Metabolites Profile of Extracts and Fractions of Erythroxylum mexicanum Kunth by UHPLC-QTOF-MS/MS and its Antibacterial, Cytotoxic and Nitric Oxide Inhibitory Activities.

The present study shows the untargeted metabolite profiling and in vitro antibacterial, cytotoxic, and nitric oxide (NO) inhibitory activities of the methanolic leaves extract (MLE) and methanolic stem extract (MSE) of Erythroxylum mexicanum, as well as the fractions from MSE. Using ultra-high performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), a total of 70 metabolites were identified; mainly alkaloids in the MLE, while the MSE showed a high abundance of diterpenoids. The MSE fractions exhibited differential activity against Gram-positive bacteria. Notably, the hexane fraction (HSF) against Streptococcus pyogenes ATCC 19615 (MIC=62.5 µg/mL) exhibited a bactericidal effect. The MSE fractions exhibited cytotoxicity against all cancer cell lines tested, with selectivity towards them compared to a noncancerous cell line. Particularly, the HSF and chloroform fraction (CSF) showed the highest cytotoxicity against prostate cancer (PC-3) cells, with IC50 values of 19.9 and 18.1 µg/mL and selectivity indexes of 3.8 and 4.2, respectively. Both the HSF and ethyl acetate (EASF) fractions of the MSE inhibited NO production in RAW 264.7 macrophages, with NO production percentages of 50.0 % and 51.7 %, respectively, at a concentration of 30 µg/mL. These results indicated that E. mexicanum can be a source of antibacterial, cytotoxic, and anti-inflammatory metabolites.

UPLC-QTOF-MS analysis of cytotoxic and antibacterial extracts of Hechtia glomerata Zucc.

Hechtia glomerata, a Mexican medicinal plant employed against bacterial infections and as food, is taxonomically related to the genus Tillandsia which has anticancer activity. Organic and aqueous extracts of H. glomerata leaves were prepared and tested for cytotoxic and antibacterial activity. UPLC-QTOF-MS analysis determined the chemical composition of active extracts to find cytotoxic and antibacterial compounds. Hexane extract was cytotoxic against HepG2, Hep3B and MCF7 (IC50: 24-28 µg/mL), whereas CHCl3/MeOH extract against PC3 and MCF7 (IC50: 25 and 32 µg/mL). CHCl3/MeOH extract showed antibacterial activity against Staphylococcus aureus and Enterococcus faecium (MIC: 125 and 62.5 µg/mL). Hexane extract cytotoxic compounds were ß-sitosterol, stigmasterol, phytol and ursolic acid. CHCl3/MeOH extract antibacterial and/or cytotoxic compounds were daucosterol, oleanolic acid, resveratrol, quercetin, kaempferol, apigenin, cyanidin, p-coumaric acid and caffeic acid. This plant could be useful against bacterial infections and cancer. However, in vivo studies are needed to determine its toxicity and therapeutic efficacy.

Cytotoxic Fractions from Hechtia glomerata Extracts and p-Coumaric Acid as MAPK Inhibitors.

Preliminary bioassay-guided fractionation was performed to identify cytotoxic compounds from Hechtia glomerata, a plant that is used in Mexican ethnomedicine. Organic and aqueous extracts were prepared from H. glomerata's leaves and evaluated against two cancer cell lines. The CHCl3/MeOH (1:1) active extract was fractionated, and the resulting fractions were assayed against prostate adenocarcinoma PC3 and breast adenocarcinoma MCF7 cell lines. Active fraction 4 was further analyzed by high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry analysis to identify its active constituents. Among the compounds that were responsible for the cytotoxic effects of this fraction were flavonoids, phenolic acids, and aromatic compounds, of which p-coumaric acid (p-CA) and its derivatives were abundant. To understand the mechanisms that underlie p-CA cytotoxicity, a microarray assay was performed on PC3 cells that were treated or not with this compound. The results showed that mitogen-activated protein kinases (MAPKs) that regulate many cancer-related pathways were targeted by p-CA, which could be related to the reported effects of reactive oxygen species (ROS). A molecular docking study of p-CA showed that this phenolic acid targeted these protein active sites (MAPK8 and Serine/Threonine protein kinase 3) at the same binding site as their inhibitors. Thus, we hypothesize that p-CA produces ROS, directly affects the MAPK signaling pathway, and consequently causes apoptosis, among other effects. Additionally, p-CA could be used as a platform for the design of new MAPK inhibitors and re-sensitizing agents for resistant cancers.

Achillin Increases Chemosensitivity to Paclitaxel, Overcoming Resistance and Enhancing Apoptosis in Human Hepatocellular Carcinoma Cell Line Resistant to Paclitaxel (Hep3B/PTX).

Multidrug resistance (MDR) has become a major obstacle in the treatment of cancer, and is associated with mechanisms such as increased drug outflow, reduction of apoptosis, and/or altered drug metabolism. These problems can be mitigated by the coadministration of agents known as chemosensitizers, as they can reverse resistance to anticancer drugs and eventually resensitize cancer cells. We explore the chemosensitizing effect of Achillin, a guaianolide-type sesquiterpene lactone isolated from the Mexican medicinal plant Artemisia ludovisiana, to reverse MDR in Hep3B/PTX cells of hepatocellular carcinoma, which present resistance to paclitaxel (PTX). Achillin showed an important effect as chemosensitizer; indeed, the cytotoxic effect of PTX (25 nM) was enhanced, and the induction of G2/M phase cell cycle arrest and apoptosis were potentiated when combining with Achillin (100 µM). In addition, we observed that Achillin decreases P-gp levels and increases the intracellular retention of doxorubicin in Hep3B/PTX cells; in addition, homology structural modeling and molecular docking calculations predicted that Achillin interacts in two regions (M-site and R-site) of transporter drug efflux P-glycoprotein (P-gp). Our results suggest that the chemosensitizer effect demonstrated for Achillin could be associated with P-gp modulation. This work also provides useful information for the development of new therapeutic agents from guaianolide-type sesquiterpene lactones like Achillin.

16-Hydroxy-Lycopersene, a Polyisoprenoid Alcohol Isolated from Tournefortia hirsutissima, Inhibits Nitric Oxide Production in RAW 264.7 Cells and Induces Apoptosis in Hep3B Cells.

Three polyisoprenoid alcohols were isolated from the leaves of Tournefortia hirsutissima by a bioassay-guided phytochemical investigation. The compounds were identified as 16-hydroxy-lycopersene (Compound 1), (Z8,E3,ω)-dodecaprenol (Compound 2) and (Z9,E3,ω)-tridecaprenol (Compound 3). Compound 1, an unusual polyisoprenoid, was characterized by 1D and 2D NMR. We also determined the absolute configuration at C-16 by the modified Mosher's method. The in vitro antiproliferative and anti-inflammatory activities of the isolated compounds were evaluated. Among isolates, Compound 1 moderately inhibited the nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. On the other hand, Compound 1 displayed selective antiproliferative activity against HeLa, PC3, HepG2 and Hep3B cancer cells and was less potent against IHH non-cancerous cells. Compound 1 in Hep3B cells showed significant inhibition of cell cycle progression increasing the sub-G1 phase, suggesting cell death. Acridine orange/ethidium bromide staining and Annexin V-FITC/PI staining demonstrated that cell death induced by Compound 1 in cells Hep3B was by apoptosis. Further study showed that apoptosis induced by Compound 1 in Hep3b cells is associated with the increase of the ratio of Bax/Bcl-2, and caspase 3/7 activation. These results suggest that Compound 1 induce apoptotic cell death by the mitochondrial pathway. To our knowledge, this is the first report about the presence of polyprenol Compounds 1-3 in T. hirsutissima, and the apoptotic and anti-inflammatory action of Compound 1.

Gallic acid sensitizes paclitaxel-resistant human ovarian carcinoma cells through an increase in reactive oxygen species and subsequent downregulation of ERK activation.

Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N­acetyl-L­cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC. Taken together, these results indicate that GA sensitizes PTX-resistant ovarian carcinoma cells via the ROS­mediated inactivation of ERK, and suggest that GA could represent a useful co-adjuvant to PTX in ovarian carcinoma treatment.

Phenolic Compounds Isolated from Caesalpinia coriaria Induce S and G2/M Phase Cell Cycle Arrest Differentially and Trigger Cell Death by Interfering with Microtubule Dynamics in Cancer Cell Lines.

Caesalpinia coriaria (C. coriaria), also named cascalote, has been known traditionally in México for having cicatrizing and inflammatory properties. Phytochemical reports on Caesalpinia species have identified a high content of phenolic compounds and shown antineoplastic effects against cancer cells. The aim of this study was to isolate and identify the active compounds of a water:acetone:ethanol (WAE) extract of C. coriaria pods and characterize their cytotoxic effect and cell death induction in different cancer cell lines. The compounds isolated and identified by chromatography and spectroscopic analysis were stigmasterol, ethyl gallate and gallic acid. Cytotoxic assays on cancer cells showed different ranges of activities. A differential effect on cell cycle progression was observed by flow cytometry. In particular, ethyl gallate and tannic acid induced G2/M phase cell cycle arrest and showed interesting effect on microtubule stabilization in Hep3B cells observed by immunofluorescence. The induction of apoptosis was characterized by morphological characteristic changes, and was supported by increases in the ratio of Bax/Bcl-2 expression and activation of caspase 3/7. This work constitutes the first phytochemical and cytotoxic study of C. coriaria and showed the action of its phenolic constituents on cell cycle, cell death and microtubules organization.