction, including euphol and friedelin, while ellagic acid was identified as main phenolic compound in aqueous extract. Therefore, the greater cytotoxic activity of the ethanol extract of the aerial parts of Euphorbia tirucalli for gastric cancer, when compared to latex, seems to originate from the antiproliferative effects of ellagic acid and triterpenes. Graphical Abstract


Introduction
Euphorbia tirucalli L. (Euphorbiaceae) is traditionally used in Brazil for the treatment of cancer and, recently, also for gastric ulcers.Two popular preparations are used.One of them is prepared by addition of six drops of latex in two liters of water and the other is an ethanol extract with the aerial parts of the plant.Chemically, the plant is known to contain triterpenes, diterpenes (de Souza et al. 2019), maloyl glucans (Kuster et al. 2015), flavonoids (de Lima et al. 2021) and hydrolyzable tannins (Yoshida et al. 1991) as major classes of secondary metabolites.Euphol and tirucallol are the major triterpenes of E. tirucalli, frequently evaluated for antinociceptive and anticancer activities on breast, glioblastoma, and gastric tumors (Lin et al. 2012;Wang et al. 2013;Chen et al. 2015;Dutra et al. 2015;Silva et al. 2018).A previous study by our research group evaluated the in vitro cytotoxic activity of the (3:1) dichloromethane/ methanol extract of latex, which showed selective toxicity on gastric cancer cells (AGS), but not significant cytotoxicity on macrophage cells (RAW 264.7).In addition, it showed important immunomodulatory activity (de Souza et al. 2019).This study stimulated us to evaluate the cytotoxic activity and the chemical composition of the extract of the aerial parts and, in this way, to determine the most cytotoxic popular product.

Cytotoxicity of E. tirucalli ethanol extract and fractions
The ethanol extract obtained from the aerial parts of E. tirucalli, as well as its fractions obtained by liquid/liquid extractions, were submitted to the cytotoxic assay against AGS cells (gastric adenocarcinoma).As shown in Table S1, the cytotoxic activity of the ethanol extract (11.73 ± 0.31 µg/mL) was six times higher than that obtained for the latex of the plant (69.43 ± 1.29 µg/mL)(de Souza et al. 2019).Such cytotoxicity was higher for the hexane (10.33 ± 2.01 µg/mL) and aqueous (13.08 ± 0.99 µg/mL) fractions, among the most lipophilic and hydrophilic substances, respectively.Therefore, we will show below the chemical composition of these fractions to correlate it with the antiproliferative activity.

Chemical profile of hexane and dichloromethane fractions by GC-MS
Table 1 shows the substances identified in the hexane and dichloromethane fractions by GC-MS (Figure S1 and S2), which contains fatty acids and triterpenes at mostly.Euphol (62.3%/32.5%),tirucallol (16.5%/10.7%)and lupen-3-one (8.0%/3.1%)were the major triterpenes, while lupeol, glutinol and friedelin were the minor ones, in addition to two other unidentified ones.Glutinol is a pentacyclic triterpene with a taraxane nucleus previously characterized in aerial parts of E. tirucalli from Pakistan and Japan (Khan et al. 1987;Uchida et al. 2010), while friedelin has a friedelane nucleus, and was found in other species of the genus Euphorbia (Farozi et al. 2015), but in Euphorbia tirucalli, it is described for the first time.

Chemical profile of ethyl acetate and aqueous fractions by (-)-ESI-FT-ICR MS and (-)-ESI-LTQ MS/MS
Among the hydrophilic substances found in E. tirucalli, flavonoids and hydrolysable tannins stand out.As phenolic compounds, they are ionized as negative ions and therefore can be identified through their exact masses by high resolution mass spectrometry in the negative electrospray source (FT-ICR MS) and by mass fragmentation experiments (LTQ-MS/ MS) (Figures S3-S6).Table 2 shows the main phenolic compounds found in the ethyl acetate and aqueous fractions.Flavonoids, such as rutin (46.9%) identified by signal at m/z 609.14 as well as by its MS 2 fragments at m/z 463.08 (loss of rhamnose) and m/z 301.08 (loss of rutinose) (Figure S4), quercitrin (100%) by signal at m/z 447.09 and fragments at m/z 301.08 (loss of rhamnose) (Figure S5) (de Lima et al. 2021), hydrolysable tannins such as corilagin (83.2%, signal at 633.07, and fragments at m/z 463.17 -loss of gallic acid, and at m/z 300.92 -loss of galloylglucose), pedunculagin/casuariin (10.8%) and among others, are abundant in the ethyl acetate fraction (Figure S3).However, they are absent or with low intensity peaks in the aqueous fraction, where ellagic acid (47.2%) and 2,3-hexahydroxydiphenoyl-D-glucose (or isomer) (24.9%) were found as intense signals.Both last molecules are usually the result of the hydrolysis of tannins.Corilagin fragments at m/z 463.17 and m/z 301.00 were observed to occur in losses of gallic acid and glucose, respectively, by (-) ESI-LTQ-MS/MS (Figure S6).For ellagic acid, the major fragment at m/z 256.86 is the result of a decarboxylation (Figure S7).Fragments at m/z 301.00 characterized the identification of 2,3-hexahydroxydiphenoyl-D-glucose or isomers (m/z 481.42), resulting from glucose loss and ellagic acid formation (Figure S8).As we are comparing phenolate ions, which have similar pK a values for their phenolics, the signal intensities in parentheses can be comparative quantitative values of the compounds in the extracts.

of the cytotoxic evaluation of E. tirucalli fractions with their chemical composition
Euphol/tirucalol tetracyclic triterpenes and pentacyclic triterpenes (lupeol, lupenone, friedelin, and glutinol) detected by GC-MS in hexane and dichloromethane fractions are known for their cytotoxic activity on different tumor cells (Lin et al. 2012;de Oliveira et al. 2021).Lupeol, for example, inhibited the proliferation of BGC823, N87, and HGC27 gastric cancer cells with significant inhibition rates from 50 µg/mL to 200 µg/mL, in addition to increase NK cell proliferation and lupenone had anti-cancer activity against breast cancer cells MCF-7 (IC 50 8.07 µg/mL) (Wu et al. 2013;Xu et al. 2018).The higher relative concentration of these triterpenes in the hexane fraction seems to be the reason for their greater cytotoxicity on AGS cells when compared to the dichloromethane fraction.
As reviewed by Mohammadinejad et al. (2021) ellagic acid contains anticancer activity as an interesting molecular mediator in signaling pathways in different human cancers (Mohammadinejad et al. 2021).In relation to gastric cancer, Lim et al. (2019) have suggested that ellagic acid suppresses acidity-enhanced migration and invasion of gastric cancer cells via inhibition of the expression of multiple factors (COX 1, COX2, snail, twist 1, and c-myc) (Lim et al. 2019).They concluded ellagic acid may be an effective agent for cancer treatment under acidosis.The cytotoxic activity of the ethyl acetate fraction (120.9 ± 2.21 µg/mL) was about ten times lower than that of the aqueous fraction (13.08 ± 0.99 µg/mL).Although it is abundant in phenolic compounds (tannins and flavonoids), of which hydrolyzable tannins are known for their cytotoxicity, the presence of ellagic acid in the aqueous fraction was important for the antiproliferative activity in gastric cancer.
Therefore, the greater cytotoxic activity of the ethanol extract of the aerial parts of Euphorbia tirucalli for gastric cancer, when compared to latex, seems to originate from the antiproliferative effects of ellagic acid and triterpenes.

Experimental
Experimental part is provided as supplemental material

Conclusion
For gastric adenocarcinoma, E. tirucalli is a source of antiproliferative substances.The ethanol extract of the aerial parts was more cytotoxic when compared to the latex extracted from the plant, probably due to a synergistic effect of triterpenes and ellagic acid.The clinical potential of these two products for the treatment of gastric diseases should be investigated.
index on hP-5 Ms. b n.i: not identified.

Table 1 .
chemical composition of the hexane/dichloromethane fractions of the aerial parts of E. tirucalli.

Table 2 .
More intense peaks of phenolic compounds from ethyl acetate and aqueous fractions of E. tirucalli aerial parts.